The Interactive Fly

Genes involved in tissue and organ development

Midgut

Anterior midgut Transcription factors achaete brinker CrebA CrebB-17A daughterless dimmed dmyc Ecdysone-induced protein 74EF Ecdysone-induced protein 75B Ecdysone receptor Eip93F Ets at 21C extra-extra extra machrochaete forkhead Fos-related antigen GATAe Hairless Helix loop helix protein 106 Hormone receptor like/Hr46 (common alternative name: DHR3) Hormone receptor-like in 96 hindsight homothorax huckebein modulo Neurotactin odd-skipped p53 PAR-domain protein 1 pdm1 polyhomeotic Pox meso Ptx1 schnurri scute serpent short gastrulation single minded snail (indirectly from mesoderm) spalt tango teashirt tinman (indirectly from mesoderm) tramtrack twist vrille X-box-binding protein 1 Other Anktm1 (alternative name: dTRPA1) Autophagy-related 8a big brain Calcium/calmodulin dependent protein kinase 1 COP9 complex homolog subunit 5 crossveinless c death executioner Bcl-2 homologue decapentaplegic (indirect from mesoderm) Delta disc proliferation abnormal Diuretic hormone 31 Drosulfakinin Ecdysone-induced protein 63E Epidermal growth factor receptor fear of intimacy Ferritin 1 heavy chain homologue and Ferritin 2 light chain homologue Focal adhesion kinase-like geminin Glial Lazarillo hoka homer Inositol 1,4,5,-tris-phosphate receptor inscuteable (preferred name: not enough muscle) kakapo Keren lethal (2) giant larvae Lipid storage droplet-2 Moesin-like Mothers against dpp misato myoblast city Myosin 31DF myospheroid Nedd2-like caspase not enough muscle pebble Pez pitchoune Polypeptide N-Acetylgalactosaminyltransferase 4 Ptp52F Rac1 refractory to sigma P Rheb rhomboid rolling pebbles scrawny shotgun skittles Syntaxin 1A takeout Talin Toll tolloid related 1 Transforming growth factor beta at 60A vein visceral mesodermal armadillo-repeats

Posterior midgut Transcription factors achaete asense brachyenteron caudal cubitus interruptus daughterless dmyc E(spl) region transcript m7 extra-extra forkhead Hairless huckebein invected labial (cooper cells) miranda optomotor-blind-related-gene-1 PAR-domain protein 1 pdm1 schnurri scute serpent short gastrulation single minded snail spalt tailless tinman (indirect) tramtrack T-related gene (preferred name - brachyenteron) vnd/NK2 Other arc big brain Calcium/calmodulin dependent protein kinase 1 canoe COP9 complex homolog subunit 5 decapentaplegic (indirect) Delta disc proliferation abnormal fear of intimacy folded gastrulation Gliotactin hodor (preferred name: pH-sensitive chloride channel 2) inscuteable myospheroid pitchoune pH-sensitive chloride channel 2 (common alternative name: Hodor) Notch Snakeskin Src oncogene at 42A shotgun Syntaxin 1A Toll tolloid related 1 wunen and wunen-2

Midgut

Transcription factors achaete cryptocephal daughterless defective proventriculus E2F transcription factor 2 engrailed extradenticle extra machrochaete fruitless grain grainyhead Hepatocyte nuclear factor 4 hopscotch knirps labial mastermind moira MTF-1 NK1 (synonym: S59) O-GlcNAc transferase (preferred name: super sex combs) pangolin period prospero punt ribbon scab schnurri scute seven in absentia (sina) short gastrulation Signal-transducer and activator of transcription protein at 92E slow border cells Sox21a Sox100b Suppressor of Hairless

Other Adenosine receptor ahuizotl Apc-like Autophagy-specific gene 9 bark beetle (also known as Anakonda) basket (also known as JNK) Buffy Calmodulin Clathrin heavy chain corkscrew crumbs domeless double parked dystrophin enabled Fasciclin2 fat facets frayed frazzled GART trifunctional enzyme headcase I'm not dead yet Inositol-requiring enzyme-1 Integrin linked kinase kramer Laminin A Laminin B1 Licorne Matrix metalloproteinase 1 Merlin mesh Myd88 Myosin 61F myospheroid NADPH oxidase Niemann-Pick Type C-1 Niemann-Pick Type C-2 Notch Ornithine decarboxylase antizyme outstretched pancreatic eIF-2α kinase p38b Peptidoglycan recognition protein LA Peptidoglycan recognition protein LC Peptidylglycine-alpha-hydroxylating monooxygenase PNGase-like rhomboid rugose Scab Secreted decoy of InR (midgut mesoderm) shotgun slit slowpoke small wing alpha Spectrin spinster Synaptobrevin Tie-like receptor tyrosine kinase trio WD repeat domain 62 wee windpipe wishful thinking

Origin of endoderm and the midgut

The midgut is derived from the anterior and posterior midgut primordia during the process of gastrulation [Images]. It should be kept in mind that the most terminal aspects of the embryo are fated to become gut endoderm. The terminal system (torso), regulating tailless and huckebein are responsible for this fate determination. Gastrulation is the defining event of gut morphogenesis. The anterior midgut is formed from the anterior midgut primordium; the posterior midgut is derived from the posterior midgut primordium, and the midgut proper is derived from endodermal cells that migrate from both anterior and posterior primordia. The gut is enshrouded in mesoderm which forms vascular musculature around the gut, and is also responsible for creating the gastric ceca. Overlying mesoderm communicates with the gut by secreted factors and through contact.

Dpp has a prime function during endoderm induction in Drosophila. Dpp is secreted from the outer cell layer of the embryonic midgut (the visceral mesoderm) where its main source of expression in parasegment ps7 depends directly on the homeotic gene Ultrabithorax. In the same cell layer, Dpp stimulates expression of another extracellular signal, Wingless (Wg), in a neighboring parasegment that, in turn, feeds back to ps7 to stimulate Ubx expression. Thus, Dpp is part of a "parautocrine" feedback loop for Ubx (i.e., an autocrine feedback loop based partly on paracrine action that sustains its own expression through Dpp and Wg). Dpp also spreads to the inner layer of the embryonic midgut, the endoderm, where it synergizes with Wg to induce expression of the homeotic gene labial (lab). To achieve this, Dpp locally elevates the endodermal expression levels of Drosophila D-Fos with which it cooperates to induce lab. Differentiation of various cell types in the larval gut depends on these inductive effects of Dpp and Wg (Bienz, 1997 and references).

A secondary signal has been discovered with a permissive role in this process; it comes from Vein, a neuregulin-like ligand that stimulates the Epidermal growth factor receptor (Egfr) and Ras signaling. Dpp and Wg up-regulate vein expression in the midgut mesoderm in two regions overlapping the Dpp sources. Experiments based on lack of function and ectopic stimulation of Dpp and Egfr signaling show that these two pathways are functionally interdependent and that they synergize with one another other, revealing functional intertwining. The transcriptional response elements for the Dpp signal in midgut enhancers from homeotic target genes are bipartite, comprising CRE sites as well as binding sites for the Dpp signal-transducing protein Mad. Of these sites, the CRE seems to function primarily in the response to Ras. Since up-regulation of vein requires dpp and wg, Vein is considered a secondary signal of Dpp and Wg. Vein stimulates homeotic gene expression in both cell layers of the midgut (Szüts, 1998).

Physiological and stem cell compartmentalization within the Drosophila midgut

The Drosophila midgut is maintained throughout its length by superficially similar, multipotent intestinal stem cells that generate new enterocytes and enteroendocrine cells in response to tissue requirements. This study found that the midgut shows striking regional differentiation along its anterior-posterior axis. At least ten distinct subregions differ in cell morphology, physiology and the expression of hundreds of genes with likely tissue functions. Stem cells also vary regionally in behavior and gene expression, suggesting that they contribute to midgut sub-specialization. Clonal analyses showed that stem cells generate progeny located outside their own subregion at only one of six borders tested, suggesting that midgut subregions resemble cellular compartments involved in tissue development. Tumors generated by disrupting Notch signaling arose preferentially in three subregions and tumor cells also appeared to respect regional borders. Thus, apparently similar intestinal stem cells differ regionally in cell production, gene expression and in the ability to spawn tumors (Marianes, 2013).

These experiments significantly expand previous knowledge of regional variation within the Drosophila midgut. At the levels of cell morphology, cell behavior and gene expression, the midgut is much more highly organized than a uniform cellular tube containing an acidic middle region or 'stomach'. Regionalization likely supports the complex metabolic tasks carried out by the midgut. Ingested food goes through multiple intermediate stages during digestion and these steps may be most efficient if carried out in a controlled sequence. Some of these steps are associated with an array of bacterial species that constitute the normal intestinal microbiome. This work will allow the function of genes, pathways, cells and regions within the midgut to be tested in digestion, tissue maintenance, microbiome function and immunity (Marianes, 2013).

After this work was submitted for publication (Buchon et al. (2013) also described midgut regionalization; they classified five major midgut divisions (R1–R5), 13 total subregions (e.g., R1a and R1b), and 2 microregions (B_R2–3, B_R3–4). Converting counts of cell number within the 10 regions described in this to the fractional length coordinates used by (Buchon, 2013) suggests a close correspondence between the studies: A1 = R1a + R1b; A2 = R2a + R2b; A3 = R2c; Cu = R3a + R3b; LFC = R3c; Fe = B_R3–R4, P1 = R4a + R4b; P2 = R4c; P3 = R5a; P4 = R5b. In the case of single regions in the current system that Buchon split in two, differences in gene expression were also usually noted; for example, between the anterior and posterior Cu region Buchon (2013) or the subregions of A2 and P1 that differ in lipid accumulation. Enterocyte morphology and qualitative gene expression differences were noted in defining subregions. In contrast to the current study, Buchon (2013) views constrictions to be of primary importance and define two constrictions as micro-regions in their own right. This study found that enterocyte subregions abut directly, and favors the view that constrictions are mesodermal features, not microregions (Marianes, 2013).

The results argue strongly that the striking regionalization of structure and gene expression within the midgut is maintained at least in part by regional differences between their resident stem cells. In the midgut subregions surrounding five different boundaries, no single stem cell was found that produced differentiated cells on the opposite side of the boundary, that is from a region different from the one in which it resided. All the ‘non-crossing’ clones contacted the regional boundary, and in 78% the founder stem cell was located on or within one cell of the boundary, such that progeny cells could have reached the adjacent region prior to differentiation. In contrast, boundary clones with the same general properties almost always crossed the LFC/Fe boundary, showing that ‘non-crossing’ behavior would not occur by chance. Consistent with the existence of epigenetic differences in stem cells that limit trans-regional differentiation, clones frequently pushed into adjacent regions at the boundary, but retained their autonomous identity based on marker expression. Mechanical and/or adhesive forces may also contribute to maintaining some regional boundaries. Indeed, the tendency of tumor cells to respect regional boundaries suggests that cell-cell interactions at boundaries are likely to be important as well as stem cell programming (Marianes, 2013).

The behavior of ISC clones at regional boundaries is reminiscent of the behavior of clones in developing imaginal discs at 'compartment' boundaries. In the developing Drosophila embryo and imaginal discs, the engrailed gene and hedgehog signaling play important roles in defining posterior compartments. No expression of engrailed or the closely related gene invected was seen in any midgut region, and expression of hedgehog pathway components was similar on both sides of non-crossing boundaries such as Fe/P1. Dorsal ventral compartments in the developing wing are mediated by apterous and by Notch signaling. Apterous was expressed only at very low levels throughout all regions and Serrate was found at significant levels only in posterior regions 2–4 where the gene is dispensable for cell differentiation. In the developing vertebrate brain, Hox genes are important in specifying developmental compartments. However, Hox genes are only expressed at very low levels in endodermal cells during embryogenesis and these genes were very weakly expressed in the RNAseq studies, perhaps due to expression in non-endodermal cells within these samples. Consequently, the genetic basis for adult midgut compartmentalization probably differs from previously studied examples of tissue regionalization (Marianes, 2013).

The homeotic transcription factor labial (lab) is an outstanding candidate for a regional regulatory factor. In the embryonic and larval gut, lab is required for Cu cell specification, differentiation and maintenance. The gene is expressed in copper cells, but not elsewhere in the larval midgut, and similar specificity of lab expression was seen in the adult. When lab is mis-expressed during embryonic development in other midgut regions, the copper region can expand. Endodermal cell identity along the a/p axis may be determined by signals from adjacent mesoderm during embryogenesis, and then fixed by the induction of secondary factors such as lab. Gene expression within the midgut muscles might play a similar role in the adult midgut, however, expression boundaries of muscle genes were frequently offset with respect to endodermal regions. Whether this bears any relationship to the documented offset in homeotic gene expression between the ectoderm and visceral endoderm in embryonic development remains unclear. A key question is whether individual or combinations of differentiation regulators analogous to lab specify other midgut subregions in which the ISCs fail to generate cells across regional boundaries. The RNAseq data should provide a valuable resource in identifying such factors. For example, one potential candidate, the homeotic gene defective proventriculus (dve), functions in copper cells and its expression was observed to fall sixfold between Fe and P1 (Marianes, 2013).

One remaining question is whether a pre-existing pattern of larval midgut subdivision plays any role in the origin of adult midgut organization. The larval gut has a middle acidic region containing copper cells and an iron region like the adult tissue, and EM studies show additional morphological differences. However, it is not known whether regions analogous to other eight midgut domains described in this study exist in larvae. The larval midgut contains nests of diploid intestinal precursors that proliferate following pupariation to build the adult gut and establish its ISCs. Larval midgut domains might serve as a template for adult regionalization if gut precursor cells within each region already differ autonomously and do not mix during pupal development. However, cells do cross boundaries between the hindgut and midgut during pupal gut development. Identical regionalization within larval and adult guts might be disadvantageous to species with very different larval and adult diets, hence many adult midgut regions are likely to be established de novo or to be re-specified during pupal development (Marianes, 2013).

Many mammalian tissues such as skin, muscle, lung, liver, and intestine contain thousands of spatially dispersed stem cells, like the Drosophila midgut. The current studies raise the question of whether these tissues exhibit finer grained regional patterns of gene expression than has been previously recognized, patterns that might be supported by small autonomous differences in their stem cells. Currently, the strongest indication for such regionalization comes from studies of the intestine. Lineage labeling shows that similar stem cells expressing Lgr5 exist along the mammalian gut despite the fact that enterocytes, enteroendocrine cells and bacterial symbionts differ regionally. For example, iron absorption in mammals takes place primarily in the duodenum, a specialized subregion of the small intestine located just downstream from the acidic stomach. This is similar to the position of the midgut iron region just downstream from the acid-producing parietal cells of the Cu and LFC regions. The antibacterial lectin RegIIIγ, which like Drosophila PGRPs recognizes bacterial peptidoglycans, is expressed most prominently in the distal region of the small intestine. The existence of tissue and stem cell regionalization in other mammalian tissues deserves further detailed investigation (Marianes, 2013).

The human large intestine is much more prone to cancer than the small intestine. The current studies suggest that regional differences in the properties of apparently similar stem cells and tissue cells contribute to such differences. The midgut zones most favorable for the expansion of Notch-deficient cells showed pre-existing differences in Notch signaling within the early enterocyte lineage. Delta expression did not decrease shortly after ISC division, as in other regions, and Notch signaling persisted throughout enterocyte development. Curiously, same tumor-prone regions with persistent Notch signaling also were enriched in lipid droplets. At present it is not clear how the altered signaling, regional metabolic activity and tumor susceptibility are related. Additionally, regional differences in the microbiome, as suggested by the observation of domain-specific expression of PGRP proteins, may also influence the occurrence of cancer. Gastric bacteria such as Helicobacter pylori contribute to stomach cancer, while colonic Bacteroides fragilis likely promote gut cell DNA damage and colon cancer. Regional tissue differences likely also affect rates of tumor progression and metastasis. These observations emphasize the importance of understanding tissues region by region (Marianes, 2013).

In sum, the Drosophila midgut provides an outstanding tissue in which to explore and understand the significance of intrinsic stem cell differences. GAL4 drivers were identified that allow gene expression to be manipulated in all intestinal cell types, including cells such as circular muscle and enteric neurons that are thought to contribute to niche function. Will altering the expression of genes that normally differ between regions cause ISCs to generate cells with heterotypic characteristics? Such studies might eventually make it possible to stimulate medically useful responses from the endogenous stem cells that remain within a diseased tissue (Marianes, 2013).

Local control of intestinal stem cell homeostasis by enteroendocrine cells in the adult Drosophila midgut

Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream. This study reports a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts (beyond its known roles in development) as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2 (Rickets), the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP. This study has therefore identified a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells (Scopelliti, 2014).

Bursicon, also known as the tanning hormone, has been studied for decades due its essential role as the last hormone in the cascade of Ecdysis. In all invertebrate metazoa, this endocrine cascade is fundamental to coordinate molting events during animal lifetime, and in holometabolous insects, such as Drosophila, it control metamorphosis. Fly gene expression data suggest that the endocrine hormones and their cognate receptors involved in key stages of development may have other roles during adult animal life. However, these functional roles are largely unknown. This study is the first to demonstrate a role of Bursicon beyond development (Scopelliti, 2014).

A model is suggested in which Bursicon from enteroendocrine cells in the posterior midgut acts through DLGR2 to increase the production of cAMP within the VM, a mesenchymal ISC niche. This signaling limits the production of niche-derived, EGF-like Vein, leading to ISC quiescence. Burs protein expression was detected via immunolabeling in approximately 50% of the enteroendocrine cells of the posterior midgut, which appeared in stochastic spatial distribution within the most posterior segment of the adult midgut. Given that the percentage of enteroendocrine cells expressing Burs remained constant, it is likely that Burs expression might label a subtype of enteroendocrine cells within the midgut (Scopelliti, 2014).

The evidence indicates that burs mRNA levels are upregulated in the midgut during the phase of relative ISC quiescence in mature animals under homeostatic conditions. Conversely, during the phase of growth of the young immature gut or the dysplastic phase of the aging gut (both characterized by relative high rates of ISC proliferation) burs levels were relatively low, and burs overexpression was sufficient to suppress ISC proliferation. Therefore, the results provide the first demonstration of a tissue-intrinsic role of enteroendocrine cells, which drives homeostatic stem cell quiescence in the adult Drosophila midgut. Future studies should further characterize the upstream mechanisms controlling Burs production in the midgut, which might be linked to the yet undefined events involved in the regulation of overall tissue size and proliferation (Scopelliti, 2014).

Enteroendocrine cells are well known for their ability to mediate interorgan communication via hormone secretion into the bloodstream. The results demonstrate a novel, local role for enteroendocrine cells as paracrine regulators of stem cell proliferation. Such a mechanism could be phylogenetically conserved and take place in the mammalian intestine and other tissues of the gastrointestinal tract. This may therefore represent an unappreciated but yet important function of these cells beyond their conventional endocrine role (Scopelliti, 2014).

Mammalian LGRs are thought to drive ISC proliferation acting as receptors for the Wnt agonists R-spondins, which are unrelated to Bursicon and absent in the fly genome. Accordingly, no changes were detected in either Wg levels or signaling in burs or rk mutant midguts (Scopelliti, 2014).

Unexpectedly for a Wnt agonists and positive regulators of ISC proliferation, recent studies suggest that LGRs can act as tumor suppressors in colorectal cancer. Moreover, mammalian LGRs have also been shown to be activated by alternative ligands and promote cAMP signaling after the binding of yet unknown ligand(s). Therefore, it is likely that an unidentified functional homolog of Bursicon may act as an additional LGR ligand in mammals, driving ISC quiescence by regulating mitogenic signals from the surrounding niche as described here. Remarkably, DLGR2 shows closer sequence homology to the still poorly characterized LGR4, which (consistent with the Drosophila data) is expressed by the murine intestinal smooth-muscle layers and can signal via cAMP production. Consistent with the model, a recent study correlates loss-of-function mutations in LGR4 with multiple types of human epithelial carcinomas. Therefore, the results uncovered a novel biological role for LGRs, which is likely to impact mammalian stem cell research by providing a mechanistic framework for the so far correlative mammalian evidence toward a potential role of LGRs as tumor suppressor genes (Scopelliti, 2014).

Altogether, these results demonstrate a novel paradigm in the regulation of intestinal homeostasis involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells, which may provide insights into other stem cell based systems (Scopelliti, 2014).

The adult Drosophila gastric and stomach organs are maintained by a multipotent stem cell pool at the foregut/midgut junction in the cardia (proventriculus)

Stomach cancer is the second most frequent cause of cancer-related death worldwide. Thus, it is important to elucidate the properties of gastric stem cells, including their regulation and transformation. To date, such stem cells have not been identified in Drosophila. Using clonal analysis and molecular marker labeling, this study has identified a multipotent stem-cell pool at the foregut/midgut junction in the cardia (proventriculus). Daughter cells migrate upward either to anterior midgut or downward to esophagus and crop. The cardia functions as a gastric valve and the anterior midgut and crop together function as a stomach in Drosophila; therefore, the foregut/midgut stem cells have been named gastric stem cells (GaSC). JAK-STAT signaling regulates GaSC proliferation, Wingless signaling regulates GaSC self-renewal, and hedgehog signaling regulates GaSC differentiation. The differentiation pattern and genetic control of the Drosophila GaSCs suggest the possible similarity to mouse gastric stem cells. The identification of the multipotent stem cell pool in the gastric gland in Drosophila will facilitate studies of gastric stem cell regulation and transformation in mammals (Singh, 2011).

This study has identified multipotent gastric stem cells at the junction of the adult Drosophila foregut and midgut. The GaSCs express the Stat92E-GFP reporter, wg-Gal4 UAS-GFP, and Ptc, and are slowly proliferating. The GaSCs first give rise to the fast proliferative progenitors in both foregut and anterior midgut. The foregut progenitors migrate downward and differentiate into crop cells. The anterior midgut progenitors migrate upward and differentiate into midgut cells. However, at this stage because of limited markers availability and complex tissues systems at cardia location, it is uncertain how many types of cells are produced and how many progenitor cells are in the cardia. Clonal and molecular markers analysis suggest that cardia cells are populated from gastric stem cells at the foregut/midgut (F/M) junction; however, it cannot be ruled out that there may be other progenitor cells with locally or limited differential potential that may also take part in cell replacement of cardia cells. Nevertheless, the observed differentiation pattern of GaSCs in Drosophila may be similar to that of the mouse gastric stem cells. Gastric stem cells in the mouse are located at the neck-isthmus region of the tubular unit. They produce several terminally differentiated cells with bidirectional migration, in which upward migration towards lumen become pit cells and downward migration results in fundic gland cells (Singh, 2011).

Three signal transduction pathways differentially regulate the GaSC self-renewal or differentiation. The loss of JAK-STAT signaling resulted in quiescent GaSCs; that is, the stem cells remained but did not incorporate BrdU or rarely incorporated BrdU. In contrast, the amplification of JAK-STAT signaling resulted in GaSC expansion (Singh, 2011).

These observations indicate that JAK-STAT signaling regulates GaSC proliferation. In contrast, the loss of Wg signaling resulted in GaSC loss, while the amplification of Wg resulted in GaSC expansion, indicating that Wg signaling regulates GaSC self-renewal and maintenance. Finally, the loss of Hh signaling resulted in GaSC expansion at the expense of differentiated cells, indicating that Hh signaling regulates GaSC differentiation. The JAK-STAT signaling has not been directly connected to gastric stem cell regulation in mammal. However, the quiescent gastric stem cells/progenitors are activated by interferon γ (an activator of the JAK-STAT signal transduction pathway), indicating that JAK-STAT pathways may also regulate gastric stem cell activity in mammals. Amplification of JAK-STAT signaling resulted in expansion of stem cells in germline, posterior midgut and malpighian tubules of adult Drosophila. In the mammalian system, it has been reported that activated STAT contributes to gastric hyperplasia and that STAT signaling regulates gastric cancer development and progression. Wnt signaling has an important function in the maintenance of intestinal stem cells and progenitor cells in mice and hindgut stem cells in Drosophila, and its activation results in gastrointestinal tumor development. Tcf plays a critical role in the maintenance of the epithelial stem cell. Mice lacking Tcf resulted in depletion of epithelial stem-cell compartments in the small intestine as well as being unable to maintain long-term homeostasis of skin epithelia. A recent study even demonstrates that the Wnt target gene Lgr5 is a stem cell marker in the pyloric region and at the esophagus border of the mouse stomach. Further, it has been found that overactivation of the Wnt signaling can transform the adult Lgr5^+ve stem cells in the distal stomach, indicating that Wnt signaling may also regulate gastric stem cell self-renewal and maintenance in the mammal. Sonic Hedgehog (Shh) and its target genes are expressed in the human and rodent stomach. Blocking Shh signaling with cyclopamine in mice results in an increase in the cell proliferation of gastric gland, suggesting that Shh may also regulate the gastric stem cell differentiation in mice. These data together suggest that the genetic control of the Drosophila GaSC may be similar to that of the mammalian gastric stem cells (Singh, 2011).

The potential GaSCs niche. In most stem cell systems that have been well characterized to date, the stem cells reside in a specialized microenvironment, called a niche.66 A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The niche stromal cells often secrete growth factors to regulate stem cell behavior, and the stem cell niche plays an essential role in maintaining the stem cells, which lose their stem-cell status once they are detached from the niche (Singh, 2011).

Loss of the JAK-STAT signaling results in the GaSCs being quiescent; the stem cells remain but do not proliferate or rarely proliferate. The Dome receptor is expressed in GaSCs, while the ligand Upd is expressed in adjacent cells. Upd-positive hub cells function as a germline stem cell niche in the Drosophila testis. Further, thia study demonstrated that overexpression of upd results in GaSC expansion, suggesting that the Upd-positive cells may function as a GaSC niche. Furthermore, while Stat92E-GFP expression is regulated by the JAK-STAT signaling in other systems, its expression at the F/M junction seems independent of the JAK-STAT signaling because Stat92E-GFP expression is not significantly disrupted in the Stat92E^ts mutant flies, suggesting that the GaSCs may have unique properties (Singh, 2011).

The stomach epithelium undergoes continuous renewal by gastric stem cells throughout adulthood. Disruption of the renewal process may be a major cause of gastric cancer, the second leading cause of cancer-related death worldwide, yet the gastric stem cells and their regulations have not been fully characterized. A more detailed characterization of markers and understanding of the molecular mechanisms control gastric stem cell behavior will have a major impact on future strategies for gastric cancer prevention and therapy. The information gained from this report may facilitate studies of gastric stem cell regulation and transformation in mammals (Singh, 2011).

Intestinal lipid droplets as novel mediators of host-pathogen interaction in Drosophila

Lipid droplets (LDs) are lipid carrying multifunctional organelles, which might also interact with pathogens and influence the host immune response. However, the exact nature of these interactions remains currently unexplored. This study shows that systemic infection of Drosophila adult flies with non-pathogenic E. coli, the extracellular bacterial pathogen P. luminescens or the facultative intracellular pathogen P. asymbiotica results in intestinal steatosis marked by lipid accumulation in the midgut. Accumulation of LDs in the midgut also correlates with increased whole-body lipid levels characterized by increased expression of genes regulating lipogenesis. The lipid enriched midgut further displays reduced expression of enteroendocrine secreted hormone, Tachykinin. The observed lipid accumulation requires the Gram-negative cell wall pattern recognition molecule PGRP-LC, but not PGRP-LE, for the humoral immune response. Altogether, these findings indicate that Drosophila LDs are inducible organelles, which can serve as marker for inflammation and depending on the nature of the challenge they can dictate the outcome of the infection (Harsh, 2019).

The Drosophila E78 nuclear receptor regulates dietary triglyceride uptake and systemic lipid levels

Lipid levels are maintained by balancing lipid uptake, synthesis, and mobilization. Although many studies have focused on the control of lipid synthesis and mobilization, less is known about the regulation of lipid digestion and uptake. This study show that the Drosophila E78A nuclear receptor plays a central role in intestinal lipid homeostasis through regulation of the CG17192 digestive lipase. E78A mutant adults fail to maintain proper systemic lipid levels following eclosion, with this effect largely restricted to the intestine. Transcriptional profiling by RNA-seq revealed a candidate gene for mediating this effect, encoding the predicted adult intestinal lipase CG17192. Intestine-specific disruption of CG17192 results in reduced lipid levels similar to that seen in E78A mutants. In addition, dietary supplementation with free fatty acids, or intestine-specific expression of either E78A or CG17192, is sufficient to restore lipid levels in E78A mutant adults. These studies support the model that E78A is a central regulator of adult lipid homeostasis through its effects on CG17192 expression and lipid digestion. This work also provides new insights into the control of intestinal lipid uptake and demonstrate that nuclear receptors can play an important role in these pathways (Praggastis, 2020).

A genetic framework controlling the differentiation of intestinal stem cells during regeneration in Drosophila

The speed of stem cell differentiation has to be properly coupled with self-renewal, both under basal conditions for tissue maintenance and during regeneration for tissue repair. Using the Drosophila midgut model, this study analyzed at the cellular and molecular levels the differentiation program required for robust regeneration. The intestinal stem cell (ISC) and its differentiating daughter, the enteroblast (EB), were observed to form extended cell-cell contacts in regenerating intestines. The contact between progenitors is stabilized by cell adhesion molecules, and can be dynamically remodeled to elicit optimal juxtacrine Notch signaling to determine the speed of progenitor differentiation. Notably, increasing the adhesion property of progenitors by expressing Connectin is sufficient to induce rapid progenitor differentiation. It was further demonstrated that JAK/STAT signaling, Sox21a and GATAe form a functional relay to orchestrate EB differentiation. Thus, this study provides new insights into the complex and sequential events that are required for rapid differentiation following stem cell division during tissue replenishment (Zhai, 2017).

Key questions in stem cell biology are how the pool of stem cells can be robustly expanded yet also timely contracted through differentiation to generate mature cells according to the need of a tissue, and what are the underlying mechanisms that couple stem cell proliferation and differentiation. Over the last years, the mechanisms underlying intestinal stem cell activation have been extensively studied in both flies and mammals, while the genetic control of progenitor differentiation, especially during regeneration, has only recently begun to be understood (Zhai, 2017).

The transcription factor Sox21a has recently been the focus of studies in fly intestines. Using a Sox21a-sGFP transgene, this study uncovered its dynamic expression pattern in intestinal progenitors. Higher levels of Sox21a were found in ISC during homeostatic conditions but in EB during regeneration, supporting the roles of Sox21a in both ISC maintenance and EB differentiation at different conditions. The highly dynamic expression pattern of Sox21a revealed by this sGFP-tagged transgene per se argues against accumulation and perdurance of GFP fusion protein. Indeed, immunostaining using an antibody against Sox21a also indicated stronger Sox21a expression in ISC in homeostatic condition and global activation of Sox21a in progenitors under DSS-induced regeneration. However, Chen (2016) suggested that Sox21a levels are always higher in EB than in ISC by applying another antibody against Sox21a. The inconsistency between these studies may have arisen from the differences in EB stages examined or the sensitivity of respective detection approaches (Zhai, 2017).

This study has analyzed the cellular processes required for efficient progenitor differentiation during regeneration. Three main findings are reported revealing: i) the importance of extended contact between a stem cell and its differentiating daughter, ii) the existence of specific mechanisms allowing fast differentiation during regeneration, and iii) the characterization of a genetic program instructing the transition from EB to EC. These results together led to a proposal of a molecular framework underlying intestinal regeneration that is discussed below step by step (Zhai, 2017).

By studying the mechanisms of Sox21a-induced differentiation, this study found that ISC establishes extended contact with its differentiating daughter within a progenitor pair. Increased interface contact was not only observed upon Sox21a expression but also during regeneration after bacterial infection and DSS-feeding. Since the presence of extended contact is rare in intestinal progenitors under homeostatic conditions, it is hypothesized that extended contact between progenitors is related to increased epithelial renewal as a mechanism to elicit optimal juxtacrine Notch signaling to accelerate the speed of progenitor differentiation. The observations that down-regulation of the cell adhesion molecules E-Cadherin or Connectin suppresses rapid progenitor differentiation upon regeneration, and that overexpression of Connectin is sufficient to promote differentiation, underline the importance of increased cell-cell contact in rapid differentiation. This study shows that one early role of Sox21a is to promote the formation of this contact zone, possibly through transcriptional regulation of Connectin. Further studies should identify the signals and pathways leading to the change of contact between progenitors to adjust the rate of differentiation (Zhai, 2017).

Intestinal progenitors with extended contact in non-homeostatic midguts have been observed in some studies, but their role and significance have not been analyzed. Previous studies have also shown that progenitor nests are outlined by E-Cadherin/β-Catenin complexes, yet it was not known whether different degrees of progenitor contact are associated with their ISC versus EB fate. Consistent with these results, recent modeling analyses suggested a positive correlation between the contact area of progenitor pairs and the activation of Notch signaling. Thus, it seems that an increase in the contact area between intestinal progenitors is a hallmark of progenitors that are undergoing accelerated differentiation towards ECs. Another study has suggested an inhibitory role of prolonged ISC-EB contact to restrict ISC proliferation. Collectively, these studies and the current findings suggest that the strong contact between ISC and EB promotes on one hand the efficient differentiation of EBs into mature intestinal cells while on the other hand preventing stem cells from over-dividing. Thus, it is hypothesize that alteration in the contact zone provides a mechanism for ensuring both the appropriate speed of differentiation and the timely resolution of stem cell proliferative capacity (Zhai, 2017).

A second finding of this study consists in revealing the existence of specific mechanisms accelerating differentiation for tissue replenishment. In addition to the extended contact discussed above, a difference was observed in the pattern of ISC division between homeostatic and highly regenerative intestines. The modes of ISC division in Drosophila have been the topic of intense discussion, and the general consensus is that it is associated with an asymmetric cell fate outcome, in which one cell remains an ISC and the other engages in differentiation. In line with these previous studies, the results support the notion that asymmetric cell division is the most prevalent mode of ISC division under homeostatic conditions, where the rate of epithelial renewal is low. However, use of ISC- and EB-specific markers shows that upon rapid regeneration an ISC divides into two cells both expressing the ISC marker Dl-GFP but with one cell showing weak Notch activity. Similarly to other Notch-mediated cell-fate decision systems, this study suggests that the two resulting Dl-GFP+ cells from a symmetric division stay in close contact and compete for the stem cell fate. While this study is not the first to postulate the existence of symmetric ISC division, the use of reliable ISC- and EB-specific markers allows better visualization of this process. Applying a dual-color lineage tracing system to unravel the final fate of respective cells in a Dl+-Dl+ pair could reinforce the existence of symmetric stem cell division. This is nevertheless technically challenging to apply here since all the current available lineage-tracing settings require a heat shock to initiate the labeling, which affects intestinal homeostasis (Zhai, 2017).

Importantly, this study shows that the genetic program required for fast intestinal regeneration differs from the one involved in basal intestinal maintenance. This study indicates that GATAe, Dpp signaling, and the cell adhesion molecules E-cadherin and Connectin are not critical for progenitor differentiation when the rate of epithelial renewal is low, whereas their roles become crucial upon active regeneration. It is speculated that many discrepancies in the literature can be reconciled by taking into consideration that some factors are required only for rapid differentiation but not in basal conditions. For instance, the implication of Dpp signaling in differentiation has been disputed, since one study focused on bacterial infection-induced regeneration while two other studies dealt with basal conditions. The current study points to a clear role of Dpp signaling in the differentiation process upon regeneration. Therefore, better defining the genetic program that allows adjusting the speed of differentiation would be of great interest (Zhai, 2017).

Cell fate determination and differentiation involve extensive changes in gene expression and possibly also gradual change of cell morphology. The EB to EC differentiation in the adult Drosophila intestine provides a model of choice to study this process. This transition includes changes in cell shape, an increase in cell size, DNA endoreplication leading to polyploidy and the activation of the set of genes required for EC function. This study has integrated a number of pathways (Notch, JAK/STAT and Dpp/BMP) and transcription factors (Sox21a and GATAe) into a sequential framework. It was further shown that Sox21a contributes to the EB-EC transition downstream of JAK/STAT but upstream of Dpp signaling and GATAe. The recurrent use of several factors, namely JAK/STAT, Sox21a and GATAe at different processes including ISC self-renewal and EB-EC differentiation is likely to be a general feature during cell fate determination, and somehow also complicates the study of differentiation. Future work should analyze how each of the factors interacts with the other in a direct or indirect manner. It would be interesting as well to further study how these factors shape intestinal regionalization as the gut exhibits conspicuous morphological changes along the length of the digestive tract (Zhai, 2017).

Several of the findings described in this study are likely to apply to the differentiation program that takes place in mammals. Since Notch signaling plays major roles in stem cell proliferation and cell fate specification from flies to mammals, it would be interesting to decipher whether in mammals changes in progenitor contact also impact differentiation speed and whether a specific machinery can accelerate progenitor differentiation when tissue replenishment is required (Zhai, 2017).

Drosophila MOV10 regulates the termination of midgut regeneration

The molecular mechanisms by which stem cell proliferation is precisely controlled during the course of regeneration are poorly understood. Namely, how a damaged tissue senses when to terminate the regeneration process, inactivates stem cell mitotic activity, and organizes ECM integrity remain fundamental unanswered questions. The Drosophila midgut intestinal stem cell (ISC) offers an excellent model system to study the molecular basis for stem cell inactivation. This study shows that a novel gene, CG6967 or dMOV10, is induced at the termination stage of midgut regeneration, and shows an inhibitory effect on ISC proliferation. dMOV10 encodes a putative component of the microRNA (miRNA) gene silencing complex (miRISC). The data, along with previous studies on the mammalian MOV10, suggest that dMOV10 is not a core member of miRISC, but modulates miRISC activity as an additional component. Further analyses identified direct target mRNAs of dMOV10-containing miRISC, including Daughter against Dpp (Dad), a known inhibitor of BMP/TGF-β signaling. RNAi knockdown of Dad significantly impaired ISC division during regeneration. Six miRNAs were identified that are induced at the termination stage and their potential target transcripts. One of these miRNAs, mir-1, is required for proper termination of ISC division at the end of regeneration. It is proposed that miRNA-mediated gene regulation contributes to the precise control of Drosophila midgut regeneration (Takemura, 2021).

A cell atlas of the adult Drosophila midgut

Studies of the adult Drosophila midgut have led to many insights in understanding of cell-type diversity, stem cell regeneration, tissue homeostasis, and cell fate decision. Advances in single-cell RNA sequencing provide opportunities to identify new cell types and molecular features. This study used single-cell RNA sequencing to characterize the transcriptome of midgut epithelial cells and identified 22 distinct clusters representing intestinal stem cells, enteroblasts, enteroendocrine cells (EEs), and enterocytes. This unbiased approach recovered most of the known intestinal stem cells/enteroblast and EE markers, highlighting the high quality of the dataset, and led to insights on intestinal stem cell biology, cell type-specific organelle features, the roles of new transcription factors in progenitors and regional variation along the gut, 5 additional EE gut hormones, EE hormonal expression diversity, and paracrine function of EEs. To facilitate mining of this rich dataset, a web-based resource is provided for visualization of gene expression in single cells. Altogether, this study provides a comprehensive resource for addressing functions of genes in the midgut epithelium (Hung, 2020).

Like its mammalian counterpart, the adult Drosophila midgut is a complex tissue composed of various cell types performing diverse functions, such as digestion, absorption of nutrients, and hormone production. Enterocytes (ECs) secrete digestive enzymes, and absorb and transport nutrients, whereas enteroendocrine cells (EEs) secrete gut hormones that regulate gut mobility and function in response to external stimuli and bacteria. The fly midgut is a highly regenerative organ that has been used extensively in recent years as a model system to characterize the role of signaling pathways that coordinate stem cell proliferation and differentiation in the context of homeostasis and regeneration. For example, EGFR, JAK/STAT, and Hippo signaling control intestinal stem cell (ISC) growth and proliferation, while Notch signaling regulates ISC differentiation. To maintain homeostasis, ISC proliferates and gives rise to a transient progenitor, the enteroblast (EB), defined by the expression of Su(H)GBE-lacZ, a Notch pathway activity reporter. In addition, both ISCs and EBs express the SNAIL family transcription factor escargot (esg). Polyploid ECs, characterized by the expression of Myosin31DF (Myo1A) and nubbin (also called pdm1), differentiate from EBs. In contrast, EEs, marked by the expression of prospero (pros), are derived from ISCs through distinct progenitors, called pre-EEs, that express Piezo, a cation channel that senses mechanical tension. In addition, the midgut is surrounded by visceral muscles, which control midgut movements and secrete niche signals, such as Wingless (Wg), the EGFR ligand Vein (Vn), and the JAK-STAT ligand Unpaired1 (Upd1) to control ISC activities (Hung, 2020).

Similar to the compartmentalized mammalian digestive tract, the fly midgut can be divided into regions with distinct morphological, histological, and genetic properties. For example, the middle region of the midgut, which contains a group of specialized copper cells, is acidic and resembles the mammalian stomach. In addition, EEs produce at least 10 different gut hormone peptides that are produced in specific regions: Allatostatins (AstA, AstB/Mip, AstC), Tachykinin (Tk), neuropeptide F (NPF), DH31, CCHa1, CCHa2, Orcokinin B, and Bursicon (Burs). AstA-producing EEs are located in the posterior region of the gut, whereas EEs in the anterior, middle, and first half of the posterior midgut produce AstC. Moreover, individual EEs are able to produce 2 combinations of different hormones. In particular, some NPF-producing EEs also produce Tk. The diversity and regional differences in EEs hinder the ability to comprehensively characterize subtypes of EEs using bulk RNA sequencing (RNA-seq) (Hung, 2020).

To further characterize gene expression and cell types in the adult fly midgut, single-cell RNA sequencing (scRNA-seq) was used, as it provides an unbiased approach to survey cell-type diversity, function, and define relationships between cell types. This study reveals molecular markers for each cell type, cell type-specific organelle features, regional differences among ECs, a transitional state of premature ECs, transcriptome differences between ISCs and EBs, 5 additional gut hormones, diverse hormone expression of EEs, paracrine function of EEs, a subset of EEs, and cell-type similarity between the fly and the mammalian gut. This study demonstrates how the dataset can be used to characterize new genes involved in gut cell lineage and in particular, it was demonstrated that the transcription factor klumpfuss suppresses EE formation. Finally, a web-based visualization resource was built that allows users to browse scRNA-seq data, query the expression of any genes of interest in different cell types, and compare the expression of any 2 genes in individual cells. Altogether, this study provides a valuable resource for future studies of the Drosophila midgut (Hung, 2020).

This study surveyed the cell types of the adult intestinal epithelium using scRNA-seq and identified all known cell types, 1 cell type (esg+ pros+) in the middle region of the midgut, differentiating ECs, and 5 unknown cell types (unk1, unk2, EC-like 1 to 3). This study recovered most previously known ISC/EBs and EEs markers, demonstrating the robustness of the scRNA-seq approach. Interestingly, gene expression analysis revealed that ISCs are enriched for free ribosomes and possess mitochondria with fewer cristae. Transcription factors expressed differently along the guts and cytoskeletal proteins and transcription factors preferentially expressed in the ISC/EB population were identified. In particular, this study validated that klu is specifically expressed in EBs, and knockdown of klu in ISC/EBs (with esg-Gal4) results in an increase of EEs, suggesting that klu inhibits EE differentiation. When the scRNA-seq study was performed using inDrop, a clear separation was not seen between cells that expressed Dl and cells expressing Notch downstream targets, E(spl)m3-HLH, E(spl)malpha-BFM, E(spl)mbeta-HLH, and the EB marker, klu. Thus, it was not certain whether this could be resolved using the 10x Genomics technology. Interestingly, using data from 10x Genomics this study was able to detect one subset of cells in the ISC/EB cluster that expresses Dl+ klu- (ISC) and another subset expressing Dl- klu+ (EB). Therefore, using data from 10x Genomics alone allows demarcation of the ISCs and EBs (Hung, 2020).

This study started with a small number of cells in the first sample for inDrop and 10x Genomics technologies, which recovered 344 cells and 256 cells, respectively. This allowed testing and comparison of the 2 technologies. Next, the number of cells was increased for each replicate (7,282 for inDrop and 2,723 for 10x Genomics). The 2 replicates allowed evaluation of the consistency of cell-type discovery between the 2 platforms. Indeed, all of the major cell types (cardia, ISC/EB, EE, dEC, aEC, mEC, pEC, LFC, copper, and iron cells) were detected using both approaches (Hung, 2020).

Cell morphology and digestive functions are different along the length of the Drosophila midgut. For example, ECs in the middle midgut secrete acid and absorb metal ions, whereas ECs in the posterior midgut contain lipid droplets and uptake lipid nutrients. These characteristics reflect regionalized gene-expression differences as previously shown by bulk RNA-seq analyses. Differentially expressed transcription factors were sought that could underlie regionalized gene expression and identified a number of potential candidates. In particular, vnd, odd, caup, and tup are preferentially expressed in the anterior region. Only one discrepancy (odd) was detected between these results and previously published bulk RNA-seq data (expressed in the posterior region from the Flygut-seq). Further studies will be required to resolve these differences. lab, Ptx1, CREG, apt, and dve are preferentially expressed in the middle midgut, consistent with the previous observation that the homeobox genes lab, Ptx1, and dve have been shown to be expressed in the adult middle midgut. Finally, bab2, ham, cad, Ets21C, Hnf4, and hth are preferentially expressed in the posterior midgut; the homeobox gene cad and Ets21C have been previously reported to be expressed in the posterior midgut. The expression pattern of these regionalized transcription factors from the Flygut-seq are listed in an accompanying dataset to help compare these findings. Recently, scRNA-seq of the mouse embryo identified a group of 20 transcription factors that are expressed spatially along the anterior-posterior axis of the gut tube. Interestingly, 6 out of 20 transcription factors expressed in the mouse gut have fly orthologs that are also expressed differently along the anterior-posterior axis of the fly midgut. For example, mouse Irx3 and fly caup are expressed in the anterior region, mouse Hoxb1 and fly lab are expressed in the anterior-middle region, and mouse Cdx2 and fly cad are expressed in the posterior region (Hung, 2020).

The regional expression of the transcription factors described above may also underlie the regionalization of EE populations. For example, cad, which is expressed in posterior ECs, is also highly expressed in AstA-EEs that are localized in the posterior midgut. This study also identified another transcription factor expressed in posterior EEs, Poxn, that is homologous to mouse Pax8, which is expressed regionally in the mouse gut tube. Whether Poxn is expressed in posterior EEs has not yet been experimentally tested. Similarly, stem cell morphology and proliferation activity also differ along the anterior-posterior axis of the gut. However, although previous cell-specific RNA-seq studies revealed regional differences in stem cell transcriptomes, this scRNA-seq analysis was not able to identify subgroups or regional ISC/EB clusters, despite the fact that some stem cells express some regional markers, such as lab or Ptx1. It is possible that the regional differences in ISC transcriptomes are less prominent than the regional differences in EC transcriptomes (Hung, 2020).

Regarding EEs, candidate markers and 5 additional gut hormones were identified: sNPF, ITP, Nplp2, CCAP, and CNMa. In addition, it was found that individual EEs are able to express up to 5 different hormones, in contrast to the traditional view that these cells only produce 2 hormones. Interestingly, a recent mammalian study showed that EEs express different hormones and that they can switch their hormonal repertoire depending on their tissue location. The most frequent combinations of gut hormones were AstA/AstC/Orcokinin/CCHa1/CCHa2 for AstA-EEs, AstC/Orcokinin for AstC-EEs, and NPF/Tk/Nplp2/Orcokinin for NPF-EEs. In addition, it was found that EEs may also act in a paracrine manner because NPF-EEs expressed AstC-R2, which can receive signals from AstC-EEs. Finally, it was shown that a subset of EEs expressing NPF and Tk in the middle of the midgut also expressed the esg progenitor marker (Hung, 2020).

This study provides a rich resource to further characterize the molecular signature of each cell type and gene functions in different cell types in homeostatic conditions. Further scRNA-seq of the fly gut will allow a number of questions to be addressed. These include changes in cell states, cell-type composition, and transcriptomes in the context of regeneration, aging, infection, axenic condition, different diet, various mutant backgrounds, and disease models, such as the Yorkie-induced intestine tumor model. In addition to the higher ISC proliferation activity, the female midgut is larger and longer than the male midgut. Hence, it is highly warranted to use scRNA-seq to delineate the gut at physiological and functional levels based on sex differences. Furthermore, during aging, changes in ISC proliferation, regeneration capacity, innate immune and inflammatory response, and tissue integrity occurs, which can be analyzed using scRNA-seq. Taking these data together, it is felt that future scRNA-seq will provide a fundamental understanding of the changes in cell states and interplay among cell types and disease (Hung, 2020).

EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells

Tissue-specific adult stem cells are commonly associated with local niche for their maintenance and function. In the adult Drosophila midgut, the surrounding visceral muscle maintains intestinal stem cells (ISCs) by stimulating Wingless (Wg) and JAK/STAT pathway activities, whereas cytokine production in mature enterocytes also induces ISC division and epithelial regeneration, especially in response to stress. This study shows that EGFR/Ras/ERK signaling is another important participant in promoting ISC maintenance and division in healthy intestine. The EGFR ligand Vein is specifically expressed in muscle cells and is important for ISC maintenance and proliferation. Two additional EGFR ligands, Spitz and Keren, function redundantly as possible autocrine signals to promote ISC maintenance and proliferation. Notably, over-activated EGFR signaling could partially replace Wg or JAK/STAT signaling for ISC maintenance and division, and vice versa. Moreover, although disrupting any single one of the three signaling pathways shows mild and progressive ISC loss over time, simultaneous disruption of them all leads to rapid and complete ISC elimination. Taken together, these data suggest that Drosophila midgut ISCs are maintained cooperatively by multiple signaling pathway activities and reinforce the notion that visceral muscle is a critical component of the ISC niche (Xu, 2011).

Adult stem cells commonly interact with special microenvironment for their maintenance and function. Many adult stem cells, best represented by germline stem cells in Drosophila and C. elegans, require one primary maintenance signal from the niche while additional signals may contribute to niche integrity. ISCs in the Drosophila midgut do not seem to fit into this model. Instead, they require cooperative interactions of three major signaling pathways, including EGFR, Wg and JAK/STAT signaling, for long-term maintenance. Importantly, Wg or JAK/STAT signaling over-activation is able to compensate for ISC maintenance and proliferation defects caused by EGFR signaling disruption, and vice versa. Therefore, ISCs could be governed by a robust mechanism, signaling pathways could compensate with each other to safeguard ISC maintenance. The mechanisms of the molecular interactions among these pathways in ISC maintenance remains to be investigated. In mammals, ISCs in the small intestine are primarily controlled by Wnt signaling pathways, and there are other ISC specific markers not controlled by Wnt signaling. In addition, mammalian ISCs in vitro strictly depend on both EGFR and Wnt signals, indicating that EGFR and Wnt signaling may also cooperatively control mammalian ISC fate. It is suggested that combinatory signaling control of stem cell maintenance could be a general mechanism for ISCs throughout evolution (Xu, 2011).

The involvement of EGFR signaling in Drosophila ISC regulation may bring out important implications to understanding of intestinal diseases, in which multiple signaling events could be involved. For example, in addition to Wnt signaling mutation, gain-of-function K-Ras mutations are frequently associated with colorectal cancers in humans. Moreover, activation of Wnt signaling caused by the loss of adenomatous polyposis coli (APC) in humans initiates intestinal adenoma, but its progression to carcinoma may require additional mutations. Interestingly, albeit controversial, Ras signaling activation is suggested to be essential for nuclear β-catenin localization, and for promoting adenoma to carcinoma transition. In the Drosophila midgut, loss of APC1/2 genes also leads to intestinal hyperplasia because of ISC overproliferation. Given that EGFR signaling is generally activated in ISCs, it would be interesting to determine the requirements of EGFR signaling activation in APC-loss-induced intestinal hyperplasia in Drosophila, which might provide insights into disease mechanisms in mammals and humans (Xu, 2011).

Previous studies suggest that intestinal VM structures the microenvironment for ISCs by producing Wg and Upd maintenance signals. This study identified Vn, an EGFR ligand, as another important ISC maintenance signal produced from the muscular niche. Therefore, ISCs are maintained by multiple signals produced from the muscular niche. In addition, Spi and Krn, two additional EGFR ligands, were identified that function redundantly as possible autocrine signals to regulate ISCs. These observations are consistent with a previous observation that paracrine and autocrine EGFR signaling regulates the proliferation of AMPs during larval stages, suggesting that this mechanism is continuously utilized to regulate adult ISCs for their maintenance and proliferation. The only difference is that the proliferation of AMP cells is unaffected when without autocrine Spi and Krn, due to redundant Vn signal from the VM, whereas autocrine Spi/Krn and paracrine Vn signals are all essential in adult intestine for normal ISC maintenance and proliferation. It was found that Vn and secreted form of Spi have similar roles in promoting ISC maintenance and activation, but additional regulatory or functional relationships among these ligands require further investigation, as the necessity of multiple EGFR ligands is still not completely understood. It is known that secreted/activated Spi and Krn are diffusible signals, but clonal analysis data show that Spi and Krn can display autonomous phenotypes. This observation indicates that these two ligands could behave as very short range signals in the intestinal epithelium, or they could diffuse over long distance but the effective levels of EGFR activation could only be achieved in cells where the ligands are produced. Interestingly, palmitoylation of Spi is shown to be important for restricting Spi diffusion in order to increase its local concentration required for its biological function. Whether such modification occurs in intestine is unknown, but it is speculated that Vn, Spi and Krn, along with the possibly modified forms, may have different EGFR activation levels or kinetics, and only with them together effective activation threshold could be reached and sustained in ISCs to control ISC behavior. Therefore, a working model is proposed that ISCs may require both paracrine and autocrine mechanisms in order to achieve appropriate EGFR signaling activation for ISC maintenance and proliferation.

Mechanisms of JAK/STAT signaling activation is rather complex. In addition to Upd expression from the VM, its expression could also be detected in epithelial cells with great variability in different reports, possibly due to variable culture conditions. Upon injury or pathogenic bacterial infection, damaged ECs and pre-ECs are able to produce extra cytokine signals, including Upd, Upd2 and Upd3, to activate JAK/STAT pathway in ISCs to promote ISC division and tissue regeneration. Several very recent studies suggest that EGFR signaling also mediates intestinal regeneration under those stress conditions in addition to its requirement for normal ISC proliferation. Therefore, in addition to basal paracrine and autocrine signaling mechanisms that maintain intestinal homeostasis under normal conditions, feedback regulations could be employed or enhanced under stress conditions to accelerate ISC division and epithelial regeneration (Xu, 2011).

Evidence so far has indicated a central role of N signaling in controlling ISC self-renewal. N is necessary and sufficient for ISC differentiation. In addition, the downstream transcriptional repressor Hairless is also necessary and sufficient for ISC self-renewal by preventing transcription of N targeting genes in ISCs. Therefore, N inhibition could be a central mechanism for ISC fate maintenance in Drosophila. High Dl expression in ISCs may lead to N inhibition, though how Dl expression is maintained in ISCs at the transcriptional level is not clear yet. Hyperactivation of EGFR, Wg or JAK/STAT signaling is able to induce extra Dl⁺ cells, suggesting that these three pathways might cooperatively promote Dl expression in ISCs. It is also possible that these pathways regulate Dl expression indirectly. As Dl-N could have an intrinsically regulatory loop for maintaining Dl expression and suppressing N activation, these pathways could indirectly regulate Dl expression by targeting any component within the regulatory loop. Identifying their respective target genes by these signaling pathways in ISCs would be an important starting point to address this question (Xu, 2011).

A feedback amplification loop between stem cells and their progeny promotes tissue regeneration and tumorigenesis

Homeostatic renewal of many adult tissues requires balanced self-renewal and differentiation of local stem cells, but the underlying mechanisms are poorly understood. This study identified a novel feedback mechanism in controlling intestinal regeneration and tumorigenesis in Drosophila. Sox21a, a group B Sox protein, is preferentially expressed in the committed progenitor named enteroblast (EB) to promote enterocyte differentiation. In Sox21a mutants, EBs do not divide, but cannot differentiate properly and have increased expression of mitogens, which then act as paracrine signals to promote intestinal stem cell (ISC) proliferation. This leads to a feedback amplification loop for rapid production of differentiation-defective EBs and tumorigenesis. Notably, in normal intestine following damage, Sox21a is temporally downregulated in EBs to allow the activation of the ISC-EB amplification loop for epithelial repair. It is proposed that executing a feedback amplification loop between stem cells and their progeny could be a common mechanism underlying tissue regeneration and tumorigenesis (Chen, 2016b).

Adult stem cells have important roles in maintaining tissue and organ homeostasis by their prolonged ability to produce progenitor cells that differentiate into multiple types of mature cells. Production of the progenitor cells from stem cells must be coordinated with cell differentiation and the overall tissue demand, as disruption of this coordination could lead to tissue degeneration, if cell production is not sufficient, or hyperplasia/ tumorigenesis, if the cell production is unrestricted and exceeds the pace of cell differentiation. However, the molecular mechanism that coordinates progenitor cell proliferation with cell differentiation is largely unknown (Chen, 2016b).

The adult Drosophila midgut has been established as a simple and useful system for the study of the stem cell behavior during homeostatic tissue renewal and in response to environmental changes. Like mammalian intestine, the Drosophila midgut epithelium is constantly replenished by adult intestinal stem cells (ISCs), although at a relatively slower pace. In addition, signaling pathways that regulate mammalian ISC activity, such as Wnt, JAK/STAT, EGFR/Ras, Hippo, BMP and Notch, also play important roles in regulating Drosophila ISC activity during normal homeostasis and/or stress conditions. The Drosophila ISC, which generates a relatively simple stem cell lineage, can be specifically marked by Delta (Dl), the Notch ligand. After each asymmetric division, an ISC will produce a new ISC and a committed progenitor cell named enteroblast (EB), which will further differentiate into either an enterocyte or an enteroendocrine cell, depending on the levels of Notch activation it received from ISCs. Enterocyte differentiation from EB requires high levels of Notch activation, and JAK/STAT signaling activity is required for both enterocyte and enteroendocrine cell differentiation from EB. Aside from the signaling pathways, many transcription factors have been identified as important regulators of cell differentiation. Enterocyte differentiation from EB requires downregulation of Escargot (Esg) and activation of Pdm1, whereas enteroendocrine cell differentiation from EB requires release of the inhibition by the transcriptional repressor Tramtrack and activation of acheate-scute complex (AS-C) genes and Prospero (Pros), the enteroendocrine cell determination factor. It is largely unclear how these signaling pathways and transcription factors are coordinately regulated for balanced self-renewal of ISCs and differentiation of EBs to maintain intestinal (Chen, 2016b).

Sox family transcription factors, which share a DNA binding high-mobility-group domain, are known as important regulators of cell fate decisions during development and in adult tissue homeostasis. In mouse small intestine, Sox2 is expressed in ISCs and progenitor cells and is critical for ISC maintenance and differentiation of Paneth cells. Several Sox family proteins have been identified in Drosophila, but their potential roles in the ISC lineage are unclear. This study characterized the function of a Drosophila Sox gene, Sox21a, in the ISC lineage. Sox21a is expressed in EBs and acts as a tumor suppressor in the midgut epithelium. One important function of Sox21a is to promote enterocyte differentiation by inducing Pdm1 expression. By studying its tumor suppressing function, a novel feedback amplification loop was identified between ISC and EB, which is normally suppressed by Sox21a. Temporal activation of this loop is essential for damage-induced intestinal regeneration, whereas sustained activation of this loop leads to tumorigenesis. Therefore, this study has revealed a novel mechanism that coordinates stem cell activity with progenitor cell differentiation, and connects regeneration with tumorigenesis (Chen, 2016b).

Sox family proteins in metazoan are divided into different groups based on their similarity in biochemical properties, and Sox21a belongs to the SoxB2 subgroup whose function is relatively less studied compared to the most related SoxB1 subgroup proteins, such as the pluripotency factor Sox2. By cellular and genetic analysis, this study has characterized the functions of Sox21a, a member of the SoxB2 group in Drosophila; two major roles were revealed. First, Sox21a is essential for EC differentiation from EB. Sox21a protein is mainly expressed in differentiating EBs, a pattern that is consistent with its role in EB differentiation. Loss of Sox21a causes accumulation of undifferentiated cells that fail to express Pdm1, the EC marker. The majority of these mutant cells remain as diploid EBs and some begin to show polyploidy, indicating that mechanisms controlling EC differentiation and cell ploidy can be uncoupled. On the other hand, forced transgene expression of Sox21a in EBs accelerates their differentiation into ECs as evidenced by precocious expression of Pdm1. However, forced expression of Sox21a in ISCs does not induce their differentiation, suggesting that Sox21a is necessary but not sufficient for inducing EC differentiation from ISCs. One possible explanation for this is that EC differentiation requires both Sox21a and Notch activity. Indeed, although Notch activity is known to be both necessary and sufficient for inducing EC differentiation from ISCs, Notch activated EBs that have already been presented along the length of midgut in young flies remain dormant for several days until there is a need for cell replacement. These observations indicate that activation of Notch alone is not sufficient to induce EB differentiation, but only primes EB for EC differentiation. Notch-activated EBs will stay in undifferentiated state until Sox21a is activated, which then promotes differentiation of the primed EBs into ECs (Chen, 2016b).

Another surprising role for Sox21a revealed in this study is that it provides a feedback regulation of ISC activity by suppressing mitogenic signals from differentiating EBs. This function is important for controlling the strength of the ISC-EB amplification loop for balanced self-renewal of ISCs and differentiation of EBs. Because Sox21a is also required for EB differentiation, disruption of this function will cause sustained activation of the ISC-EB amplification loop as well as blocked EB differentiation, leading to the formation of EB-like tumors. Importantly, following epithelial damage, Sox21a is quickly downregulated in EBs. This allows temporal activation of the ISC-EB loop for rapid production of progenitor cells prepared for epithelial repair. During recovery, Sox21a is then temporally upregulated in EBs. This not only stops the ISC-EB amplification loop to avoid excessive EB production, but also accelerates cell differentiation for epithelial repair. Therefore, Sox21a does not simply act as a tumor suppressor in intestine. It is dynamically regulated to control the process of epithelial regeneration in response to various environmental changes via regulating the strength of the ISC-EB amplification loop. Because Sox21a expression in intestine is dynamic during normal adult development, it is conceivable that its expression could possibly be influenced by physiological changes, such as food intake and activity of symbiotic bacteria, and fine-turning Sox21a activity could be important for maintaining regular epithelial turnover. How Sox21a expression is regulated is unclear, but signaling pathways that are implicated in regulating intestinal regeneration, such as JAK/STAT or EGFR/Ras pathways are potential candidate regulators, especially JAK/STAT, which is known to be essential for EB differentiation. During the preparation of this manuscript, two groups have reported the function of Sox21a in Drosophila midgut (Meng, 2015; Zhai, 2015). Meng did not observe the tumor suppressive function of Sox21a, possibly because they used a weak mutant allele of Sox21a in their study. Notably, Zhai observed a similar tumor suppressive role for Sox21a as reported here. Interestingly, they suggest that Sox21a is regulated by JAK/STAT signaling, as Sox21a transgene expression is able to rescue the differentiation defects caused by disrupted JAK/STAT signaling. However, the current found that Sox21a was still expressed in JAK/STAT compromised EBs. Therefore, how Sox21a is regulated during normal homeostasis and regeneration remains to be further explored, and it is possible that Sox21a could be controlled by a combination of regulators in a cell type-specific manner, with different mechanisms in ISCs and EBs. Because Sox proteins commonly function together with other cell type-specific co-factors in regulating gene transcription, Sox21a could function with different co-factors in ISCs and EBs. These are interesting questions worthy of further investigation (Chen, 2016b).

Cells in a given tumor are usually heterogeneous and based on the ability to initiate tumors, tumor cells can be divided into tumor-initiating cells and non-tumor-initiating cells. In this case, Sox21 mutant EBs can be regarded as the tumor-initiating cells in vivo. Depleting Sox21a specifically in EBs is sufficient to initiate EB-like tumors. Conversely, restoring Sox21a function specifically in EBs is sufficient to prevent tumor development in Sox21a mutant intestine. However, unlike typical tumor-initiating cells, Sox21a mutant EBs are post-mitotic cells. In addition, their ability to initiate tumors depends on the activity of local ISCs. Therefore, this study also reveals a novel example of tumor-initiating cells in vivo that do not divide themselves, but can 'propagate' themselves by utilizing local stem cells (Chen, 2016b).

In short, by studying the function of Sox21a in Drosophila ISC lineages, this study identified a novel feedback amplification loop between stem cells and their progeny that mediates epithelial regeneration and tumorigenesis. It has long been suggested that tissue regeneration and tumorigenesis are intimately associated, although the mechanistic connection is still obscure. The Sox21a-Spi mediated- ISC-EB amplification loop revealed in this study may provide a simple example of potential mechanisms that could connect tissue regeneration with tumorigenesis: transient activation of the stem cell- progeny amplification loop promotes regeneration, whereas sustained or irreversible activation of the amplification loop promotes tumorigenesis. It is proposed that this could be a general mechanism underlying tissue regeneration and tumorigenesis in other tissues, including that in mammals and humans (Chen, 2016b).

Aneuploidy in intestinal stem cells promotes gut dysplasia in Drosophila

Aneuploidy is associated with different human diseases including cancer. However, different cell types appear to respond differently to aneuploidy, either by promoting tumorigenesis or causing cell death. This study examined the behavior of adult Drosophila melanogaster intestinal stem cells (ISCs) after induction of chromosome missegregation either by abrogation of the spindle assembly checkpoint or through kinetochore disruption or centrosome amplification. These conditions induce moderate levels of aneuploidy in ISCs, and no evidence of apoptosis was found. Instead, a significant accumulation was found of ISCs associated with increased stem cell proliferation and an excess of enteroendocrine cells. Moreover, aneuploidy causes up-regulation of the JNK pathway throughout the posterior midgut, and specific inhibition of JNK signaling in ISCs is sufficient to prevent dysplasia. These findings highlight the importance of understanding the behavior of different stem cell populations to aneuploidy and how these can act as reservoirs for genomic alterations that can lead to tissue pathologies (Resende, 2018).

Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway

Drosophila adult midgut intestinal stem cells (ISCs) maintain tissue homeostasis by producing progeny that replace dying enterocytes and enteroendocrine cells. ISCs adjust their rates of proliferation in response to enterocyte turnover through a positive feedback loop initiated by secreted enterocyte-derived ligands. However, less is known about whether ISC proliferation is affected by growth of the progeny as they differentiate. This study shows that nutrient deprivation and reduced insulin signaling results in production of growth-delayed enterocytes and prolonged contact between ISCs and newly formed daughters. Premature disruption of cell contact between ISCs and their progeny leads to increased ISC proliferation and rescues proliferation defects in insulin receptor mutants and nutrient-deprived animals. These results suggest that ISCs can indirectly sense changes in nutrient and insulin levels through contact with their daughters and reveal a mechanism that could link physiological changes in tissue growth to stem cell proliferation (Choi, 2011).

Previous studies have focused on responses of ISC proliferation to enterocyte death, delineating a positive feedback mechanism by which ligands secreted from dying enterocytes activate ISC proliferation. The data propose a model of additional regulation where cell contact between ISCs and newly formed enteroblasts acts to inhibit ISC proliferation through a negative feedback loop (see Cell contact regulates ISC proliferation) (Choi, 2011).

Nutrient deprivation leads to decreased ISC proliferation rates and clones containing fewer cells than clones made in animals fed a rich diet. However, it is unclear why these clones fail to eventually reach the same size as wild-type clones. One possibility is that nutrient-deprived midguts contain fewer cells. Therefore, the number of cells that each ISC needs to generate to maintain tissue homeostasis would be smaller. A second possibility is built on the observation that turnover and production of 8n and 16n enterocytes is reduced in animals fed a poor diet, and this could result in the depletion of a source of promitotic ligands, thereby decreasing the need for a stem cell to divide (Choi, 2011).

Protein deprivation and reduced insulin signaling leads to an increase in the number of lower ploidy enterocyte daughters per midgut, suggesting that endoreduplication in the midgut is regulated by nutrition. Because enterocyte turnover is reduced in nutrition-deprived animals, it raises the intriguing possibility that 8n cells act to inhibit the growth and endoreduplication of 4n cells into mature enterocytes through an as-yet-unidentified signal. These similarities between nutrient-deprived clones and dInR mutant clones suggest that the effects of nutrition may be mediated in part through the insulin-signaling pathway. Consistent with a role for nutrition and the insulin-signaling pathway in growth and endoreduplication, constitutive activation of dInR in ISC clones led to enterocytes with significantly higher ploidy than normal. Interestingly, these clones were smaller than wild-type, suggesting that excessive or prolonged contact between enterocytes and ISCs may also play a role in the regulation of ISC proliferation (Choi, 2011).

The findings raise the as-yet-unexplored possibility that germ-line stem cell and neuroblast stem cell daughters might also nonautonomously regulate stem cell proliferation. When both the ISC and the enteroblast were mutant for dInR, a further increase in cell cycle arrest was observed, suggesting an autonomous role for insulin signaling in the regulation of ISC proliferation (Choi, 2011).

Significantly higher levels of DE-cadherin were found between both dInR mutant enteroblast and wild-type ISCs and dInR mutant enteroblasts and dInR mutant ISCs, demonstrating that the insulin-signaling pathway regulates the stability of the adherens junction. The results are striking because, in the ovary and testis, loss of dInR signaling in the germ-line stem cell niche leads to a decrease rather than an increase in DE–cadherin at the adherens junction (Choi, 2011).

The data presented in this study demonstrate that the enteroblast can nonautonomously regulate the rate of ISC proliferation. How might this be achieved? One possibility is that the enteroblast inhibits ISC proliferation by providing a short-range inhibitory signal whose effect is removed as the ISC and enteroblast separate. A second possibility is that separation of ISCs and enteroblasts leads to the release from a cellular compartment of a factor that can drive proliferation. The ideal candidate is β-catenin, which is not only a member of the adherens junction but also a transcriptional activator, which is required for ISC proliferation (Choi, 2011).

Recently, ISCs and enteroblast number were examined under protein-poor conditions in old animals expressing green fluorescent protein driven by the escargot promoter (esg-GFP), which is thought to be specific to ISCs and enteroblasts. A decrease in esg-GFP–positive cells was observed in 16- to 17- and 20- to 21-d-old animals fed a poor diet, leading to the conclusion that ISC maintenance is regulated by a protein-poor diet. In contrast, this study did not observe a decrease in ISC number in females fed a protein-poor diet. Presumably, the modest decrease in GFP-positive cells observed by the previous study was due to loss of the excess enteroblasts seen in aging midguts, which is consistent with recently published work showing that insulin-signaling mutants can suppress this aging phenotype (Choi, 2011).

Immunoglobulin superfamily beat-Ib mediates intestinal regeneration induced by reactive oxygen species in Drosophila

Reactive oxygen species (ROS) often injure intestinal epithelia that cause loss of damaged cells, which is mainly repaired by proliferation of intestinal stem cells (ISCs). To maintain the homeostatic state, coordination of sensing of the ROS injury and the subsequent epithelial cell loss with the replenishment by cell renewal is crucial. However, little is known about how gut epithelial cells initiate regenerative responses against ROS to maintain the tissue integrity. A genome-wide screen was carried out, by which immunoglobulin superfamily beaten path Ib (beat-Ib) as an essential gene for provoking ISC proliferation against ROS in Drosophila intestine. Interestingly, the beat-Ib function is required in differentiated enterocytes, the main targeted cells by ROS in the intestinal tract, but is dispensable in the stem cells. Moreover, beat-Ib is not involved in enterocyte apoptosis at ROS injury. These findings indicate the essential role of beat-Ib in Drosophila midgut enterocytes for initiating the non-cell-autonomous induction of ISC division in response to environmental ROS stresses (Nagai, 2020).

An Optogenetic Method to Study Signal Transduction in Intestinal Stem Cell Homeostasis

Homeostasis in adult organs involves replacement of cells from a stem cell pool maintained in specialized niches regulated by extracellular signals. This cell-to-cell communication employs signal transduction pathways allowing cells to respond with a variety of behaviors. To study these cellular behaviors, signaling must be perturbed within tissues in precise patterns, a technique recently made possible by the development of optogenetic tools. Tools have been developed to study signal transduction in vivo in an adult fly midgut stem cell model where signaling was regulated by the application of light. Activation was achieved by clustering of membrane receptors EGFR and Toll, while inactivation was achieved by clustering the downstream activators ERK/Rolled and NFkappaB/Dorsal in the cytoplasm, preventing nuclear translocation and transcriptional activation. Both pathways contribute to stem and transit amplifying cell numbers and affect the lifespan of adult flies. This study further presents new approaches to overcome overexpression phenotypes and novel methods for the integration of optogenetics into the already-established genetic toolkit of Drosophila (Bunnag, 2020).

Peroxisome Elevation Induces Stem Cell Differentiation and Intestinal Epithelial Repair

Epithelial-repair-dependent mucosal healing (MH) is associated with a more favorable prognosis for patients with inflammatory bowel disease (IBD). MH is accomplished via repair and regeneration of the intestinal epithelium. However, the mechanism underlying MH is ill defined. This study found a striking upregulation of peroxisomes in the injured crypts of IBD patients. By increasing peroxisome levels in Drosophila midguts, it was found that peroxisome elevation enhanced RAB7-dependent late endosome maturation, which then promoted stem and/or progenitor-cell differentiation via modulation of Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT)-SOX21A signaling. This in turn enhanced ISC-mediated regeneration. Importantly, RAB7 and SOX21 were upregulated in the crypts of IBD patients. Moreover, administration of drugs that increased peroxisome levels reversed the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. This study demonstrates a peroxisome-mediated epithelial repair mechanism, which opens a therapeutic avenue for the enhancement of MH in IBD patients (Du, 2020b).

Inflammatory bowel disease (IBD) encompasses a range of complex, long-lasting, and relapsing-remitting disorders, of which ulcerative colitis (UC) and Crohn's disease (CD) are the two most prevalent manifestations. Currently, the incidence of IBD is increasing globally, and the prevalence has increased to just below 0.5% in the Western population. Although genetic predisposition, inappropriate immune responses, and environmental factors have been reported to be closely associated with IBD, its exact etiology and pathogenesis remain poorly understood (Du, 2020b).

A series of recent clinical studies indicated that complete regeneration of the intestinal mucosa (also called 'mucosal healing') is associated with a more favorable prognosis for patients with IBD. In the mammal intestine, pluripotent intestinal stem cells (ISCs), located at the base of the epithelial crypts, are responsible for the repair of the damaged epithelium by differentiating into multiple epithelial progenies. Thus, a better understanding of cellular and molecular mechanisms that underlie ISC-mediated epithelial repair could not only provide insight into the etiology but also the therapeutic targets of IBD (Du, 2020b).

To identify the mechanism with which peroxisomes regulate the differentiation of ISC progenies during midgut regeneration, RNA sequencing (RNA-seq) was performed on dissected midguts from the Pex10-null and WT flies. This study showed that Sox21a, a transcription factor essential for ISC-to-EC differentiation, had a significantly lower level in mutant midguts compared with that in WT midguts after injury. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed genes, which only appeared in WT midguts but not in Pex10-null midguts during regeneration, showed significant enrichment of genes involved in the endocytosis process. A number of these endocytosis genes have been reported to regulate stem- and/or progenitor-cell differentiation in diverse animal models, such as polo, vap, Pcyt1, and Ubi-p63E. To confirm the results of the RNA-seq analyses, real-time quantitative PCR (qPCR) analyses were performed using sorted ISCs and EBs (esg-GFP+ cells). The results of real-time qPCR analyses of the selected genes, including Sox21a, vap, and rl/ERK showed similar expression patterns to those of RNA-seq analysis during regeneration. Thus, peroxisomes may regulate the ISC-to-EC differentiation by promoting the expression of Sox21a and/or that of specific endocytosis-related genes during regeneration (Du, 2020b).

Since the Sox21a mutant has a similar differentiation defect (Zhai, 2015, Zhai, 2017, Chen, 2016) to Pex10 and Pex2 mutants, first, it was verified whether peroxisomes regulate the differentiation of ISCs and EBs through Sox21a-mediated signaling. The endogenous SOX21A reporter line Sox21a-HA was generated using a CRISPR-Cas9 knockin system. As expected, prior to injury, the expression of the SOX21A-HA protein in Pex10-null ISCs and EBs was similarly weak compared with its expression in control ISCs and EBs. However, after injury, while SOX21A expression was dramatically upregulated in control ISCs and EBs, it still retained a relatively low level in Pex10-null ISCs and EBs. Flip-out RNAi clone analysis consistently showed that peroxisomes promoted SOX21A expression after injury (Du, 2020b).

More importantly, forced expression of either Sox21a or GATAe (one of the downstreams of SOX21A, which also showed a similar expression change pattern with Sox21a in Pex10-null mutant clone cells partially rescued the differentiation defect of ISC progenies in Pex10-null clones as evidenced by the generation of Pdm1+ cells. Consistently, forced expression of either Sox21a or GATAe in ISCs and EBs also partially restored the defects of esg-GFP+ cell and pH3+ cell accumulation in Pex10-null midguts (Du, 2020b).

Since previous studies have shown that the JAK and STAT signaling functions upstream of Sox21a in ISCs and EBs to promote EC formation (Zhai, 2015, 2017), this study tested the genetic interaction between peroxisomes and the JAK and STAT signaling. After injury, while the activity of the JAK and STAT signaling was significantly upregulated in control midguts as indicated by the mRNA expression of the STAT target Socs36E and the 10XSTAT-GFP transcriptional reporter, it still retained a relatively low level in Pex10-null midguts. More importantly, forced expression of a constitutively active version of JAK (hopscotch [hop]) (UAS-hopTUM) significantly restored the expression defect of SOX21A in Pex10-depleted cells and the differentiation defect of ISCs and EBs in Pex10 RNAi clones as evidenced by the generation of Pdm1+ cells. In addition, depletion of PEX10 did not affect the activity of Notch signaling, and overexpression of constitutively active Notch (Notchintra) did not show obvious rescue of the ISC-to-EC differentiation defect of Pex10 mutant flies (Du, 2020b).

Similar to the human intestine, the adult midgut in Drosophila uses resident ISCs to replenish damaged and lost epithelial cells after injury. The Drosophila ISCs divide to self-renew and produce non-dividing enteroblasts (EBs) or enteroendocrine mother cells (EMCs) depending on Notch activity. Post-mitotic EBs with high levels of Notch signaling further differentiate into absorptive enterocytes (ECs). The EMCs divide once to produce a pair of secretory enteroendocrine cells (EEs). In response to Drosophila midgut injury, ISCs transiently increase their proliferation rates and initiate the production of differentiated ECs to compensate for the damaged and lost cells. This mechanism lasts for a few days until the damaged midgut recovers its normal morphology. In addition to the activation of numerous signals, intestinal damage results in clear changes of cellular structures including the organelles of stem and/or progenitor cells. Although the signaling requirements for the stem cell function in intestinal regeneration have been reported in detail, the contribution of organelle dynamics in stem cells to intestinal repair remains poorly understood (Du, 2020b).

Among different organelles, peroxisomes are remarkably plastic with the capability to change their composition, abundance, and morphology in response to environmental stimuli, which in turn may play a role in intestinal repair. By analyzing samples from human patients, utilizing the Drosophila midgut model, and leveraging the mouse IBD model, this study elucidates a conserved molecular pathway with which peroxisome proliferation induces stem-cell-mediated intestinal repair. Furthermore, a new therapeutic approach for the treatment of IBD has been suggested (Du, 2020b).

To meet various cellular requirements, organelles in one cell do not function as isolated or static units but rather form dynamic contacts between each other. The findings of this study show that peroxisomes likely modulate the maturation of late endosomes via modulation of vascular transportation, which had been reported to regulate several types of signaling, such as Notch and JAK and STAT signaling. Further studies should aim to understand the detailed mechanism of how peroxisomes modulate the maturation of late endosomes during tissue regeneration (Du, 2020b).

This study showed that administration of peroxisome-proliferating agents, NaPB and fenofibrate, which are two drugs approved by the food and drug administration (FDA), effectively reversed the symptoms of DSS-induced colitis in mice. Since NaPB and fenofibrate have a long history of use for disease treatment in human patients, they can be applied to determine their therapeutic effect of enhancing mucosal healing in patients with IBD. Since the peroxisome enhances intestinal repair by promoting stem cell differentiation, it will likely not cause stem cell over-proliferation and tumor formation. These findings not only have important implications for deciphering the functions of the peroxisome in stem cells, tissue regeneration, and injury-induced human diseases but also suggest the peroxisome as a promising therapeutic target of IBD (Du, 2020b).

Petsakou, A., Liu, Y., Liu, Y., Comjean, A., Hu, Y., Perrimon, N. (2023). Cholinergic neurons trigger epithelial Ca(2+) currents to heal the gut. Nature, 623(7985):122-131. PubMed ID: 37722602

Cholinergic neurons trigger epithelial Ca(2+) currents to heal the gut

A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the aetiology of chronic disorders such as inflammatory bowel diseases and cancer. This study used the Drosophila midgut to investigate this and discovered that during regeneration a subpopulation of cholinergic neurons triggers Ca(2+) currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. It was found that downregulation of the conserved cholinergic enzyme Acetylcholine esterase in the gut epithelium enables acetylcholine from specific Eiger (TNF in mammals)-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca(2+), which spreads in the epithelium through Innexin2-Innexin7 gap junctions, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki (YAP in humans) activation, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel disease. Altogether, the conserved cholinergic pathway facilitates epithelial Ca(2+) currents that heal the intestinal epithelium. These findings demonstrate nerve- and bioelectric-dependent intestinal regeneration and advance current understanding of how a tissue returns to homeostasis after injury (Petsakou, 2023).

A high-fat diet induces a microbiota-dependent increase in stem cell activity in the Drosophila intestine

Over-consumption of high-fat diets (HFDs) is associated with several pathologies. Although the intestine is the organ that comes into direct contact with all diet components, the impact of HFD has mostly been studied in organs that are linked to obesity and obesity related disorders. Drosophila was used as a simple model to disentangle the effects of a HFD on the intestinal structure and physiology from the plethora of other effects caused by this nutritional intervention. A HFD, composed of triglycerides with saturated fatty acids, was shown to trigger activation of intestinal stem cells in the Drosophila midgut. This stem cell activation was transient and dependent on the presence of an intestinal microbiota, as it was completely absent in germ free animals. Moreover, major components of the signal transduction pathway have been elucidated. In this study, JNK (basket) in enterocytes was necessary to trigger synthesis of the cytokine upd3 in these cells. This ligand in turn activated the JAK/STAT pathway in intestinal stem cells. Chronic subjection to a HFD markedly altered both the microbiota composition and the bacterial load. Although HFD-induced stem cell activity was transient, long-lasting changes to the cellular composition, including a substantial increase in the number of enteroendocrine cells, were observed. Taken together, a HFD enhances stem cell activity in the Drosophila gut and this effect is completely reliant on the indigenous microbiota and also dependent on JNK signaling within intestinal enterocytes (von Frieling, 2020).

A switch in tissue stem cell identity causes neuroendocrine tumors in Drosophila gut

Intestinal stem cells (ISCs) are able to generate gut-specific enterocytes, as well as neural-like enteroendocrine cells. It is unclear how the tissue identity of the ISC lineage is regulated to confer cell-lineage fidelity. This study shows that, in adult Drosophila midgut, loss of the transcriptional repressor Tramtrack in ISCs causes a self-renewal program switch to neural stem cell (NSC)-like, and that switch drives neuroendocrine tumor development. In Tramtrack-depleted ISCs, the ectopically expressed Deadpan acts as a major self-renewal factor for cell propagation, and Sequoia acts as a differentiation factor for the neuroendocrine phenotype. In addition, the expression of Sequoia renders NSC-specific self-renewal genes responsive to Notch in ISCs, thus inverting the differentiation-promoting function of Notch into a self-renewal role as in normal NSCs. These results suggest an active maintenance mechanism for the gut identity of ISCs, whose disruption may lead to an improper acquisition of NSC-like traits and tumorigenesis (Li, 2020).

In addition to the nervous system, neuroendocrine (NE) cells are found in many non-neural tissues and can develop neoplasias that are known as NE tumors (NETs). The NE cells in non-neural tissues display characteristics that are typical of neurons, such as membrane excitability and hormone secretion, yet many of these NEs are generated from adult stem cells of endodermal origin. It is, thus, intriguing how the tissue identity of stem cells is regulated and controlled to safeguard cell-lineage fidelity (Li, 2020).

The intestinal epithelium in adult Drosophila midgut is maintained by intestinal stem cells (ISCs)-the multipotent cells that are capable of generating both absorptive enterocytes (ECs) and secretary enteroendocrine cells (EEs). EEs are neural-like cells and are able to secrete sets of hormone peptides that are similar to those secreted by NE cells in the Drosophila brain. While the initiation of EC generation is driven by Delta (Dl)/Notch-mediated lateral inhibition between the two immediate stem cell daughters, the initiation of EE generation occurs at the stem cell level, with a transient expression of the proneural gene Scute (Sc) that induces ISCs to self-renew and to generate an EE progenitor cell (EEP). Each EEP then divides one more time before terminal differentiation to yield a pair of EEs. Sc encodes a basic helix-loop-helix (bHLH) transcription factor and belongs to the achaete-scute gene complex (AS-C), a Drosophila proneural gene cluster that is expressed in neural progenitor cells and is important for the development of the embryonic central nervous system and sensory organs of both larva and adult. Thus, it appears that there is a transient activation of neural-like programs in ISCs that directs EE generation from ISCs (Li, 2020).

Tramtrack (Ttk, or Ttk69 isoform), which encodes a BTB-domain-containing transcriptional repressor, acts as a master repressor of the differentiation of EEs from ISCs. Depletion of ttk in ISCs causes derepression of AS-C genes including Sc and Asense (Ase). The continuous expression of Sc and Ase directs ISCs to continuously generate EEs, leading to the formation of EE-like tumors, or NETs. One intriguing observation from the ttk-depleted ISCs is that continuous derepression of the differentiation-promoting factors does not compromise ISC maintenance, but continuous overexpression of Sc in normal ISCs will cause regional ISC loss over time. This study characterized the ttk-depleted ISCs and, surprisingly, found that the original self-renewal program of ISCs had switched to a neuroblast-like self-renewal program that is responsible for NET tumorigenesis (Li, 2020).

The results reveal an ISC-to-NB switch in the tissue stem cell self-renewal program that drives NET development from ttk- depleted ISCs. Loss of ttk causes the derepression of NB-specific transcription factors, including dpn and seq, and the concomitant loss of ISC-specific factors. The ectopically expressed Dpn acts as a major self-renewal factor for the self-duplication of tumor cells, while Seq has a 'selector' function in selecting Notch target genes by recruiting Su(H) to the enhancer regions of NB-specific genes, thereby rendering these genes responsive to Notch in the tumor cells. In addition, Seq also has a role in NE differentiation by inducing AS-C gene expression. The cooperative function of Dpn and Seq leads to the activation of a NB-like self-renewal program as well as a NE differentiation program and the continuous activation of these two programs leading to NET development from ISCs (Li, 2020).

There are two types of NBs in the central brain of Drosophila larva: type I and type II. Dpn is specifically expressed in the type II but not the type I NBs. Interestingly, Notch appears to be more important in type II NBs than in type I NBs. Thus, it appears that the ectopically activated self-renewal program in ISCs described in this study is more similar to that used in the type II NBs. Compared to the type I NB lineage, the type II NB lineage goes through one extra type of transient amplification progenitors before terminal cell fate specification, indicating that this type II-like self-renewal program, if hijacked by tumor cells, could potentially be more potent to initiate tumorigenesis (Li, 2020).

As the loss of a single factor, Ttk, in ISCs is sufficient for the switch of tissue stem cell program and for the subsequent development of NETs in the midgut, Ttk could be viewed as a specific class of stem cell factors, which is proposed in this study as a tissue identity maintenance (TIM) factor; such factors function to safeguard the tissue identity of stem cells. ISCs not only give rise to ECs that function to digest and absorb nutrients but also give rise to neural-like EE cells. Conceivably, ISCs may need to use a basal or transient neural-like program in order to endow their capacity to generate EEs, as the proneural factor Sc is transiently expressed in ISCs, and this transient expression initiates EE generation. In this context, a stem cell identity factor like Ttk may be necessary to enable ISCs to maintain a gut identity and thereby prevent excessive acquisition of NSC-like traits (Li, 2020).

The Ttk protein is characterized by having a BTB domain in addition to a zinc-finger DNA-binding domain, and although there is no direct protein ortholog of Ttk in mammals, BTB-domain-containing transcription factors are found in all eukaryotes, including mammals. Moreover, alteration of Notch activity as well as increased expression of proneural genes are also known to occur in mammalian models of NETs and in human NETs. Thus, it is possible that there are TIM factors that function in stem cells in other tissues and organisms and that 'stem cell identity switch' could be a common mechanism underlying NET formation and, possibly, other stem-cell-mediated tumorigenesis (Li, 2020).

Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction

The age-associated decline of adult stem cell function is closely related to the decline in tissue function and age-related diseases. However, the underlying mechanisms that ultimately lead to the observed functional decline of stem cells still remain largely unexplored. This study investigated Drosophila midguts and found a continuous downregulation of lipoic acid synthase, which encodes the key enzyme for the endogenous synthesis of alpha-lipoic acid (ALA). upon aging. Importantly, orally administration of ALA significantly reversed the age-associated hyperproliferation of intestinal stem cells (ISCs) and the observed decline of intestinal function, thus extending the lifespan of Drosophila. This study reports that ALA reverses age-associated ISC dysfunction by promoting the activation of the endocytosis-autophagy network, which decreases in aged ISCs. Moreover, this study suggests that ALA may be used as a safe and effective anti-aging compound for the treatment of ISC-dysfunction-related diseases and for the promotion of healthy aging in humans (Du, 2020a).

A Drosophila model of oral peptide therapeutics for adult Intestinal Stem Cell tumors

Peptide therapeutics, unlike small molecule drugs, display crucial advantages of target-specificity and the ability to block large interacting interfaces such as those of transcription factors. The transcription co-factor of the Hippo pathway, YAP/Yki, has been implicated in many cancers, and is dependent on its interaction with the DNA-binding TEAD/Sd proteins via a large Ω-loop. In addition, the mammalian Vestigial Like (VGLL) protein, specifically its TONDU domain, competitively inhibits YAP-TEAD interaction, resulting in arrest of tumor growth. This study shows that either overexpression of the TONDU peptide or its oral uptake leads to suppression of Yorkie (Yki)-driven intestinal stem cell (ISC) tumors in the adult Drosophila midgut. In addition, comparative proteomic analyses of peptide-treated and untreated tumors, together with ChIP analysis, reveal that integrin pathway members are part of the Yki-oncogenic network. Collectively, these findings establish Drosophila as a reliable in vivo platform to screen for cancer oral therapeutic peptides and reveal a tumor suppressive role for integrins in Yki-driven tumors (Bajpai, 2020).

Coordination of tumor growth and host wasting by tumor-derived Upd3

yki-induced gut tumors in Drosophila are associated with host wasting, including muscle dysfunction, lipid loss, and hyperglycemia, a condition reminiscent of human cancer cachexia. This model has been used to identify tumor-derived ligands that contribute to host wasting. To identify additional molecular networks involved in host-tumor interactions, PathON, a web-based tool analyzing the major signaling pathways in Drosophila was developed, and the Upd3/Jak/Stat axis was uncovered as an important modulator. yki-gut tumors were found to secrete Upd3 to promote self-overproliferation and enhance Jak/Stat signaling in host organs to cause wasting, including muscle dysfunction, lipid loss, and hyperglycemia. It was further revealed that Upd3/Jak/Stat signaling in the host organs directly triggers the expression of ImpL2, an antagonistic binding protein for insulin-like peptides, to impair insulin signaling and energy balance. Altogether, these results demonstrate that yki-gut tumors produce a Jak/Stat pathway ligand, Upd3, that regulates both self-growth and host wasting (Ding, 2021).

Microenvironmental innate immune signaling and cell mechanical responses promote tumor growth

Tissue homeostasis is achieved by balancing stem cell maintenance, cell proliferation and differentiation, as well as the purging of damaged cells. Elimination of unfit cells maintains tissue health: however, the underlying mechanisms driving competitive growth when homeostasis fails, for example, during tumorigenesis, remain largely unresolved. Using a Drosophila intestinal model, this study found that tumor cells outcompete nearby enterocytes (ECs) by influencing cell adhesion and contractility. This process relies on activating the immune-responsive Relish/NF-κB pathway to induce EC delamination and requires a JNK-dependent transcriptional upregulation of the peptidoglycan recognition protein PGRP-LA. Consequently, in organisms with impaired PGRP-LA function, tumor growth is delayed and lifespan extended. This study identifies a non-cell-autonomous role for a JNK/PGRP-LA/Relish signaling axis in mediating death of neighboring normal cells to facilitate tumor growth. It is proposed that intestinal tumors 'hijack' innate immune signaling to eliminate enterocytes in order to support their own growth (Zhou, 2021).

The intestinal epithelium separates the organism from the environment and plays essential roles in nutrient uptake and immune and regenerative processes. Intestinal renewal requires dynamic regulation of cell-cell contacts between enterocytes, and this is achieved by highly proliferative stem cells, proper differentiation, and cell loss by cell extrusion and apoptosis. Dysregulation of cell death in the intestinal epithelium can lead to pathologies such as intestinal bowel diseases and cancer (Zhou, 2021).

Studies in Drosophila have made important contributions toward an understanding of intestinal homeostasis, innate immunity, and aging. In adult flies, intestinal stem cells (ISCs) self-renew and produce progenitor cells called enteroblasts (EBs). These EBs can differentiate into either enteroendocrine cells (EEs) or enterocytes (ECs). The intestinal epithelium undergoes rapid stem cell division and differentiation to continuously replace damaged ECs and ensure tissue integrity and homeostasis, similar to mammalian intestines. Previous studies have shown that bacterial infection induces ISC proliferation and elimination of damaged ECs, thereby leading to remodeling of the intestinal epithelium. Enteric infection also triggers the evolutionarily conserved NF-ΚB pathway through the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), leading to the production of antimicrobial peptides (AMPs) for host immune defense (Zhou, 2021).

In addition to the role of the NF-κB pathway in AMP production in different cell types, several studies have identified non-immune functions. For example, constitutive activation of NF-κB reduces animal lifespan, NF-κB activity has been implicated in age-related neurodegenerative diseases, and NF-κB regulates Mef2 to coordinate its immune functions with metabolism. Further evidence links NFκB to Ras/MAPK and JAK/STAT signaling pathways. This allows for the proper balance of immune responses with cell growth and proliferation. Moreover, it has been recently reported that the NF-κB pathway in Drosophila is involved in infection-induced EC shedding, which facilitates maintenance of barrier function during intestinal regeneration (Zhou, 2021).

The Drosophila BMP2/4 homolog Decapentaplegic (Dpp) is involved in multiple developmental processes. The Dpp signal is transduced by the type I receptor Thickveins (Tkv) and type II receptor Punt that phosphorylate Drosophila Smad transcriptional factors such as Mothers against Dpp (Mad), Medea (Med), and the coregulator Schnurri (Shn) to regulate gene expression. Inactivation of BMP signaling components in the Drosophila intestine leads to intestinal tumor formation resembling juvenile polyposis syndrome (JPS). In humans, loss of BMP signaling leads to JPS, which has been associated with increased risks of developing gastrointestinal cancer (Zhou, 2021).

Cell replenishment and rearrangement are common mechanisms to sustain tissue homeostasis, which is also essential for development. Tissue growth requires dynamic cell rearrangements including cell elimination by mechanical competition. For example, epithelial cells can be eliminated by cell extrusion to maintain tissue homeostasis. Recent studies also suggested that tumor cells outcompete and eliminate their neighboring cells to clear space for their expansion. However, the underlying mechanisms and how tumor cells eliminate normal cells in the tumor microenvironment remain largely unknown (Zhou, 2021).

This study demonstrates a critical role for mechanical competition in the tumor microenvironment to promote tumorigenesis. Mechanistically, it was shown that tumor induces DE-cadherin- and myosin-dysregulation-associated mechanical stresses to nearby ECs. These processes trigger the ROCK-associated JNK signaling and subsequent activation of PGRP-LA/NF-κrB/ Relish in surrounding ECs to regulate the expression of pro- apoptotic genes and thereby promote cell delamination and apoptosis. The dying ECs then induce paracrine JAK/STAT signaling to trigger regeneration and further promote tumorigenesis. Importantly, tumors with associated activation of JNK/ PGPR-LA/Relish cascades can be inhibited by preventing apoptosis or by administering ROCK inhibitors. These results thus establish a tumor-cell-driven inflammatory feedback mechanism for competitive growth (Zhou, 2021).

This study demonstrates a non-cell-autonomous feedback mechanism that facilitates tumor development. First, tumor growth outcompetes its microenvironment by inducing mechanical forces. This triggers stress related ROCK/JNK signaling and induces EC elimination through activation of PGRP-LA/Relish signaling and downstream pro-apoptotic genes. Subsequently, dying ECs produce cytokines that activate JAK/STAT signaling in tumor cells to further promote tumor growth, thereby establishing a positive amplification loop between the tumor and its microenvironment (Zhou, 2021).

Cells undergoing rapid proliferation will push on their neighbors, which leads to local increase in mechanical pressures and triggers cell delamination. Previous studies have shown that the expansion of tumor cells triggers cell competition, which involves mechanical interactions. For instance, ectopic expression of Ras oncogene drives mechanical competitive growth and induces delamination of nearby wild-type cells in Drosophila. Hence, these data are in line with the current interpretation that mechanical competition also drives tumor competitive growth in the Drosophila intestine. In the epithelium, the mechanical modulation of surface tension is regulated by the actomyosin complex, counterbalanced by the Cadherin-dependent cell-cell adhesions. This study shows that tumors interact with their microenvironment and trigger ROCK/ JNK related cell death. A similar mechanism was observed in mammalian Madin-Darbin canine kidney (MDCK) cells, which, when deficient in the polarity gene scribble, are eliminated by mechanical cell competition, a process that requires the activation of the ROCK-p38-p53 pathway (Zhou, 2021).

The intestinal epithelium requires homeostatic mechanisms to counterbalance stem cell division and elimination of damaged or unfit cells. Dysregulation of either of these homeostatic programs can lead to tumor development. This study found that tumor cells induce immune-responsive PGRP- LA/Relish signaling for cell delamination and apoptosis. The role of NF-κB in the regulation of apoptosis has been discussed before for Drosophila Imd and mammalian TNFR1 pathways, which share key components to regulate NF-κB-related immune response and caspase-dependent apoptosis. Several studies have shown that tumor cells displace the nearby ECs through activation of Hippo and JNK signaling for tumor progression. In addition, JNK acts in parallel with NF-κB to control EC shedding during intestinal regeneration. In mammals, intestinal TNFR1 signaling is also required for EC detachment and apoptosis (Zhou, 2021).

Previous studies revealed a role for NF-κB signaling in cell death of outcompeted cells during development. In Drosophila wing discs, Myc-induced cell competition triggers Imd/Relish-related activation of the pro-apoptotic gene Hid for cell death. The Toll-signaling transcription factors Dorsal and Dif have been suggested to be required for Minute-induced cell competition by inducing Reaper-dependent apoptosis of outcompeted cells. However, further evidence showed axenic conditions abolished Toll-inhibition-induced competitive growth in the outcompeted cells. This suggested that infection contributes to Toll pathway inhibition induced cell competition. The current experiments indicate that axenic conditions failed to abolish the Imd activation in the tumor-surrounding ECs, suggesting a tumor-associated role of Imd/Relish induced EC cell death. Furthermore, a recent study discovered that cells with growth advantages, such as high protein synthesis, induce NF-κB-dependent autophagy to eliminate neighboring unfit cells in developing tissues. NF-κB and its upstream activating receptor are also required for salivary gland degradation through autophagy. Eye disc tumors also trigger a cell-autonomous feedback loop to promote proliferation by activation of JNK, Yki, and JAK/STAT signaling. In a distinct organ with a high rate of turnover, the data suggest that the NF-κB/Rel-dependent EC cell death cooperates with compensatory stem cell proliferation through the non-cell-autonomous activation of JNK and JAK/ STAT signaling for tumor progression (Zhou, 2021).

PGRPs are known as immune modulators of NF-κB signaling through binding and recognizing bacterial peptidoglycans. Several PGRPs have been implicated in other important biological processes beyond immunity such as host-microbe homeostasis, systemic inflammatory response, tissue integrity, and aging. PGRPs contain a RIP RHIM domain which has been proposed to activate NF-κB signaling. However, unlike in mammals, the Drosophila RHIMs may not be required for cell death. Consistently, it was found that the PGRP-LAD containing the RHIM domain does not induce cell death. However, PGRP-LAF lacking RHIM drives EC delamination and apoptosis. Previous studies suggested a regulatory role of PGRP-LA in controlling NF-κB activity rather than binding to peptidoglycan. In this study, PGRP-LA depletion extends the lifespan of tumor-bearing flies. However, the expression of PGRP-LA is low in the intestine during normal homeostasis, suggesting PGRP-LA may not be involved in normal aging and intestinal homeostasis. Whether the programed cell death pathways impact metazoan lifespan remains unknown, and this requires further research (Zhou, 2021).

This study revealed that tumor induces cytoplasmic enrichment of DE-cadherin::GFP and activates p-Myosin signal in nearby ECs with elongated cell morphology. The DE-cadherin reporter and p-Myosin staining have been previously used as mechano-transduction sensors in Drosophila. The results therefore suggest that the tumor induces DE-cadherin- and myosin-dysregulation-associated mechanical stresses. However, changes in mechanical tension in cells adjacent to tumor cells is a rapid process and more evidence will be required to illustrate mechanical competition, e.g., by monitoring mechanosensors in live tissue. Unfortunately, this remains technically difficult in the Drosophila intestine because of constraints on live imaging and a lack of molecular markers. Moreover, how cells sense and respond to mechanical stress in the context of tumor growth requires further investigations (Zhou, 2021).

Control of intestinal cell fate by dynamic mitotic spindle repositioning influences epithelial homeostasis and longevity

Tissue homeostasis depends on precise yet plastic regulation of stem cell daughter fates. During growth, Drosophila intestinal stem cells (ISCs) adjust fates by switching from asymmetric to symmetric lineages to scale the size of the ISC population. Using a combination of long-term live imaging, lineage tracing, and genetic perturbations, this study demonstrates that this switch is executed through the control of mitotic spindle orientation by Jun-N-terminal kinase (JNK) signaling. JNK interacts with the WD40-repeat protein Wdr62 at the spindle and transcriptionally represses the kinesin Kif1a to promote planar spindle orientation. In stress conditions, this function becomes deleterious, resulting in overabundance of symmetric fates and contributing to the loss of tissue homeostasis in the aging animal. Restoring normal ISC spindle orientation by perturbing the JNK/Wdr62/Kif1a axis is sufficient to improve intestinal physiology and extend lifespan. These findings reveal a critical role for the dynamic control of SC spindle orientation in epithelial maintenance (Hu, 2019).

This study directly demonstrates that cell fate and spindle orientation are tightly linked and identifies a function for JNK signaling in promoting symmetric lineages through the realignment of the mitotic spindle. The data support a model in which the mutual recruitment of phosphorylated JNK (pJNK) and Wdr62 to the spindle, as well as the JNK-dependent transcriptional repression of Kif1a, is required for spindle positioning toward a planar orientation. Because the activation of JNK also prevents cortical localization of Mud, it is proposed that JNK activity disrupts engagement of the spindle with cortical determinants of spindle orientation and limits the force exerted on astral microtubules by repressing Kif1a expression (Hu, 2019).

Live long-term lineage tracing results reveal that planar spindles result in symmetric division outcomes, whereas oblique spindles precede asymmetric outcomes. As such, changes in spindle orientation (after paraquat, short-term refeeding, and age) reflect changes in division modes. Although live imaging is a powerful tool to directly visualize spindle orientation and fate outcomes, the lower resolution compared with fixed imaging could potentially cause a wider error range in spindle angle measurements. Nonetheless, the ability to clearly visualize the vector bisecting the segregation of the two cell bodies during telophase and the vector lining the basal region of neighboring stem cells helps alleviate this issue. Another potential caveat in this analysis is that fates of ISC daughter cells may have been mis-scored because of a delay in Su(H) activation. However, an asymmetric outcome was never observed to derive from planar spindles, and division outcomes were scored as symmetric only if Su(H) activity was not observed at the end of the time-lapse recording, which was ~4 h after Su(H) activation was first observed in divisions with outcomes scored as asymmetric. In paraquat-exposed animals that overexpressed Kif1a in ISCs, Su(H) expression was detected in outcomes scored as asymmetric at roughly the same time frame as in control conditions, suggesting that stress conditions like paraquat exposure do not grossly perturb regulation of Su(H) expression (Hu, 2019).

The spindle angle that separates symmetric and asymmetric divisions is ~15°, and it is unclear whether cell fate specification during divisions with spindle orientation around that angle is deterministic or stochastic. A small subset of spindle orientations above 20° (2 of 22 examples) resulted in 2 YFP+ cells rather than 1 YFP+ cell and 1 YFP+/mCherry+ cell. It is possible that these divisions still result in an asymmetric outcome but may have generated an mCherry- EE cell rather than an mCherry+ EB cell. The rare occurrence of these events is consistent with the smaller population of EEs compared with EB/ECs in the intestine, and spindle orientation during EE fate specification may be important to segregate prospero (Hu, 2019).

Although the results are thus compatible with a deterministic model for cell fate specification, they do not rule out a role for neutral drift. In a neutral drift model, the stem cell pool is maintained by a balance of ISC loss (by generating two differentiated cells) and duplication. It is unknown how regulation of spindle orientation affects neutral drift and whether spindle orientation differs between divisions leading to two ISCs or two EBs. Addressing these issues will be important for comprehensive understanding of cell fate determination in this system (Hu, 2019).

The disparity between spindle behaviors after paraquat treatment and those after Ecc15 infection shows that the nature of the environmental trigger is critical. Although both stresses induce strong proliferative responses, their effects on spindle orientation and the corresponding cell fate outcome are different. Based on the data in this study, this disparity is likely caused by differing levels of JNK activity. JNK is activated by oxidative stress and is thus strongly induced by paraquat exposure. Ecc15 infection, in turn, promotes ISC proliferation by predominantly stimulating JAK/signal transducer and activator of transcription (STAT) activation in ISCs and only transiently activating JNK. JNK was shown to be activated immediately after Ecc15 infection (30 min post-infection), but the genes encoding components of the JNK pathway were no longer upregulated as early as 4 h post-infection. These observations are consistent with analysis 16-20 h post-infection, particularly the absence of phosphorylated JNK at the mitotic spindle in Ecc15-infected animals. However, a possible role of JNK on spindle orientation at earlier time points after infection cannot be ruled out (Hu, 2019).

Previous studies have reported that similar to the current observations with Ecc15, infection of another strain of bacteria, Pseudomonas entomophila, largely promoted asymmetric fate outcomes. However, JNK activity was still detected in the entire gut 2 days post-infection, although the specific cell type (stem cells versus differentiated cells) in which JNK was activated was not examined. The possibility that JNK is activated in ISCs after P. entomophila infection was not ruled out. The difference in pathology of P. entomophila-which is lethal, unlike Ecc15-may contribute to a different response in JNK activation. One hypothesis is that although JNK may be activated after P. entomophila infection in ISCs, it is not recruited to the mitotic spindle and therefore would not affect spindle orientation. Future studies are needed to test this hypothesis and explore possible mechanisms of a pathogen-specific difference (Hu, 2019).

In recruitment to the spindle, pJNK and Wdr62 depend mutually on each other. Although JNK clearly plays a critical role in this process, the data do not rule out a role for other kinases that have been reported to recruit Wdr62 to the centrosome, including Aurora A and Polo-like kinase. Unlike other reports in neural stem cells, this study did not find an obvious role for Wdr62 in maintaining bipolar spindles. Reports have also identified roles for Wdr62 in stabilizing microtubules and centrosomes in interphase neural stem cells, and although the effects of Wdr62 depletion during interphase was not tested in this study, the absence of gross mitotic defects suggests that in Drosophila ISCs, Wdr62 may function selectively in spindle orientation. However, somewhat smaller clone sizes were observed of ISC lineages deficient for Wdr62, and therefore a function for interphase Wdr62 cannot be ruled out. Disruption of Wdr62 activity during interphase may also contribute to the inconsistent effect on lifespan observed after Wdr62 depletion, despite the restoration of oblique spindles in ISCs of old flies (Hu, 2019).

The consequences of the loss of Pins and Mud seem to vary depending on the tissue: disrupting Pins and Mud in Drosophila neuroblasts randomizes the mitotic spindle, but in the mammalian skin, basal stem cells with depleted LGN favor planar spindles, similar to observations in Drosophila ISCs. A loss of cortical Mud was observed after JNK activation, supporting the notion that JNK regulates the interaction between the astral microtubules and the cell cortex to promote planar spindles. The extent to which JNK or Wdr62 interacts directly with Mud is an important question for further study (Hu, 2019).

The mechanism by which Kif1a promotes oblique spindle orientation in ISCs is unclear. Khc-73, a kinesin in the same Kinesin-3 family, is believed to interact with Pins or Disc Large in Drosophila S2 cells and neuroblasts to orient astral microtubules to the cell cortex, and Kif1a may play similar roles in ISCs. Although the data suggest that JNK regulates Kif1a levels transcriptionally, it is possible that JNK also regulates Kif1a at the protein level and may direct its possible interaction with the spindle recruitment machinery (Hu, 2019).

The data reveal how a physiological role for JNK signaling in regulating spindle positioning during periods of tissue resizing becomes hijacked under stress and age, limiting tissue homeostasis and shortening lifespan. It remains unclear how JNK is activated in ISCs during starvation-refeeding, but insulin signaling has been implicated in promoting symmetric outcomes during adaptive resizing of the Drosophila intestine. It will be interesting to test whether insulin signaling and JNK interact to regulate spindle positioning in ISCs, because elevated insulin signaling activity may also contribute to the age-related chronic activation of JNK. The age-related bias toward planar spindle orientations is reminiscent of the changes in spindle orientation of germline stem cells in old male flies, and restoring oblique spindle orientation in aged ISCs by increasing Kif1a expression is sufficient to improve intestinal physiology and extend lifespan. Understanding the exact mechanisms and consequences of ISC spindle positioning will be critical to identifying new intervention strategies to allay age-related dysfunction in barrier epithelia (Hu, 2019).

Such interventions are likely to have significant clinical relevance, because barrier epithelia in mammals regenerate and age through mechanisms that are similar to the Drosophila intestinal epithelium. However, although SC fate determination by changes in spindle orientation is observed in multiple mammalian tissues during development, the extent to which similar mechanisms determine cell fate in adult mammalian tissues is unclear. Mouse ISCs within the adult intestine use different mechanisms to establish cell fate, because spindle orientation is largely planar, and extrinsic cues preferentially differentiate one of the daughter cells. In the mouse trachea, however, it has been reported that spindle orientation fluctuates in basal stem cells in response to injury and may affect cell fate specification. Given the variation in lineage, cell composition, and organization in different adult tissues, it is likely that the importance of spindle orientation in cell specification differs among tissues. Determining the tissues in which spindle orientation is linked with cell fate, and testing whether the role of JNK in the regulation of spindle orientation in these SCs is conserved, will provide important insight into regenerative biology (Hu, 2019).

The MicroRNA miR-277 Controls Physiology and Pathology of the Adult Drosophila Midgut by Regulating the Expression of Fatty Acid beta-Oxidation-Related Genes in Intestinal Stem Cells

This study tested whether stem and progenitor cell types might have a distinctive metabolic profile in the intestinal lineage. This study tested that hypothesis and investigated the metabolism of the intestinal lineage from stem cell (ISC) to differentiated epithelial cell in their native context under homeostatic conditions. An initial in silico analysis of single cell RNAseq data and functional experiments identify the microRNA miR-277 as a posttranscriptional regulator of fatty acid beta-oxidation (FAO) in the intestinal lineage. Low levels of miR-277 are detected in ISC and progressively rising miR-277 levels are found in progenitors during their growth and differentiation. Supporting this, miR-277-regulated fatty acid beta-oxidation enzymes progressively declined from ISC towards more differentiated cells in pseudotime single-cell RNAseq analysis and in functional assays on RNA and protein level. In addition, in silico clustering of single-cell RNAseq data based on metabolic genes validates that stem cells and progenitors belong to two independent clusters with well-defined metabolic characteristics. Furthermore, studying FAO genes in silico indicates that two populations of ISC exist that can be categorized in mitotically active and quiescent ISC, of which the latter relies on FAO genes. In line with an FAO dependency of ISC, forced expression of miR-277 phenocopies RNAi knockdown of FAO genes by reducing ISC size and subsequently resulting in stem cell death. This study also investigated miR-277 effects on ISC in a benign and a newly developed CRISPR-Cas9-based colorectal cancer model and found effects on ISC survival, which as a consequence affects tumor growth, further underlining the importance of FAO in a pathological context. Taken together, this study provides new insights into the basal metabolic requirements of intestinal stem cell on beta-oxidation of fatty acids evolutionarily implemented by a sole microRNA. Gaining knowledge about the metabolic differences and dependencies affecting the survival of two central and cancer-relevant cell populations in the fly and human intestine might reveal starting points for targeted combinatorial therapy in the hope for better treatment of colorectal cancer in the future (Zipper, 2022).

The Yun/Prohibitin complex regulates adult Drosophila intestinal stem cell proliferation through the transcription factor E2F1

Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. From an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), this study identified a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. It was demonstrated that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, this study found that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, this study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation (Zho, 2022).

Prohibitins (PHBs) are members of the conserved SPFH superfamily. PHB1 was first identified by its antiproliferative activity upon ectopic expression, which was later attributed to its 3' untranslated region instead of the PHB protein itself. The PHB complex contains two homologous members: PHB1 and PHB2. PHB1 and PHB2 are ubiquitously expressed and are present in the mitochondria, the nucleus, cytosol, and the lipid rafts of the plasma membrane. A number of studies have described a role of the PHB complex within the mitochondria, where it forms a supramacromolecular structure at the inner membrane of the mitochondria acting as a scaffold (or a chaperone) for proteins and lipids regulating mitochondrial metabolism. The PHB complex has been implicated in various cellular and developmental processes and diseases, such as mitochondrial respiration, signaling, and mitophagy depending on its cellular localization. Disruption of the PHB genes has effects ranging from decreased replicative lifespan in yeast, to larval arrest in Drosophila, and to embryonic lethality in mice. However, it remains unexplored whether they play a role in intestinal stem cell regulation in Drosophila (Zho, 2022).

Proliferation and differentiation of adult stem cells must be tightly controlled to maintain tissue homeostasis and prevent tumorigenesis. However, how stem cell proliferation is properly controlled and in particular how the cell cycle is regulated in stem cells is not fully understood. This study identified Yun as an ISC proliferation regulator from a large-scale RNAi screen. Loss of yun function in progenitors restricts them from proliferating, such that they remain in a quiescent state under normal conditions and during tissue regeneration following acute tissue damage. Yun was shown to act as a scaffold for the PHB complex, and the Yun/PHB complex is regulated by EGFR signaling and functions through E2F1 to sustain proliferation of normal stem cells for tissue homeostasis/regeneration and transformed stem cells in tumorigenesis (Zho, 2022).

In addition to EGFR signaling, the levels of the Yun/PHB complex are also elevated upon activation of JAK/STAT signaling or in the absence of Notch, indicating that the Yun/PHB complex may also be regulated by JAK/STAT and Notch signaling directly or indirectly. Previous studies and the current data show that EGFR signaling acts downstream of JAK/STAT, Notch, and Wnt signaling in ISC proliferation and that ectopic expression of yun/Phb could rescue proliferation defects in the absence of EGFR signaling. Therefore, it is proposed that EGFR signaling is the major upstream of signal of the Yun/PHB complex, although the possibilities cannot be fully excluded that the complex may also be regulated by the other signaling pathways directly or indirectly. The EGFR/MAPK pathway and E2F1 are differentially required for stem cell proliferation (mitosis) and differentiation (endoreplication). How E2F1 is differentially regulated during these two processes is not clear. The regulation of E2F1 levels by EGFR/MAPK signaling has been proposed to be due to increased translation or/and increased protein stability, possibly involving some unknown cytoplasmic factors. The identification of the Yun/PHB complex may account for the differential regulation of E2F1 by EGFR/MAPK signaling. The Yun/PHB complex is expressed in progenitors and mediates EGFR/MAPK signaling for ISC proliferation but not progeny differentiation, indicating that the Yun/PHB complex is more specifically required for ISC proliferation. Interestingly, a previous study identified another target of EGFR signaling, the transcription factor Sox100B/dSox9B, which has a critical role in progeny differentiation, indicating that the control of the ISC proliferation and progeny differentiation by EGFR/MAKP signaling is likely differentially mediated by different effectors (Zho, 2022).

The levels of E2F1 protein, along with the expression of PCNA-GFP, were significantly diminished in yun/Phb-defective progenitors or imaginal wing discs, suggesting that Yun affects E2F1 levels. Biochemical analysis shows that the Yun/PHB complex associates with E2F1 in vivo, indicating that the Yun/PHB complex interacts and stabilizes E2F1 protein to regulate ISC proliferation. Furthermore, ectopic expression of single components of the Yun/PHB complex increased E2F1 protein levels, which were further increased when two or three components of the Yun/PHB complex were coexpressed, supporting the notion that the Yun/PHB complex regulates E2F1 protein levels. Moreover, ectopic expression of E2F1/Dp significantly restored ISC proliferation in yun/Phb-defective intestines. Consistently, knockdown of the negative regulator of E2F1, Rb, also restored ISC proliferation in these yun/Phb-defective intestines, albeit at a weaker level than that of E2F1/Dp overexpression. Together, these data demonstrate that E2F1 acts downstream of the Yun/PHB complex for ISC proliferation. Interestingly, overexpressing PCNA alone is not sufficient to restore ISC proliferation in yun-depleted intestines, indicating that collective activation of multiple downstream targets of the E2F1/Dp complex are required to restore ISC proliferation. It has been previously proposed that PHB1 binds to Rb and functions as a negative regulator of E2F1-mediated transcription. These studies contrast with previous work suggesting that the PHB complex is required for cell proliferation. This in vivo study uncovered how E2F1 is differential regulated by EGFR/MAPK signaling and acts downstream of the Yun/PHB complex in ISC proliferation to maintain tissue homeostasis under normal and stress conditions and during tumorigenesis in Drosophila, which is in striking contrast to the proposed antiproliferation role of PHB1 (Zho, 2022).

Unlike Yun, Phb1 and Phb2 are conserved and have been reported to form a complex that localizes to the nucleus, plasma membrane, and mitochondria in mammalian cells. In mitochondria, the PHB complex functions as chaperones in mitochondria of some cell types. Although in flies, the Yun/PHB complex is partially localized in mitochondria in progenitors, the results indicate that the ISC proliferation defects observed in yun/Phb-defective progenitors are unlikely due to their roles in mitochondria. Human Phb1 and Phb2 could significantly restore ISC proliferation defects in Phb1- and Phb2-depleted intestines, respectively, and were required for the proliferation of different human cancer cell lines, indicating that the function of the PHB complex in proliferation is conserved. Finally, the observations that 1) Yun acts as a scaffold of PHBs for their proper function; 2) the Yun/PHB complex acts downstream of EGFR/MAPK signaling; and 3) PHBs and EGFR/MAPK signaling are evolutionarily conserved, suggest that a functional counterpart of Yun exists in mammals, which is different in primary sequence but possibly similar in structure (Zho, 2022).

MicroRNA mediated regulation of the onset of enteroblast differentiation in the Drosophila adult intestine

Somatic adult stem cell lineages in high-turnover tissues are under tight gene regulatory control. Like its mammalian counterpart, the Drosophila intestine precisely adjusts the rate of stem cell division with the onset of differentiation based on physiological demand. Although Notch signaling is indispensable for these decisions, the regulation of Notch activity that drives the differentiation of stem cell progenies into functional, mature cells is not well understood. This study reports that commitment to the terminally differentiated enterocyte (EC) cell fate is under microRNA control. An intestinally enriched microRNA, miR-956, fine-tunes Notch signaling activity specifically in intermediate, enteroblast (EB) progenitor cells to control EC differentiation. This study further identified insensitive mRNA as a target of miR-956 that regulates EB/EC ratios by repressing Notch activity in EBs. In summary, this study highlights a post-transcriptional gene-regulatory mechanism for controlling differentiation in an adult intestinal stem cell lineage (Mukherjee, 2022).

Coordinated repression of pro-differentiation genes via P-bodies and transcription maintains Drosophila intestinal stem cell identity

The role of processing bodies (P-bodies), key sites of post-transcriptional control, in adult stem cells remains poorly understood. This paper reports that adult Drosophila intestinal stem cells, but not surrounding differentiated cells such as absorptive enterocytes (ECs), harbor P-bodies that contain Drosophila orthologs of mammalian P-body components DDX6, EDC3, EDC4, and LSM14A/B. A targeted RNAi screen in intestinal progenitor cells identified 39 previously known and 64 novel P-body regulators, including Patr-1, a gene necessary for P-body assembly. Loss of Patr-1-dependent P-bodies leads to a loss of stem cells that is associated with inappropriate expression of EC-fate gene nubbin. Transcriptomic analysis of progenitor cells identifies a cadre of such weakly transcribed pro-differentiation transcripts that are elevated after P-body loss. Altogether, this study identifies a P-body-dependent repression activity that coordinates with known transcriptional repression programs to maintain a population of in vivo stem cells in a state primed for differentiation (Buddika, 2022).

This study molecularly and functionally characterized stem cell mRNPs that are concluded to be P-bodies based on three observations: (1) they contained colocalized protein complexes that include fly orthologs of proteins known to localize to P-bodies in mammalian and yeast cells, (2) these mRNP granules were significantly larger than and show no colocalization with intestinal progenitor stress granules (IPSG) protein foci under controlled conditions, and (3) acute stresses increased the size of these mRNPs and promoted colocalization with IPSGs. A targeted genetic screen identified 39 previously known and 64 new genes that influenced P-body morphology, including six required for P-body formation. To examine stem cell P-body function, one of this latter class was characterized, PATR-1, an evolutionary conserved protein with both translational repression and mRNA decay functions that is necessary for proper P-body assembly in Saccharomyces cerevisiae. Depletion of P-bodies in progenitor cells upregulated the expression of pro-differentiation genes, including nubbin. Loss of stem cell P-bodies, either by genetic depletion or differentiation, led to the increased translation as well as the cytoplasmic, but not nuclear, abundance of such transcripts. It is therefore proposed that mature P-bodies are necessary for stem cell maintenance by post-transcriptionally enforcing the repression of transcriptional programs that promote differentiation (Buddika, 2022).

Quantitative super-resolution microscopy was used to visualize substructures present within mature P-bodies. Consistent with the proposed 'core-shell' structure of stress granules, P-bodies exhibit 'cores' with high protein concentrations and 'shells' with low protein concentration. Notably, Drosophila intestinal progenitor P-bodies have an ~125 nm diameter, as compared to larger P-bodies in HEK293 cells, which have a ~500 nm diameter. In addition, intestinal progenitors contain ~35-45 mature P-bodies while HEK293 cells contain only ~4-7 granules per cell, indicating that the size and number of mature P-bodies depend on cell type and species and may scale with overall cell size (Buddika, 2022).

A recent study documented the presence of P-bodies in cultured human pluripotent stem cells and suggested their presence in adult stem cells. This analysis of DDX6, the ortholog of Drosophila Me31B, showed that DDX6-dependent P-bodies could both promote and repress stem cell identity, depending on context. For example, loss of DDX6 expanded endodermally derived Lgr5+ ISCs or ectodermally derived neural progenitor cell populations but promoted the differentiation of other progenitor cell populations, including mesodermally derived progenitors. The presence of mature P-bodies in adult progenitor populations was confirmed but it was shown that they repress differentiation rather than increasing their proliferation, as in Lgr5+ ISCs. A few possible explanations could reconcile these results. Most simply, Drosophila intestinal progenitors behave more like mesodermally derived mammalian progenitors rather than endo- or ectodermally derived mammalian progenitors. Alternatively, the stem cell function of DDX6 might be affected by its roles in surrounding cells, because DDX6 was targeted in cells throughout mouse intestinal organoids, whereas PATR-1 was specifically targeted in progenitor cells in this study. Finally, DDX6-mediated P-body function might be modulated by signaling that is not fully recapitulated in in vitro derived stem cell models (Buddika, 2022).

The exact molecular function of P-bodies is a matter of current debate. Consistent with other recent studies, this analysis suggests intestinal progenitor P-bodies have both translational repressive and mRNA degradatory functions. Pdm protein was absent in progenitors despite the weak expression of nub mRNA, suggesting P-body-dependent translational repression. In addition, RNAscope analysis showed that cytoplasmic nub transcript abundance was increased in Patr-1 mutant progenitors without an indication of nuclear transcription, suggesting the stabilization of transcripts that are targeted for degradation via P-bodies. Dual P-body roles in mRNA repression and degradation are also suggested in human cultured stem cells. For instance, P-bodies influence the translation of transcripts encoding fate-instructive transcription and chromatin factors in cultured embryonic and in vitro derived adult stem cells. In addition, P-body proteins DDX6 and EDC3 are known to destabilize differentiation-inducing mRNAs, such as KLF4 in human epidermal progenitor cells, although it is important to note that P-bodies have not been reported in these cells. Notably, the mammalian homolog of nub, OCT1/POU2F1, is one of the top 30 most enriched mRNAs of P-bodies in HEK293 cells, indicating evolutionary conservation of P-body targets (Buddika, 2022).

It is propose that weak transcription of pro-differentiation genes likely maintains progenitors in a state primed for differentiation. The transcriptional repression of differentiation genes by the transcription factor esg is a key regulatory step of intestinal stem cell maintenance. The loss of esg gene expression or inability to localize the Esg protein to target genes promotes progenitor loss via premature ISC-to-EC differentiation. Similar to Patr-1 RNAi, knocking down esg itself as well as either vtd, Nipped-B, or polo, all of which are necessary for recruiting Esg to target promoters, markedly upregulated the expression of Pdm1 in intestinal progenitors. Notably, transcriptomic profiling showed that the transcript level of neither esg nor any of the Esg-targeting proteins, vtd, Nipped-B, or polo, were changed by the absence of mature P-bodies. These observations suggest that Esg protein level, its proper promoter targeting, and its transcriptional repression of EC genes are all unlikely to be affected by the loss of PATR-1 (Buddika, 2022).

In addition to identifying 64 new genes affecting P-body morphology, it is expected that the tissue-based stem cell P-body system identified and described in this study will prove critically useful in screening for chemicals, diet conditions, and stress conditions that alter P-body assembly as well as performing larger, genome-wide screens to comprehensively characterize the molecular pathways that control P-body assembly. Moreover, similar approaches can be used to identify systemic signals that promote P-body disassembly during the onset of differentiation as well as to identify molecular players of P-body disassembly (Buddika, 2022).

The RNA binding protein Swm is critical for Drosophila melanogaster intestinal progenitor cell maintenance

The regulation of stem cell survival, self-renewal, and differentiation is critical for the maintenance of tissue homeostasis. Although the involvement of signaling pathways and transcriptional control mechanisms in stem cell regulation have been extensively investigated, the role of post-transcriptional control is still poorly understood. This study shows that the nuclear activity of the RNA-binding protein Second Mitotic Wave Missing (Swm) is critical for Drosophila melanogaster intestinal stem cells (ISCs) and their daughter cells, enteroblasts (EBs), to maintain their progenitor cell properties and functions. Loss of swm causes ISCs and EBs to stop dividing and instead detach from the basement membrane, resulting in severe progenitor cell loss. swm loss is further characterized by nuclear accumulation of poly(A)+ RNA in progenitor cells. Swm associates with transcripts involved in epithelial cell maintenance and adhesion, and the loss of swm, while not generally affecting the levels of these Swm-bound mRNAs, leads to elevated expression of proteins encoded by some of them, including the fly ortholog of Filamin. Taken together, this study indicates a nuclear role for Swm in adult stem cell maintenance, raising the possibility that nuclear post-transcriptional regulation of mRNAs encoding cell adhesion proteins ensures proper attachment of progenitor cells (Ariyapala, 2022).

MicroRNA mediated regulation of the onset of enteroblast differentiation in the Drosophila adult intestine

Somatic adult stem cell lineages in high-turnover tissues are under tight gene regulatory control. Like its mammalian counterpart, the Drosophila intestine precisely adjusts the rate of stem cell division with the onset of differentiation based on physiological demand. Although Notch signaling is indispensable for these decisions, the regulation of Notch activity that drives the differentiation of stem cell progenies into functional, mature cells is not well understood. This study reports that commitment to the terminally differentiated enterocyte (EC) cell fate is under microRNA control. An intestinally enriched microRNA, miR-956, fine-tunes Notch signaling activity specifically in intermediate, enteroblast (EB) progenitor cells to control EC differentiation. This study further identified insensitive mRNA as a target of miR-956 that regulates EB/EC ratios by repressing Notch activity in EBs. In summary, this study highlights a post-transcriptional gene-regulatory mechanism for controlling differentiation in an adult intestinal stem cell lineage (Mukherjee, 2023).

Drosophila Caliban preserves intestinal homeostasis and lifespan through regulating mitochondrial dynamics and redox state in enterocytes

Precise regulation of stem cell activity is crucial for tissue homeostasis. In Drosophila, intestinal stem cells (ISCs) maintain the midgut epithelium and respond to oxidative challenges. However, the connection between intestinal homeostasis and redox signaling remains obscure. This study found that Caliban (Clbn), a component of the ribosome quality control complex (RQC), functions as a regulator of mitochondrial dynamics in enterocytes (ECs) and is required for intestinal homeostasis. The clbn knock-out flies have a shortened lifespan and lose the intestinal homeostasis. Clbn is highly expressed and localizes to the outer membrane of mitochondria in ECs. Mechanically, Clbn mediates mitochondrial dynamics in ECs and removal of clbn leads to mitochondrial fragmentation, accumulation of reactive oxygen species, ECs damage, activation of JNK and JAK-STAT signaling pathways. Moreover, multiple mitochondria-related genes are differentially expressed between wild-type and clbn mutated flies by a whole-genome transcriptional profiling. Furthermore, loss of clbn promotes tumor growth in gut generated by activated Ras in intestinal progenitor cells. These findings reveal an EC-specific function of Clbn in regulating mitochondrial dynamics, and provide new insight into the functional link among mitochondrial redox modulation, tissue homeostasis and longevity (Cai, 2020).

Context-dependent responses of Drosophila intestinal stem cells to intracellular reactive oxygen species

Stem cells have unique Reactive oxygen species (ROS) regulation while cancer cells frequently show a constitutive oxidative stress that is associated with the invasive phenotype. Antioxidants have been proposed to forestall tumor progression while targeted oxidants have been used to destroy tumor cells. This study used Drosophila midgut intestinal stem cell (ISCs) to tackle this question. The ROS levels of ISCs remained low in comparison to that of differentiated cells and increased with ageing, which was accompanied by elevated proliferation of ISCs in aged Drosophila. Neither upregulation nor downregulation of ROS levels significantly affected ISCs, implicating an intrinsic homeostatic range of ROS in ISCs. Interestingly, similar moderately elevated ROS levels were observed in both tumor-like ISCs induced by Notch (N) depletion and extracellular matrix (ECM)-deprived ISCs induced by β-integrin (mys) depletion. Elevated ROS levels further promoted the proliferation of tumor-like ISCs while reduced ROS levels suppressed the hyperproliferation phenotype; on the other hand, further increased ROS facilitated the survival of ECM-deprived ISCs while reduced ROS exacerbated the loss of ECM-deprived ISCs. However, N- and mys-depleted ISCs, which resembled metastatic tumor cells, harbored even higher ROS levels and were subjected to more severe cell loss, which could be partially prevented by ectopic supply of antioxidant enzymes, implicating a delicate pro-surviving and proliferating range of ROS levels for ISCs. Taken together, these results revealed stem cells can differentially respond to distinct ROS levels (Chen, 2021).

Defective Proventriculus Regulates Cell Specification in the Gastric Region of Drosophila Intestine

The adult Drosophila midgut and the mammalian intestine share a high degree of conservation between such signaling pathways. The gastric region located in the Drosophila middle-midgut coincides with the region containing fewest number of stem cells. It is also known as the copper cell (CC) region since it is composed of specialized groups of acid-secreting CCs, along with interstitial cells and enteroendocrine cells. The generation and maintenance of these cell populations are determined by the bone morphogenic protein-like Decapentaplegic (Dpp) signaling pathway. The morphogenic gradient of the Dpp signaling activity induces differential expression of specific transcription factors labial (lab) and defective proventriculus (dve), which are required for the generation of various cell types specific to this region. This study investigated the role of Dve in regulation of tissue homeostasis in the CC region. These studies reveal that ectopic expression of dve in stem cells suppresses their self-renewal throughout the intestine. It was further demonstrated that Dve is not required for generation of CCs. Higher levels of Dve can alter cell specification by inhibition of cut expression, which in turn prevents CC formation during homeostasis (Mehrotra, 2020).

Transiently "Undead" Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut

This study reveals surprising similarities between homeostatic cell turnover in adult Drosophila midguts and "undead" apoptosis-induced compensatory proliferation (AiP) in imaginal discs. During undead AiP, immortalized cells signal for AiP, allowing its analysis. Critical for undead AiP is the Myo1D-dependent localization of the initiator caspase Dronc to the plasma membrane. This study shows that Myo1D functions in mature enterocytes (ECs) to control mitotic activity of intestinal stem cells (ISCs). In Myo1D mutant midguts, many signaling events involved in AiP (ROS generation, hemocyte recruitment, and JNK signaling) are affected. Importantly, similar to AiP, Myo1D is required for membrane localization of Dronc in ECs. It is proposed that ECs destined to die transiently enter an undead-like state through Myo1D-dependent membrane localization of Dronc, which enables them to generate signals for ISC activity and their replacement. The concept of transiently "undead" cells may be relevant for other stem cell models in flies and mammals (Amcheslavsky, 2020).

Dynamic adult tracheal plasticity drives stem cell adaptation to changes in intestinal homeostasis in Drosophila

Coordination of stem cell function by local and niche-derived signals is essential to preserve adult tissue homeostasis and organismal health. The vasculature is a prominent component of multiple stem cell niches. However, its role in adult intestinal homeostasis remains largely understudied. This study has uncover a previously unrecognised crosstalk between adult intestinal stem cells in Drosophila and the vasculature-like tracheal system, which is essential for intestinal regeneration. Following damage to the intestinal epithelium, gut-derived reactive oxygen species activate tracheal HIF-1α and bidirectional FGF/FGFR signalling, leading to reversible remodelling of gut-associated terminal tracheal cells and intestinal stem cell proliferation following damage. Unexpectedly, reactive oxygen species-induced adult tracheal plasticity involves downregulation of the tracheal specification factor trachealess (trh) and upregulation of IGF2 messenger RNA-binding protein (IGF2BP2/Imp). These results reveal an intestine-vasculature inter-organ communication programme that is essential to adapt the stem cell response to the proliferative demands of the intestinal epithelium (Perochon, 2021).

White regulates proliferative homeostasis of intestinal stem cells during ageing in Drosophila

Tissue integrity is contingent on maintaining stem cells. Intestinal stem cells (ISCs) over-proliferate during ageing, leading to tissue dysplasia in Drosophila melanogaster. This study describes a role for white, encoding the evolutionarily conserved ATP-binding cassette transporter subfamily G, with a particularly well-characterized role in eye colour pigmentation, in ageing-induced ISC proliferation in the midgut. ISCs increase expression of white during ageing. ISC-specific inhibition of white suppresses ageing-induced ISC dysregulation and prolongs lifespan. Of the proteins that form heterodimers with White, Brown mediates ISC dysregulation during ageing. Metabolomics analyses reveal previously unappreciated, profound metabolic impacts of white inhibition on organismal metabolism. Among the metabolites affected by White, tetrahydrofolate is transported by White, is accumulated in ISCs during ageing and is indispensable for ageing-induced ISC over-proliferation. Since Thomas Morgan's isolation of a white mutant as the first Drosophila mutant, white mutants have been used extensively as genetic systems and often as controls. These findings provide insights into metabolic regulation of stem cells mediated by the classic gene white (Sasaki, 2021).

Neuroglian regulates Drosophila intestinal stem cell proliferation through enhanced signaling via the epidermal growth factor receptor

The Drosophila intestine is an excellent system for elucidating mechanisms regulating stem cell behavior. This study shows that the septate junction (SJ) protein Neuroglian (Nrg) is expressed in intestinal stem cells (ISCs) and enteroblasts (EBs) within the fly intestine. SJs are not present between ISCs and EBs, suggesting Nrg plays a different role in this tissue. This study reveals that Nrg is required for ISC proliferation in young flies, and depletion of Nrg from ISCs and EBs suppresses increased ISC proliferation in aged flies. Conversely, overexpression of Nrg in ISC and EBs promotes ISC proliferation, leading to an increase in cells expressing ISC/EB markers; in addition, an increase was observed in epidermal growth factor receptor (Egfr) activation. Genetic epistasis experiments reveal that Nrg acts upstream of Egfr to regulate ISC proliferation. As Nrg function is highly conserved in mammalian systems, this work characterizing the role of Nrg in the intestine has implications for the treatment of intestinal disorders that arise due to altered ISC behavior (Resnik-Docampo, 2021).

Evolution and genomic signatures of spontaneous somatic mutation in Drosophila intestinal stem cells

Spontaneous mutations can alter tissue dynamics and lead to cancer initiation. Although large-scale sequencing projects have illuminated processes that influence somatic mutation and subsequent tumor evolution, the mutational dynamics operating in the very early stages of cancer development are currently not well understood. To explore mutational processes in the early stages of cancer evolution, this study exploited neoplasia arising spontaneously in the Drosophila intestine. Analysing whole-genome sequencing data with a dedicated bioinformatic pipeline, this study found neoplasia formation is driven largely through the inactivation of Notch by structural variants, many of which involve highly complex genomic rearrangements. The genome-wide mutational burden in neoplasia was found to be similar to that of several human cancers. Finally, this study identified genomic features associated with spontaneous mutation, and defined the evolutionary dynamics and mutational landscape operating within intestinal neoplasia over the short lifespan of the adult fly. These findings provide unique insight into mutational dynamics operating over a short timescale in the genetic model system, Drosophila melanogaster (Riddiford, 2021).

Multiscale analysis reveals that diet-dependent midgut plasticity emerges from alterations in both stem cell niche coupling and enterocyte size

The gut is the primary interface between an animal and food, but how it adapts to qualitative dietary variation is poorly defined. This study finds that the Drosophila midgut plastically resizes following changes in dietary composition. A panel of nutrients collectively promote gut growth, which sugar opposes. Diet influences absolute and relative levels of enterocyte loss and stem cell proliferation, which together determine cell numbers. Diet also influences enterocyte size. A high sugar diet inhibits translation and uncouples intestinal stem cell proliferation from expression of niche-derived signals, but, surprisingly, rescuing these effects genetically was not sufficient to modify diet's impact on midgut size. However, when stem cell proliferation was deficient, diet's impact on enterocyte size was enhanced, and reducing enterocyte-autonomous TOR signaling was sufficient to attenuate diet-dependent midgut resizing. These data clarify the complex relationships between nutrition, epithelial dynamics, and cell size, and reveal a new mode of plastic, diet-dependent organ resizing (Bonfini, 2021).

Vinculin recruitment to α-catenin halts the differentiation and maturation of enterocyte progenitors to maintain homeostasis of the Drosophila intestine

Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by peristalsis of its variable content. Using the Drosophila intestinal epithelium, this study investigated the role of vinculin, one of the best characterised mechanoeffectors, which functions in both cadherin and integrin adhesion complexes. Vinculin was found to regulated by &alpha-catenin at sites of cadherin adhesion, rather than as part of integrin function. Following asymmetric division of the stem cell into a stem cell and an enteroblast (EB), the two cells initially remain connected by adherens junctions, where vinculin is required, only on the EB side, to maintain the EB in a quiescent state and inhibit further divisions of the stem cell. By manipulating cell tension, it was shown that vinculin recruitment to adherens junction regulates EB activation and numbers. Consequently, removing vinculin results in an enlarged gut with improved resistance to starvation. Thus, mechanical regulation at the contact between stem cells and their progeny is used to control tissue cell number (Bohere, 2022).

Age-related changes in polycomb gene regulation disrupt lineage fidelity in intestinal stem cells

Tissue homeostasis requires long-term lineage fidelity of somatic stem cells. Whether and how age-related changes in somatic stem cells impact the faithful execution of lineage decisions remains largely unknown. This study addressed this question using genome-wide chromatin accessibility and transcriptome analysis as well as single-cell RNA-seq to explore stem-cell-intrinsic changes in the aging Drosophila intestine. These studies indicate that in stem cells of old flies, promoters of Polycomb (Pc) target genes become differentially accessible, resulting in the increased expression of enteroendocrine (EE) cell specification genes. Consistently, age-related changes were found in the composition of the EE progenitor cell population in aging intestines, as well as a significant increase in the proportion of EE-specified intestinal stem cells (ISCs) and progenitors in aging flies. This study further confirmed that Pc-mediated chromatin regulation is a critical determinant of EE cell specification in the Drosophila intestine. Pc is required to maintain expression of stem cell genes while ensuring repression of differentiation and specification genes. These results identify Pc group proteins as central regulators of lineage identity in the intestinal epithelium and highlight the impact of age-related decline in chromatin regulation on tissue homeostasis (Tauc, 2021).

This study identifies a loss of lineage fidelity in ISCs of aging flies that results in an imbalance of EE vs EC differentiation and contributes to epithelial dysplasia. This loss of lineage fidelity is caused by age-related deregulation of Pc function in ISCs, resulting in de-repression and preferential expression of EE genes. It is proposed that the chronic activation of stress signaling in ISCs, triggered by local and systemic inflammatory stimuli in the aging intestine, promotes the deregulation of Pc-controlled gene activity. This is supported by the fact that genetically elevating JNK activity in ISCs disrupts lineage fidelity and causes an increase in the proportion of EEs in the gut epithelium. The increase in EE numbers contributes to epithelial dysplasia in the aging gut, as EEs can promote ISC proliferation. The stress-induced changes in lineage fidelity in ISCs thus likely set up a vicious cycle that causes progressive dysplasia and results in disruption of epithelial structure and function in the aging intestine (Tauc, 2021).

The scRNA-seq analysis of the Drosophila gut is consistent with a recent scRNA-seq study from young flies (Hung, 2020), but also captures the changes in intestinal cell states across aging. Notably, the well-characterized age-associated increase in mitotically active ISCs was observed, but a unique 'stressed ISC' cell population was identified that increases with age. The transcriptional signature distinguishing this cell population encompasses over 25% of significantly upregulated genes observed in a bulk RNA-seq study from purified old ISCs, supporting the robustness and complementarity of the two methods. This transcriptional signature was enriched in genes involved in glutathione metabolic processes, chaperone-mediated protein folding, response to heat, and regulation of cytoskeleton organization, consistent with a stressed or damaged cell state. The appearance of this 'stressed' stem cell population is further consistent with the previously described increase in inflammatory and oxidative stress in the aging intestinal epithelium, and may reflect the age-associated accumulation of oxidative, proteostatic and genomic damage in these cells. Overall, the majority of old ISCs reside in an activated cell state (~50%), whereas the 'stressed ISC' population makes up only a small percentage (<10%). How this 'stressed' population affects tissue homeostasis requires further studies. Intriguingly, the p53 and DNA repair pathways are upregulated in this cell population, while cell cycle genes are repressed, indicating that these cells may represent a correlate to mammalian senescent cells (Tauc, 2021).

The EE progenitor population within one cluster not only increases in size with age, but also upregulates pro-neural genes that are markers for neural stem cells (NSCs). It was recently shown that in a neuroendocrine tumor model, ISCs undergo an identity switch that results in the acquisition of NSC-like features (Li, 2020). This NSC gene signature was also upregulated in old ISCs analyzed by bulk RNA-seq, suggesting that a general upregulation of these genes may contribute to age-related ISC phenotypes (Tauc, 2021).

ATAC-seq data from purified ISC populations revealed only moderate changes in chromatin organization in ISCs of increasingly older animals, suggesting that ISC gene regulation is tightly controlled throughout life. At the same time, the significant increase in H3K27 dimethylation levels in aging ISCs, the fact that H3K27me2 levels are higher in EEs than in ISCs in young guts, and the observation that E(z)-mediated methylation of H3K27 is required for EE specification, all support a role for increases in H3K27me2 in skewing ISC identity towards the EE fate. Despite the high genomic abundance of H3K27me2, which accounts for up to 70% of total histone H3, the functional role of H3K27me2 remains largely uncharacterized. The broad genomic distribution of H3K27me2 was shown to suppress aberrant gene activation by controlling enhancer fidelity in mammals, and access to transcription factors and RNA Pol II to DNA in flies. If and how genomic abundance and/or distribution of H3K27me2 affects cell identity or other cellular functions has not been well explored. One study found that perturbing the ratio of H3K27me2/H3K27me3 in mouse embryonic stem cells (ESCs) affected the acquisition and repression of specific fates of these cells, indicating the importance of appropriate regulation of these marks in different cell types. The differential abundance of H3K27me2 that was observed in young ISCs and EEs further supports the importance of dynamic H3K27me2 regulation in the ISC lineage and of appropriate control of this mark to maintain lineage commitment. The loss of EE cell differentiation upon Pc or E(z) depletion in ISCs further supports a critical role for PRC in regulating H3K27 methylation status and thereby lineage fidelity. Of note, the expression of Pc, E(z) and other PcG genes, as well as the expression of trx, Trl and z, was not significantly altered in aging ISCs, suggesting that aging most likely affects their post-transcriptional regulation and/or function (Tauc, 2021).

Age-related changes in H3K27 methylation have been reported in mammalian SC populations: in aging HSCs and muscle SCs, the H3K27me3 signal exhibits broader coverage and increased intensity at transcriptional start sites and intergenic regions, indicating that there is an evolutionarily conserved effect of aging on PRC function in tissue stem cells. It would be intriguing to explore whether these alterations in H3K27me3 may underlie the age-related dysfunction in lineage potential observed in HSCs of old mice (Tauc, 2021).

Since loss of Pc induces the expression of EC genes, represses EE gene expression, and results in less accessible chromatin associated with ISC identity genes (esg, spdo) as well as pro-neural genes (dpn), it is proposed that Pc activity regulates multiple aspects of ISC specification. Despite the upregulation of EC genes after Pc depletion, ISCs did not spontaneously differentiate, ISC numbers remained normal and ISCs could still mount a proliferative response to infection. Thus, ISC function remained largely intact suggesting the primary function of Pc in ISCs is to regulate lineage commitment (Tauc, 2021).

The contrasting function of trx in EE differentiation is consistent with the known antagonism between Trx and Pc complexes and exemplifies a tightly regulated interplay of these systems in lineage commitment. In addition to upregulating EE genes, loss of trx also induced cell cycle genes and ISC proliferation, suggesting additional roles in controlling ISC function. This finding is in line with a recently published study showing TrxG factors Kismet and Trr limit ISC proliferation in the fly midgut. The fact that this group did not report changes in the EE lineage most likely reflects functional differences across TrxG complexes, the composition of which varies greatly (Tauc, 2021).

A similar function in stem cell specification has been described for PRC1 and PRC2 in the mouse, where both complexes are important to preserve ISC and progenitor cell identity in the gut, while regulating specification into specific daughter cell lineages. Loss of PRC1 function in the intestinal epithelium resulted in impairment of ISC self-renewal via de-repression non-intestinal lineage genes as well as negative regulators of the Wnt signaling pathway. Interestingly, the effects of PRC1 loss were independent of H3K27me3, revealing instead the role of H2AK119 mono-ubiquitination. PRC2 was shown to be important in ISCs only during damage-induced regeneration. In contrast, another study found significant degeneration of the SC compartment under homeostatic conditions as well. Reconciling these findings, a recent study revealed that in the absence of PRC2, mammalian cells shed H3K27me3 exclusively by replicational dilution of modified nucleosomes, and that the effects of PRC2 deletion are thus only observed in lineage progeny rather than in stem cells themselves. Both previous studies are in agreement, however, that PRC2 controls cell fate decisions, as loss of PRC2 leads to an accumulation of secretory cells, evidently due to de-repression of the secretory lineage master regulator, Atoh1, resulting in ISC differentiation. It remains unknown how aging affects PRC function and H3K27 methylation in the mammalian intestine (Tauc, 2021).

Only a few studies have rigorously investigated age-associated changes in the mammalian intestine on a histological and cell-type-specific level. One study reported changes in crypt architecture, decreased mitotic potential of ISCs and an increase in the secretory cell lineage, most likely due to increased Atoh1 expression. Given the role of PRC2 in regulating the secretory lineage in both the mammalian intestine and the fly, it is tempting to speculate a conserved age-related increase in the secretory lineage that stems from deregulation of PRC. The results support a role for increased stress signaling in driving this lineage imbalance, as overactive JNK in ISCs promotes EE differentiation. While JNK signaling has been reported to suppress Pc complex function, the data indicate that in the ISC lineage, this interaction is more complex, as both JNK activation promotes EE specification, while Pc knockdown inhibits EE specification. Further studies are needed to explore the molecular mechanisms mediating JNK/Pc interactions in the ISC lineage (Tauc, 2021).

Chronic elevation of inflammatory signaling is a well-characterized hallmark of the aging fly intestine and a hallmark of many intestinal disorders including inflammatory bowel disease (IBD), infection and colorectal cancer. Alterations in EE cell numbers and secretory activity have been reported to play a role in many diseases. In IBD, for example, EE cells were shown to contribute to pathogenesis by producing pro-inflammatory cytokines. In another study, increased numbers of EE cells were reported in human patients with chronic ulcerative colitis, potentially promoting IBD associated neoplasias. Notably, this study showed that lowering EE numbers by long-term depletion of Pc in ISCs inhibited age-induced intestinal dysplasia, supporting a pathological role for EEs in aging (Tauc, 2021).

The role of epigenetic alterations, and specifically the role of PRC, in inflammatory diseases and cancer is still under investigation. It was recently reported that suppressing EZH2 activity ameliorates experimental intestinal inflammation and delays the onset of colitis-associated cancer. However, these effects may be a consequence of EZH2 suppression in myeloid cells rather than in intestinal stem cells. Disruption in PRC2 function may also underlie human cancers, where PRC2 is often hyperactive or overexpressed. Activating EZH2 mutations, which increase total H3K27me3 levels, increase tumor survival and growth in pre-clinical models and are found in up to 24% of diffuse large B cell lymphomas. Additional work will be needed to establish whether age-related changes in PRC activity contribute to the increased onset of gastrointestinal cancers during aging (Tauc, 2021).

Taken together, these findings provide evidence for altered ISC cell states in old flies that affect intestinal homeostasis and contribute to tissue dysplasia. The results exemplify the importance of maintaining appropriate lineage decisions, as overproduction of EE cells is detrimental to the epithelium, but can be rescued by re-balancing the system towards normal EE numbers. Age-associated deregulation of lineage fidelity of ISCs due to elevated stress and misregulation of Pc are proposed as key drivers of functional decline of the intestinal epithelium. Pc group proteins may thus represent valuable therapeutic targets for age-related morbidities (Tauc, 2021).

Intravital imaging strategy FlyVAB reveals the dependence of Drosophila enteroblast differentiation on the local physiology

Aging or injury in Drosophila intestine promotes intestinal stem cell (ISC) proliferation and enteroblast (EB) differentiation. However, the manner the local physiology couples with dynamic EB differentiation assessed by traditional lineage tracing method is still vague. Therefore, this study developed a 3D-printed platform "FlyVAB" for intravital imaging strategy that enables the visualization of the Drosophila posterior midgut at a single cell level across the ventral abdomen cuticle. Using ISCs in young and healthy midgut and enteroendocrine cells in age-associated hyperplastic midgut as reference coordinates, ISC-EB-enterocyte lineages were traced with Notch signaling reporter for multiple days. The results reveal a "differentiation-poised" EB status correlated with slow ISC divisions and a "differentiation-activated" EB status correlated with ISC hyperplasia and rapid EB to enterocyte differentiation. This FlyVAB imaging strategy opens the door to long-time intravital imaging of intestinal epithelium (Tang, 2021).

Immune regulation of intestinal-stem-cell function in Drosophila

Intestinal progenitor cells integrate signals from their niche, and the gut lumen, to divide and differentiate at a rate that maintains an epithelial barrier to microbial invasion of the host interior. Despite the importance of evolutionarily conserved innate immune defenses to maintain stable host-microbe relationships, little is known about contributions of stem-cell immunity to gut homeostasis. Drosophila was used to determine the consequences of intestinal-stem-cell immune activity for epithelial homeostasis. Loss of stem-cell immunity greatly impacted growth and renewal in the adult gut. In particular, it was found that inhibition of stem-cell immunity impeded progenitor-cell growth and differentiation, leading to a gradual loss of stem-cell numbers with age and an impaired differentiation of mature enteroendocrine cells. These results highlight the importance of immune signaling in stem cells for epithelial function in the adult gut (Shin, 2022).

ClC-c regulates the proliferation of intestinal stem cells via the EGFR signalling pathway in Drosophila

Adult stem cells uphold a delicate balance between quiescent and active states, which is crucial for tissue homeostasis. Whereas many signalling pathways that regulate epithelial stem cells have been reported, many regulators remain unidentified. This study used flies to generate tissue-specific gene knockdown and gene knockout. qRT-PCR was used to assess the relative mRNA levels. Immunofluorescence was used to determine protein localization and expression patterns. Clonal analyses were used to observe the phenotype. RNA-seq was used to screen downstream mechanisms. It is reported that a member of the chloride channel family, ClC-c, which is specifically expressed in Drosophila intestinal stem/progenitor cells and regulates intestinal stem cell (ISC) proliferation under physiological conditions and upon tissue damage. Mechanistically, it was found that the ISC loss induced by the depletion of ClC-c in intestinal stem/progenitor cells is due to inhibition of the EGFR signalling pathway. These findings reveal an ISC-specific function of ClC-c in regulating stem cell maintenance and proliferation, thereby providing new insights into the functional links among the chloride channel family, ISC proliferation and tissue homeostasis (Huang, 2022).

Non-canonical Wnt signaling promotes directed migration of intestinal stem cells to sites of injury

Tissue regeneration after injury requires coordinated regulation of stem cell activation, division, and daughter cell differentiation, processes that are increasingly well understood in many regenerating tissues. How accurate stem cell positioning and localized integration of new cells into the damaged epithelium are achieved, however, remains unclear. This study shows that enteroendocrine cells coordinate stem cell migration towards a wound in the Drosophila intestinal epithelium. In response to injury, enteroendocrine cells release the N-terminal domain of the PTK7 orthologue, Otk, which activates non-canonical Wnt signaling in intestinal stem cells, promoting actin-based protrusion formation and stem cell migration towards a wound. This migratory behavior is closely linked to proliferation, and that it is required for efficient tissue repair during injury. These findings highlight the role of non-canonical Wnt signaling in regeneration of the intestinal epithelium, and identify enteroendocrine cell-released ligands as critical coordinators of intestinal stem cell migration (Hu, 2021).

A neuronal relay mediates a nutrient responsive gut/fat body axis regulating energy homeostasis in adult Drosophila

The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. This study shows that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon alpha, which signals via its neuronal receptor DLgr2/Rickets. Bursicon alpha/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon alpha/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, this work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis (Scopelliti, 2018).

Maintaining systemic energy homeostasis is crucial for the physiology of all living organisms. A balanced equilibrium between anabolism and catabolism involves tightly coordinated signaling networks and the communication between multiple organs. Excess nutrients are stored in the liver and adipose tissue as glycogen and lipids, respectively. In times of high energy demand or low nutrient availability, nutrients are mobilized from storage tissues. Understanding how organs communicate to maintain systemic energy homeostasis is of critical importance, as its failure can result in severe metabolic disorders with life-threatening consequences (Scopelliti, 2018).

The intestine is a key endocrine tissue and central regulator of systemic energy homeostasis. Enteroendocrine (ee) cells secrete multiple hormones in response to the nutritional status of the organism and orchestrate systemic metabolic adaptation across tissues. Recent work reveals greater than expected diversity, plasticity, and sensing functions of ee cells. Nevertheless, how ee cells respond to different environmental challenges and how they coordinate systemic responses is unclear. A better understanding of ee cell biology will directly impact understanding of intestinal physiopathology, the regulation of systemic metabolism, and metabolic disorders (Scopelliti, 2018).

Functional studies on inter-organ communication are often challenging in mammalian systems, due to their complex genetics and physiology. The adult Drosophila midgut has emerged as an invaluable model system to address key aspects of systemic physiology, host-pathogen interactions, stem cell biology and metabolism, among other things. As in its mammalian counterpart, the Drosophila adult intestinal epithelium displays multiple subtypes of ee cells with largely unknown functions. Recent work has demonstrated nutrient-sensing roles of ee cells (Scopelliti, 2018 and references therein).

The role of Bursicon/DLgr2 signaling has long been restricted to insect development, where the heterodimeric form of the hormone Bursicon, made by α and β subunits, is produced by a subset of neurons within the CNS during the late pupal stage and released systemically to activate its receptor DLgr2 in peripheral tissues to drive post-molting sclerotization of the cuticle and wing expansion. A recent study demonstrated a post-developmental activity for the α subunit of Bursicon (Bursα), which is produced by a subpopulation of ee cells in the posterior midgut, where it paracrinally activates DLgr2 in the visceral muscle (VM) to maintain homeostatic intestinal stem cell (ISC) quiescence (Scopelliti, 2014; Scopelliti, 2016; Scopelliti, 2018).

This study reports an unprecedented systemic role for Bursα regulating adult energy homeostasis. This work identifies a novel gut/fat body axis, where ee cells orchestrate organismal metabolic homeostasis. Bursα is systemically secreted by ee cells in response to nutrient availability and acts through DLgr2+ neurons to repress adipokinetic hormone (AKH)/AKH receptor (AKHR) signaling within the fat body/adipose tissue to restrict the use of energy stores. Impairment of systemic Bursα/DLgr2 signaling results in exacerbated oxidative metabolism, strong lipodystrophy, and organismal hypersensitivity to nutrient deprivation. This work reveals a central role for ee cells in sensing organismal nutritional status and maintaining systemic metabolic homeostasis through coordination of an intestinal/neuronal/adipose tissue-signaling network (Scopelliti, 2018).

This study shows that ee cells secrete Bursα in the presence of plentiful nutrients, while caloric deprivation reduces its systemic release and consequently results in hormone accumulation within ee cells. Interestingly, it was observed that conditions leading to the latter scenario are accompanied by reduced bursα transcription. The reasons underlying the inverse correlation between midgut bursα mRNA and protein levels are unclear and may represent part of a negative feedback mechanism for ultimate control of further protein production. A similar phenomenon is described during the regulation of the secretion of other endocrine hormones, such as DILPs (Scopelliti, 2018).

The results show that Bursα within ee cells is preferably regulated in response to dietary sugars. This is further supported by the function of Glut1 as at least one of the transmembrane sugar transporters connecting nutrient availability to Bursα signaling. Glut1 is the closest homolog of the mammalian regulator of ee incretin secretion SLC2A2, and it has been shown to positively regulate the secretion of peptide hormones in flies (Park, 2014). Whether Glut1 is a central sensor of dietary sugars and hormone secretion by ee cells remains to be addressed. However, it is likely that, in the face of challenges, such as starvation, multiple mechanisms of nutrient sensing and transport converge to allow a robust organismal adaptation to stressful environmental conditions (Scopelliti, 2018).

Reduction of systemic Bursα/DLgr2 signaling induces a complex metabolic phenotype, characterized by lipodystrophy and hypoglycemia, which is accompanied by hyperphagia. These phenotypes are not due to poor nutrient absorption or uptake by tissues or impaired synthesis of energy stores but are rather a consequence of increased catabolism. This is supported by a higher rate of glucose-derived 13C incorporation into TCA cycle intermediates, accompanied by increased mitochondrial respiration and body-heat production (Scopelliti, 2018).

While glucose tracing experiments help explain the hypoglycemic phenotype of Bursα/DLgr2-compromised animals even in the context of hyperphagia, they do not directly address the reduction in fat body triacylglycerides (TAGs). The latter would require 13C6-palmitate tracing for assessment of the rate of lipid oxidation and incorporation into the TCA cycle. This was precluded by overall poor uptake of 13C6-palmitate into adult animals even after prolonged periods of feeding. However, the depletion of fat body TAG stores in the presence of normal de novo lipid synthesis in Bursα/DLgr2-impaired animals strongly suggests that at least part of the increased rate of O₂ consumption in those animals results from increased lipid breakdown via mitochondrial fatty acid oxidation. Consistently, increased O₂ consumption rates and the thermogenic phenotype of Bursα/DLgr2-deficient animals are attenuated upon reduction of AKH/AKHR signaling. Finally, the functional role of Hormone-sensitive lipase (dHSL) in the fat body further supports the regulation of lipid breakdown by AKH/AKHR signaling as at least one of the key aspects mediating the role of Bursα/DLgr2 signaling in adult metabolic homeostasis (Scopelliti, 2018).

Previous work revealed that ee Bursα is required to maintain homeostatic ISC quiescence in the adult Drosophila midgut; that is, in the midgut of unchallenged and well-fed animals (Scopelliti, 2014, Scopelliti, 2016). Such a role of Bursα is mediated by local or short-range signaling through DLgr2 expressed within the midgut VM (Scopelliti, 2014). This study demonstrates a systemic role of Bursα that does not involve VM-derived DLgr2 but rather signals through its neuronal receptor. In that regard, the paracrine and endocrine functions of Bursα/DLgr2 are uncoupled. However, the regulation of ee-derived Bursα by nutrients is likely to affect local as well as systemic Bursα/DLgr2 signaling. Retention of Bursα within ee as observed in conditions of starvation may impair the hormone's signaling into the VM, which, in principle, would lead to ISC hyperproliferation (Scopelliti, 2014). In fact, under full nutrient conditions, genetic manipulations impairing systemic Bursα signaling, such as ee Glut1 knockdown or osbp overexpression, lead to ISC hyperproliferation comparable with that observed upon bursα knockdown (Scopelliti, 2014). This represents an apparent conundrum, as ISC proliferation is not the expected scenario in the context of starvation. However, starvation completely overcomes ISC proliferation in Bursα-impaired midguts. This is consistent with recent evidence showing that restrictive nutrient conditions, such as the absence of dietary methionine or its derivative S-adenosyl methionine, impair ISC proliferation in the adult fly midgut, even in the presence of activated mitogenic signaling pathways (Obata, 2018). Altogether, these data support a scenario in which starvation, while preventing systemic and local Bursα/DLgr2 signaling, would not result in induction of ISC proliferation as a side effect (Scopelliti, 2018).

Drosophila DLgr2 is the ortholog of mammalian LGR4, -5, and -6 with closer homology to LGR4. While LGR5 and 6 are stem cell markers in several tissues, such as small intestine and skin, LGR4 depicts broader expression patterns and physiological functions. LGR4, -5, and -6 are best known to enhance canonical Wnt signaling through binding to R-spondins. However, several lines of evidence support a more promiscuous binding affinity for LGR4, which can act as a canonical G-protein coupled receptor inducing iCa2+ and cyclic AMP signaling (Scopelliti, 2018).

Interestingly, an activating variant of LGR4 (A750T) is linked to obesity in humans, while the nonsense mutation c.376C>T (p.R126X) is associated with reduced body weight. Recent reports show that LGR4 homozygous mutant (LGR4m/m) mice display reduced adiposity and are resistant to diet- or leptin-induced obesity. These phenotypes appear to derive from increased energy expenditure through white-to-brown fat conversion and are independent of Wnt signaling. The tissue and molecular mechanisms mediating this metabolic role of LGR4 remain unclear. Therefore, the current paradigm may lead to a better understanding of LGR4's contribution to metabolic homeostasis and disease. Importantly, the results highlight the intestine and ee cells in particular as central orchestrators of metabolic homeostasis and potential targets for the treatment of metabolic dysfunctions (Scopelliti, 2018).

Bursicon is an insect-specific hormone. Therefore, direct mammalian translation of the signaling system presented in this study is unlikely. However, given the clear parallels between the metabolic functions of DLgr2 and LGR4, analysis of enteroendocrine cell-secreted factors in mammalian systems may reveal new and unexpected ligands for LGR4 (Scopelliti, 2018).

Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila

Feeding behavior is essential for growth and survival of animals; however, relatively little is known about its intrinsic mechanisms. This study demonstrates that Gart is expressed in the glia, fat body, and gut and positively regulates feeding behavior via cooperation and coordination. Gart in the gut is crucial for maintaining endogenous feeding rhythms and food intake, while Gart in the glia and fat body regulates energy homeostasis between synthesis and metabolism. These roles of Gart further impact Drosophila lifespan. Importantly, Gart expression is directly regulated by the CLOCK/CYCLE heterodimer via canonical E-box, in which the CLOCKs (CLKs) in the glia, fat body, and gut positively regulate Gart of peripheral tissues, while the core CLK in brain negatively controls Gart of peripheral tissues. This study provides insight into the complex and subtle regulatory mechanisms of feeding and lifespan extension in animals (He, 2023).

Feeding is a necessary behavior for animals to grow and survive, with a characteristic of taking food regularly. The quality and quantity of feeding directly impact the normal growth and development of animals. Time-restricted feeding or fasting is beneficial for preventing obesity, alleviating inflammation, and attenuating cardiac diseases and even has antitumor effects. Metabolic syndrome has become a global health problem. Shift work and meal irregularity disrupt circadian rhythms, with an increased risk of developing metabolic syndrome. The maintenance of the daily feeding rhythm is very important in metabolic homeostasis.Irregular feeding perturbs circadian metabolic rhythms and results in adverse metabolic consequences and chronic diseases (He, 2023).

Most behaviors in animals are synchronized to a ~24 h (circadian) rhythm induced by circadian clocks in both the central nervous system and peripheral tissues. Circadian rhythmic behaviors, such as feeding and locomotion, are involved in complex connections and specific output pathways under the control of the circadian clock. Although the core clock feedback loop has been well established in recent decades, the crucial genes responsible for rhythmic feeding regulation as well as for the interrelation between the core clocks and feeding are still unclear (He, 2023).

To increase the understanding of how the circadian clock regulates feeding and metabolism, this study sought to identify the output genes in the circadian feeding and metabolism control network, in which the model animal Drosophila provides special advantages to explore the mechanistic underpinnings and the complex integration of these primitive responses. Previous studies confirmed that one of juvenile hormone receptors, methoprene tolerance (Met), is important for the control of neurite development and sleep behavior in Drosophila. Many genes related to metabolic regulation have attracted attention in the transcriptome data from Met²⁷, a Met-deficient fly line, in which this study focused on the target genes regulated by CLOCK/CYCLE (CLK/CYC). As a basic Helix-Loop-Helix-Per-ARNT-Sim (bHLH-PAS) transcription factor with a canonical binding site “CACGTG," the CLK/CYC heterodimer is a crucial core oscillator that regulates circadian rhythms (He, 2023).

The Gart trifunctional enzyme, a homologous gene of adenosine-3 in mammals, is a trifunctional polypeptide with the activities of phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase (Tiong, 1990). Gart in astrocytes of vertebrates plays a role in the lipopolysaccharide-induced release of proinflammatory factors (Zhang, 2014), and Gart expressed in the liver and heart is required for de novo purine synthesis. However, there is no information yet for Gart's functions in feeding rhythm. In this study, Gart was identified as a candidate that was controlled by the core clock gene CLK/CYC heterodimer and was found to be related to feeding behavior in Drosophila. Thus this study focused Gart studies on the role of feeding rhythms and further regulatory mechanisms. This study provides a critical foundation for understanding the complex feeding mechanism. (He, 2023).

In animals, hundreds of genes exhibit daily oscillation under clock regulation, and some of them are involved in metabolic functions. This study identified a CLK/CYC-binding gene, Gart, which is involved in feeding rhythms and energy metabolism independent of locomotor rhythms. Previous research reported that blocking CLK in different tissues yields different phenotypes. This study found that MET, like CYC, can combine with CLK to regulate the transcription of Gart. Knocking down Gart in different tissues exhibits different phenotypes, and Gart in different tissues can rescue the phenotype caused by CLK deletion; thus, the phenomenon caused by CLK deletion is due to the change in Gart (He, 2023).

CLK regulates the feeding rhythms of Drosophila, and its loss can cause disorders of feeding rhythms and abnormal energy storage. Tim⁰¹, Cry⁰¹, and Per01 mutants have significantly lower levels of truactkglycerides (TAGs). The maintenance of energy homeostasis is achieved by a dynamic balance of energy intake (feeding), storage, and expenditure (metabolic rate), which are crucial factors for longevity and resistance to adverse environments in Drosophila. Additionally, studies have shown that mutations of Timeless and per shorten the adult lifespan of Drosophila. This study further reveals that peripheral CLKs control the oscillation of Gart among different peripheral tissues; however, core CLKs in the brain can negatively regulate Gart expression in peripheral tissues, indicating that a complex and refined network regulatory system exists between CLK and Gart in the brain and in different peripheral tissues to coordinate feeding behavior and energy homeostasis in Drosophila and that it further affects sensitivity to starvation and longevity. These novel findings enrich the network of regulatory mechanisms by the clocks-Gart pathway on feeding, energy homeostasis, and longevity (He, 2023).

Glial cells have vital functions in neuronal development, activity, plasticity, and recovery from injury. This study reveals that flies lacking Gart in glial cells display a significant decline in the viability of Drosophila under starvation, caused by a decrease in energy storage that puts flies under a state of energy deficit. This discovery extends the functions of glial cells in feeding, energy storage, and starvation resistance controlled by Gart (He, 2023).

The fat body is the primary energy tissue for the storage of fuel molecules, such as TAG and glycogen, which play an important role in the regulation of metabolic homeostasis and provide the most energy during starvation. Indeed, functional defects of the fat body increase starvation sensitivity in Drosophila. In this study, flies lacking Gart in the fat body led to decreased energy storage, which reduces the survival rate and the survival time under starvation conditions. However, flies lacking gut Gart still maintain normal energy storage, which is not sensitive to food shortage or starvation. In addition, this study found that although high temperature can stimulate the food intake of Drosophila, which is consistent with previous reports, it does not affect the feeding rhythm (He, 2023).

This study reveals that Gart in the glia and the fat body collectively regulate the homeostasis of energy intake, storage, and expenditure, thereby influencing the viability of flies under starvation stress. Although Gart in the gut strongly influences feeding behavior, it does not play similar functions as the glia and the fat body in adversity resistance. This occurs possibly because the gut has vital roles in digestion and absorption, while the fat body has crucial functions in energy metabolism. In addition, Gart in the glia and the fat body has biased roles in the synthesis of glycogen and TAG, despite having similar functions in energy storage. The biased role of the glia and the fat body may be coordinated to provide effective energy homeostasis. These findings provide new insight into how specific circadian coordination of various tissues modulates adversity resistance and aging (He, 2023).

Such robust findings in Drosophila suggest that a decrease in lifespan and an increase in sensitivity to starvation in Drosophila is a faithful readout of disordered feeding rhythms and abnormal metabolism. Gart effects on metabolism in both glia cells and the fat body indicate the intricacy of the circadian network and energy homeostasis. It is crucial for animals to have a well-organized network to coordinate and ensure that these various tissue regions are in a normal state (He, 2023).

This study has demonstrated that CLK regulates feeding, energy homeostasis, and longevity via Gart. Even though attempts were made to explore more comprehensively how Gart coordinates and regulates the physiological functions in different tissues of D. melanogaster, there are still some limitations. For instance, it is still unclear that how Gart achieves functional differentiation in different tissues, as well as whether Gart regulates lifespan through autophagy and/or bacterial content or not, which are two critical factors related to lifespan. These future studies are of great significance for understanding the relationship between feeding and longevity regulated by Gart (He, 2023).

Stem cell intrinsic hexosamine metabolism regulates intestinal adaptation to nutrient content

The intestine is an organ with an exceptionally high rate of cell turnover, and perturbations in this process can lead to severe diseases such as cancer or intestinal atrophy. Nutrition has a profound impact on intestinal volume and cellular architecture. However, how intestinal homeostasis is maintained in fluctuating dietary conditions remains insufficiently understood. By utilizing the Drosophila midgut model, this study reveals a novel stem cell intrinsic mechanism coupling cellular metabolism with stem cell extrinsic growth signal. The results show that intestinal stem cells (ISCs) employ the hexosamine biosynthesis pathway (HBP) to monitor nutritional status. Elevated activity of HBP promotes Warburg effect-like metabolic reprogramming required for adjusting the ISC division rate according to nutrient content. Furthermore, HBP activity is an essential facilitator for insulin signaling-induced ISC proliferation. In conclusion, ISC intrinsic hexosamine synthesis regulates metabolic pathway activities and defines the stem cell responsiveness to niche-derived growth signals (Mattila, 2018).

Transcriptional control of stem cell maintenance in the Drosophila intestine

Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. This paper describes how transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance; genes of the Enhancer of split complex [E(spl)-C] are identified as the major targets of this repression. In addition, the bHLH transcription factor Daughterless was found to be essential to maintain ISC identity, and bHLH binding sites promote ISC-specific enhancer activity. It is proposed that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation (Bardin, 2010).

Adult stem cells self-renew and, at the same time, give rise to progeny that eventually differentiate. This work provides evidence that one of the strategies used to maintain the identity of ISCs in Drosophila is to repress the expression of Notch target genes. Consistent with this finding, the loss of a general regulator of transcriptional repression, the Histone H2B ubiquitin protease Scrawny, gives a similar phenotype to Hairless. Additionally, several recent studies indicate that transcriptional repression of differentiation genes may be a central hallmark of stem cells in general (Bardin, 2010).

Two models have been proposed for Hairless activity. One proposes that Hairless competes with NICD for interaction with Su(H), thereby preventing transcriptional activation of Notch target genes by low-level Notch receptor activation. A second, non-exclusive, model proposes that Hairless antagonizes the transcriptional activation of Notch target genes by tissue-specific transcription factors other than Notch. Since the loss of Su(H) can suppress the phenotype of Hairless on ISC clone growth, it is proposed that Hairless promotes ISC maintenance by repressing the transcription of genes that would otherwise be activated by Notch signaling in ISCs. Thus, Hairless appears to set a threshold level to buffer Notch signaling in ISCs. In the absence of this repression, the expression of E(spl)-C genes and other Notch targets would lead to loss of the ISC fate. Importantly, the current findings suggest a mechanism for how the transcriptionally repressed state is turned off and activation of the differentiation program is initiated: high activation of Notch in enteroblasts (EBs) displaces Hairless from Su(H) and leads to expression of the E(spl)-C genes (Bardin, 2010).

E(spl)-C bHLH repressors act in part through their ability to inhibit bHLH activators. The data demonstrate that Da is also essential to maintain ISC fate and that E-box Da-binding sites are required to promote ISC-specific enhancer activity. Thus, it is proposed that activation of E(spl)-C genes by Notch in EBs downregulates Da bHLH activity and thereby contributes to turning off ISC identity in the differentiating cell. The specificity of ISC-specific E-box expression might be due to the ISC-specific expression of a bHLH family member. Although array analysis raised the possibility that Scute may be specifically expressed in ISCs, genetic analysis indicates that scute function is not essential for ISC maintenance. Alternatively, specificity of gene expression might result from inhibition of bHLH activity in the EB and differentiating daughters, possibly by E(spl)-bHLH factors, rather than by the ISC-specific expression of a Da partner. It is also possible that a non-bHLH, ISC-specific factor restricts the Da-dependent bHLH activity to ISCs in a manner similar to the synergism observed in wing margin sensory organ precursors (SOPs) between the Zn-finger transcription factor Senseless and Da (Bardin, 2010).

Recently, a role for the Da homologs E2A (Tcf3) and HEB (Tcf12) has been found in mammalian ISCs marked by the expression of Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (Lgr5) and, in this context, E2A and HEB are thought to heterodimerize with achaete-scute like 2 (Ascl2), which is essential for the maintenance and/or identity of Lgr5⁺ ISCs (van der Flier, 2009). In Drosophila, however, AS-C genes are not essential for ISC maintenance, but appear to play a role in enteroendocrine fate specification. The observation that Da bHLH activity is required for the identity of both Drosophila ISCs and mammalian Lgr5⁺ ISCs suggests that there might be conservation at the level of the gene expression program. Additionally, the bHLH genes Atoh1 (Math1) and Neurog3 are both important for differentiation of secretory cells in the mammalian intestine. Clearly, further analysis of the control of Da/E2A bHLH activity, as well as of the gene networks downstream of Da/E2A, will be of great interest (Bardin, 2010).

The data suggest that ISC fate is promoted both by inhibition of Notch target genes through Hairless/Su(H) repression and by activation of ISC-specific genes through bHLH activity. How then is asymmetry in Notch activity eventually established between the two ISC daughters to allow one cell to remain an ISC and one cell to differentiate? Three types of mechanism can be envisioned that would allow for asymmetry of Notch signaling (Bardin, 2010).

First, the binary decision between the ISC and EB fates might result from a competition process akin to lateral inhibition for the selection of SOPs. In this process, feedback loops establish directionality by amplifying stochastic fluctuations in signaling between equivalent cells into a robust unidirectional signal. The finding that the Da activator and E(spl)-bHLH repressors are important to properly resolve ISC/EB fate is consistent with this type of model. Activation of the Notch pathway in one of the daughter cells could then lead to the changes in nuclear position previously noted (Bardin, 2010).

Second, the asymmetric segregation of determinants could bias Notch-mediated cell fate decisions. The cell fate determinants Numb and Neur are asymmetrically segregated in neural progenitor cells to control Notch signaling. However, this study found no evidence for the asymmetric segregation of these proteins in dividing ISCs. Additionally, the data indicate that Numb is not important to maintain ISC fate. It cannot be excluded, however, that another, unknown Notch regulator is asymmetrically segregated to regulate the fate of the two ISC daughters (Bardin, 2010).

A third possibility is that after ISC division, one of the two daughter cells receives a signal that promotes differential regulation of Notch. Indeed, it has been noted that the axis of ISC division is tilted relative to the basement membrane, resulting in one of the progeny maintaining greater basal contact than the other. An extracellular signal coming either basally or apically could bias the Notch-mediated ISC versus EB fate decision. For instance, Wg secreted by muscle cells could act as a basal signal to counteract Notch receptor signaling activity in presumptive ISCs. This could be accomplished by Wg promoting bHLH activity or gene expression. Indeed, Wg has been demonstrated to promote proneural bHLH activity in Drosophila (Bardin, 2010).

These models are not mutually exclusive, however, and proper control of ISC and differentiated cell fates during tissue homeostasis might involve multiple mechanisms (Bardin, 2010).

Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut

Homeostasis of the intestine is maintained by dynamic regulation of a pool of intestinal stem cells. The balance between stem cell self-renewal and differentiation is regulated by the Notch and insulin signaling pathways. Dependence on the insulin pathway places the stem cell pool under nutritional control, allowing gut homeostasis to adapt to environmental conditions. This study presents evidence that miR-305 is required for adaptive homeostasis of the gut. miR-305 regulates the Notch and insulin pathways in the intestinal stem cells. Notably, miR-305 expression in the stem cells is itself under nutritional control via the insulin pathway. This link places regulation of Notch pathway activity under nutritional control. These findings provide a mechanism through which the insulin pathway controls the balance between stem cell self-renewal and differentiation that is required for adaptive homeostasis in the gut in response to changing environmental conditions (Foronda, 2014).

Drosophila midgut homeostasis involves neutral competition between symmetrically dividing intestinal stem cells

The Drosophila adult posterior midgut has been identified as a powerful system in which to study mechanisms that control intestinal maintenance, in normal conditions as well as during injury or infection. Early work on this system has established a model of tissue turnover based on the asymmetric division of intestinal stem cells (ISC). From the quantitative analysis of clonal fate data, this study shows that tissue turnover involves the neutral competition of symmetrically dividing stem cells, mediated by Dl/N signaling. This competition leads to stem-cell loss and replacement, resulting in neutral drift dynamics of the clonal population. As well as providing new insight into the mechanisms regulating tissue self-renewal, these findings establish intriguing parallels with the mammalian system, and confirm Drosophila as a useful model for studying adult intestinal maintenance (de Navascues, 2012).

This study has analysed long- and short-term lineage progression by size, survival and composition, and shows that Drosophila midgut is maintained by population asymmetry. This contrasts with a general notion in the field that homeostasis is based on the fate asymmetry of the ISC offpsring. The results reveal that ISCs divide symmetrically in response to the differentiation and subsequent loss of a neighbouring ISC (or vice versa), which leads to neutral drift of the clonal population. It is estimated that, in a context of fast turnover (two or three divisions per day), 2 in 10 divisions result in stem-cell loss and replacement. The results further indicate that the fate of the ISC daughters might not be specified on division, but rather resolved through competition between proximate cells following division (de Navascues, 2012).

The observation of neutral competition calls for the elucidation of its underlying molecular mechanism. By the nature of the process of neutral competition in this system, the associated mechanism must play a fundamental role in ISC self-renewal and enteroblast (EB) commitment and be able to implement, non-cell autonomously, the stochastic resolution of binary fate decisions. Dl-N signalling fulfils both requirements: numerous experiments underpin its central role in the choice of commitment versus self-renewal in the midgut, and studies of its function in neurogenesis have established an intrinsic ability to resolve stochastically binary choices of cell fate through the process of 'lateral inhibition', which involves reciprocal signalling between equivalent cells (de Navascues, 2012).

The model of population asymmetry involves neutral competition between proximate cells (therefore based on extrinsic signals), which implies that ISC daughters are intrinsically equivalent at birth, and that fate is resolved after ISC division. Therefore, the ISC daughters are functionally equivalent and a good substrate for lateral inhibition, This is compatible with the observations that ISC daughter cells express N and they segregate Dl symmetrically. Furthermore, evidence is provided that this situation leads to mutual signalling. The excess of ISCs found in null and hypomorphic conditions for N could be interpreted, in the framework of a unidirectional Dl signal from the ISC towards the EB, as a failure to implement the EB fate and a lapse into the 'default' ISC fate. However, this interpretation cannot account for the increased number of cells committing to EB fate in +/l(1)N^B and +/N^MCD1 midguts. These alleles exhibit Dl-dependent increased N signalling which, in a scenario of unidirectional signalling, should not affect the balance of EB commitment. Rather, their phenotype suggests that both ISC daughters can receive Dl signal and reach the threshold of N activity for commitment. This is further highlighted by the parallel between the effects of these alleles on the ISC/EB ratio and on the development of peripheral nervous system (PNS) of Drosophila, a classical model for lateral inhibition. In the PNS, a reduction of N activity, as in +/N^55e11, leads to neurogenic phenotypes with supernumerary sensory bristles, whereas excess of N activity, as in +l(1)N^B and +/N^MCD1, results in antineurogenic phenotypes, with reduced number of bristles and more cells adopting the epidermal fate. It is noteworthy that the molecular machinery that participates in Dl/N-mediated lateral inhibition in other contexts is also involved in ISC/EB fate decision. With these elements, it is proposed that ISCs divide symmetrically, and the fate of the progeny is resolved through lateral inhibition mediated by Dl/N signalling (de Navascues, 2012).

Lateral inhibition thus provides a straightforward way of implementing neutral competition. In many cases, Dl/N interaction will be restricted to sibling cells, the progeny of a single ISC division, and these cases will resolve into an ISC/EB asymmetric pair. However, if in the course of tissue turnover the division of two ISCs occurred in close proximity, non-sibling cells could interact via Dl/N and engage in competition for the ISC fate through lateral inhibition. This in turn may lead to sibling cells adopting identical fates and therefore in ISC loss and replacement. Such behaviour would translate precisely to the coupled events of loss and replacement implicit in the model, with the value of 2r weighting, combined, the chance of this contact and of its resolution into ISC loss and replacement. In this regard, the rich variety of esg⁺ cell nest composition in EB and ISC cells, as well as the frequent proximity of ISC pairs (19% of nearest pairs), suggests that such encounters are possible in space. This may require nests to drift in position, but it is significant that, unlike other Drosophila adult stem cells, ISCs are not associated with niches having hub-like anatomical properties (de Navascues, 2012).

Although the current observations can, in principle, be explained solely as a result of lateral inhibition, other mechanisms cannot be ruled out. In particular, the tissue could allow for a combination of either symmetric or intrinsically asymmetric divisions such that part of the ISC divisions that result in asymmetric fate could derive from either neutral competition or an intrinsic regulatory process. However, to conform with the observation that ISCs are equipotent, the stem-cell progeny of an intrinsically determined asymmetric cell division will have the same chance for loss and replacement in subsequent divisions as any other ISC. In other words, when facing their next division, all ISCs irrespective of their lineage history, would have to decide, stochastically, whether to undergo intrinsically asymmetric division, and, if they do not, the fate outcome of the division would be resolved again, stochastically, by extrinsically driven neutral competition. It is difficult to conceive of a scenario for the molecular regulation of such a system (de Navascues, 2012).

Although the model provides an excellent fit to the early time course, the departure of the model at day 16 is significant. Although the clone size distribution conforms closely with the predicted scaling form, consistent with the same underlying pattern of ISC fate, the overall average size of the surviving clones is smaller than predicted by a simple extrapolation of the fits to the earlier time point data with the same ISC loss/replacement rate. The departure of theory and experiment may reflect a breakdown of homeostasis due to ageing. In particular, at this point of the clone chase the flies are 21-23 days old, an age at which ageing-related non-homeostatic phenotypes can be detected in the midgut. Indeed, such behaviour might be explained by a shift towards uncompensated loss of terminally differentiated cells, consistent with the fact that the clone density continues to fall, and in a manner consistent with theory. Alternatively, it is also possible that the heat shock, which does not produce damage leading to detectable alteration of the tissue size or cell density, instead triggers the acceleration of tissue turnover, an effect that would last at least 8 days, but less than 16. This effect could be similar, but milder, to that observed in the recently described Drosophila gastric adult stem cells, which show a sharp activation of homeostatic turnover in response to heat shock. This view is supported by the observation that the mitotic index in the posterior midgut is, after heat-based clonal induction, higher than in untreated organs (de Navascues, 2012).

There are similarities between the Drosophila midgut and mammalian intestine at the levels of cell biology and genetics. This study adds a new parallel from the perspective of their strategy for homeostatic maintenance. In humans, studies of methylation patterns point at stem-cell loss and replacement in the intestinal crypt and in the mouse, using an approach similar to that employed in this study, recent studies have shown that the intestinal crypt is maintained by an equipotent Lgr5⁺ stem-cell population in which the loss of cells from the stem-cell compartment is compensated by the symmetric multiplication of neighbours. Further evidence suggests that stem-cell competence in the small intestine is ensured by proximity to Paneth cells, which aggregate throughout the crypt base. As tissue is turned over, Lgr5⁺ cells undergo neutral competition resulting in a progression towards crypt monoclonality. At longer timescales, crypts undergo fission, leading to a further 'coarsening' of the clonal population. It is speculated that in Drosophila, the esg⁺ nests fulfil a function analogous to intestinal crypts, playing host to a much smaller equipotent cell population, and undergoing fission with a far greater frequency. However, in contrast with intestinal crypt, it appears that ISC competence in Drosophila does not require a separate niche-supporting cell (de Navascues, 2012).

In summary, these studies show that, in Drosophila, adult midgut homeostasis follows a pattern of population asymmetry involving an equipotent population of ISCs. ISC division may result in any of all three possible fate outcomes leading to asymmetric fate (ISC/EB), symmetric duplication (two ISCs), or symmetric differentiation (two EBs), the latter two balanced in frequency. These findings point at a mechanism involving lateral inhibition and provide a natural framework to explain regeneration following injury or infection (de Navascues, 2012).

Niche appropriation by Drosophila intestinal stem cell tumours

Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. This study investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch(N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis (Patel, 2015).

This paper described a step-wise series of events during the earliest stage of tumour development in a stem cell niche. First, the combination of environmentally triggered mitogenic signalling and a mutation that compromises differentiation generates small clusters of differentiation-defective stem-like cells. Autocrine (Spi/EGFR) signalling between these cells then promotes their expansion into clusters, which quickly reach a size capable of physically disrupting the surrounding epithelium and driving the detachment and apical extrusion of surrounding epithelial cells (that is, ECs). This loss of normal cells seems to involve tumour cell/epithelial cell competition through integrin-mediated adhesion. Subsequently, the loss of epithelial integrity (specifically, EC detachment) triggers stress signalling (JNK, Yki/YAP) in the surrounding epithelium and underlying VM, and these stressed tissues respond by producing cytokines (Upd2,3) and growth factors (Vn, Pvf, Wg, dILP3). These signals are normally used within the niche to activate stem cells for epithelial repair, but in this context they further stimulate tumour growth in a positive feedback loop. It is noteworthy that in this example a single mutation that blocks differentiation is sufficient to drive early tumour development, even without secondary mutations in growth signalling pathways that might make the tumour-initiating cells growth factor- and niche-independent (for example, Ras, PTEN). Thus, tumour cell-niche interactions can be sufficient to allow tumour-initiating cells to rapidly expand, increasing their chance to acquire secondary mutations that might enhance their growth or allow them to survive outside their normal niche. This study highlights the importance of investigating the factors that control paracrine stem cell mitogens and survival signals in the niche environment. Tumour-niche interactions may be important to acquire a sizable tumour mass before the recruitment of a tumour-specific microenvironment that supports further tumour progression. A careful analysis of similar interactions in other epithelia, such as in the lung, skin or intestine could yield insights relevant to the early detection, treatment and prevention of cancers in such tissues (Patel, 2015).

Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila

Tissue-specific stem cells are maintained by both local secreted signals and cell adhesion molecules that position the stem cells in the niche microenvironment. In the Drosophila midgut, multipotent intestinal stem cells (ISCs) are located basally along a thin layer of basement membrane that composed of extracellular matrix (ECM), which separates ISCs from the surrounding visceral musculature: the muscle cells constitute a regulatory niche for ISCs by producing multiple secreted signals that directly regulate ISC maintenance and proliferation. This study shows that integrin-mediated cell adhesion, which connects the ECM and intracellular cytoskeleton, is required for ISC anchorage to the basement membrane. Specifically, the alpha-integrin subunits including alphaPS1 encoded by mew and alphaPS3 encoded by scb, and the beta-integrin subunit encoded by mys are richly expressed in ISCs and are required for the maintenance, rather than their survival or multiple lineage differentiation. Furthermore, ISC maintenance also requires the intercellular and intracellular integrin signaling components including Talin, Integrin-linked kinase (Ilk), and the ligand, Laminin A. Notably, integrin mutant ISCs are also less proliferative, and genetic interaction studies suggest that proper integrin signaling is a prerequisite for ISC proliferation in response to various proliferative signals and for the initiation of intestinal hyperplasia after loss of adenomatous polyposis coli (Apc). These studies suggest that integrin not only functions to anchor ISCs to the basement membrane, but also serves as an essential element for ISC proliferation during normal homeostasis and in response to oncogenic mutations (Lin, 2013).

Rac1 drives intestinal stem cell proliferation and regeneration

Adult stem cells are responsible for maintaining the balance between cell proliferation and differentiation within self-renewing tissues. The molecular and cellular mechanisms mediating such balance are poorly understood. The production of reactive oxygen species (ROS) has emerged as an important mediator of stem cell homeostasis in various systems. Recent work demonstrates that Rac1-dependent ROS production mediates intestinal stem cell (ISC) proliferation in mouse models of colorectal cancer (CRC). This study used the adult Drosophila midgut and the mouse small intestine to directly address the role of Rac1 in ISC proliferation and tissue regeneration in response to damage. The results demonstrate that Rac1 is necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent manner. The data point to an evolutionarily conserved role of Rac1 in intestinal homeostasis and highlight the value of combining work in the mammalian and Drosophila intestine as paradigms to study stem cell biology (Myant, 2013b).

The epithelium of the posterior adult Drosophila midgut is replenished by ISCs. Each ISC proliferates to give rise to an uncommitted enteroblast (EB), which will differentiate into either an enterocyte (EC) or an enteroendocrine cell (ee). ISCs are the only proliferative cells within the adult fly posterior midgut (Myant, 2013b).

Recent work shows that deletion of Rac1 suppresses intestinal hyperproliferation and ROS production in Apc-deficient mice (Myant, 2013a). It was therefore first asked whether Rac1 is sufficient to drive ROS production within ISCs in the Drosophila midgut. The UAS/Gal4 system was used to specifically overexpress Drosophila Rac1 in ISCs/EBs (progenitor cells) using the temperature-controlled escargot-gal4, UAS-gfp; tubulin-gal80ts driver (esgts > gfp). Overexpression of Rac1 resulted in a dramatic expansion of the esg > gfp cell population and increased ROS production in the midgut. These results suggest that Rac1 overexpression in progenitor cells is sufficient to drive ROS production within the intestinal epithelium (Myant, 2013b).

The epithelium of the adult posterior Drosophila midgut has a remarkable regenerative capacity. Damage induced by agents such as bacterial infection, Bleomycin, or dextran sodium sulfate (DSS) treatment leads to activation of ISC proliferation to regenerate the damaged intestinal epithelium. Previous work demonstrated that ROS production is essential for damaged-induced ISC proliferation in the fly midgut (Buchon, 2009). It was therefore asked whether ROS upregulation was important for the phenotype resulting from Rac1 overexpression in the midgut. Consistent, with the previous report preventing ROS production by NAC impaired ISC proliferation in posterior midguts from flies infected with the pathogenic bacteria Pseudomonas entomophila (Pe). Importantly, NAC treatment strongly suppressed ISC hyperproliferation in Rac1-overexpressing midguts (). These results suggest that ROS production is essential for Rac1-dependent ISC hyperproliferation in the intestine (Myant, 2013b).

It was finally asked whether Rac1 was necessary to drive ISC proliferation in response to damage. This is a question, which also derives from previous work in the mammalian intestine. A genetic approach was used to knockdown Rac1 within progenitors cells of the Drosophila midgut by RNA interference (RNAi) (esgts > Rac1-IR). Knockdown of Rac1 by 2 independent RNAi lines resulted in almost complete suppression of ISC proliferation in regenerating posterior midguts subject to Pe infection. Similar to the Drosophila midgut, the mammalian intestine displays a remarkable regenerative capacity following damage. Therefore the conservation of the requirement for Rac1 during intestinal regeneration across these species was addressed. Rac1 was conditionally deleted from the mouse intestinal epithelium using the vil-Cre-ERT2, and the effect of Rac1 loss on tissue regeneration upon DNA damage was tested. Consistent with the results in the fly midgut, Rac1 deletion significantly suppressed regeneration in the mouse intestinal epithelium (Myant, 2013b).

Altogether, this work results suggest a central conserved role for the small GTPase RAC1 as a driver of ISC proliferation through the production of ROS. These data highlight RAC1 as key player and potential therapeutic target for conditions linked to oxidative stress such as cancer and aging (Myant, 2013b).

Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling

Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. This study shows that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped-Yorkie transcriptional complex, and the Hippo (Hpo) signaling pathway directly restricts ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promotes ISC proliferation. These findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation (Jin, 2013).

SWI/SNF complex subunits regulate the chromatin structure by shutting off or turning on the gene expression during differentiation. Recently, the findings from several research reports based on the stem cell system reveal important roles of chromatin remodeling complex in stem cell state maintenance. The current study suggests that the chromatin remodeling activity of Brm complex is required for the proliferation and differentiation of Drosophila ISCs. Based on these findings, it is proposed that Brm is critical for maintaining Drosophila intestinal homeostasis. High levels of Brm in the ISC nucleus represent high proliferative ability and are essential for EC differentiation; low levels of Brm in the EC nucleus may be a response for homeostasis. Changes in Brm protein levels result in the disruption of differentiation and deregulation of cell proliferation. In line with previous findings in human, the cell-type-specific expression of Drosophila homologs BRG1 and BRM are also detected in adult tissues. BRG1 is mainly expressed in cell types that constantly undergo proliferation or self-renewal, whereas BRM is expressed in other cell types. These observations indicate that Brm may act similarly to BRG1 and BRM in controlling proliferation and differentiation (Jin, 2013).

The Hpo pathway restricts cell proliferation and promotes cell death at least in two ways: inhibiting the transcriptional co-activator Yki and inducing activation of pro-apoptotic genes such as caspases directly. This study has identified a novel regulatory mechanism of the Hpo pathway in maintaining intestinal homeostasis. In this scenario, Brm activity is regulated by the Hpo pathway. In normal physiological conditions, under the control of Hpo signaling, the function of Yki–Sd to promote ISC proliferation is restricted and the pro-proliferation of target genes such as diap1 that inhibits Hpo-induced caspase activity cannot be further activated. Therefore, Hpo signaling normally functions to restrict cell numbers in the midgut by keeping ISC proliferation at low levels. Yki is enriched in ISCs, but predominantly inactivated in cytoplasm by the Hpo pathway. The knockdown of Yki in ISCs did not cause any phenotype in the midgut, suggesting that Yki is inactivated in ISCs under normal homeostasis. During an injury, Hpo signaling is suppressed or disrupted, Yki translocates into the nuclei to form a complex with Sd, which may allow Yki–Sd to interact with Brm complex in the nucleus to activate transcriptional targets. Of note, the loss-of-function of Brm resulted in growth defect of ISCs, suggesting that Brm is required for ISC homeostasis and possessing a different role of Brm from Yki in the regulation of ISCs. It is possible that the function of Brm on ISC homeostasis is regulated via other signaling pathways by recruiting other factors. Therefore, different phenotypes induced by the loss-of-function of Brm and Yki in midgut might be due to different regulatory mechanisms. Despite its unique function cooperating with Yki in midgut, that Brm complex is essential for Yki-mediated transcription might be a general requirement for cell proliferation. While this manuscript was under preparation, Irvine lab reported a genome-wide association of Yki with chromatin and chromatin-remodeling complexes (Oh, 2013). These results support the model developed in this paper (Jin, 2013).

The current results also suggest that the interaction between Brm and Yki–Sd transcriptional complex is under tight regulation. The loss of Hpo signaling stabilizes Brm protein, whereas the active Hpo pathway restricts Brm levels by activating Drosophila caspases to cleave Brm at the D718 site and inhibiting downstream target gene diap1 transcription simultaneously. In addition, overexpression of Brm complex components induces only a mild enhancement on midgut proliferation. One possibility is that overexpressing only one of the Brm complex components does not provide full activation of the whole complex; the other possibility is that due to the restriction of the Hpo signaling, as overexpressing Brm^D718A mutant protein in ISCs/EBs exhibits a stronger phenotype than expressing the wild-type Brm and coexpression of Brm^D718A completely rescues the impairment of Hpo-induced ISC proliferation. D718A mutation blocks the caspase-dependent Brm cleavage and exhibits high activity in promoting ISC proliferation. This study has defined a previously unknown, yet essential epigenetic mechanism underlying the role of the Hpo pathway in regulating Brm activity (Jin, 2013).

It is a novel finding that Brm protein level is regulated by the caspase-dependent cleavage. To focus on the function of Brm cleavage in the presence of cell death signals, attempts were made to examine the activities of the cleaved Brm fragments. Although in vivo experiments did not show strong activity of Brm N- and C-cleavage products in promoting proliferation of ISCs, the C-terminal fragment of Brm that contains the ATPase domain exhibits a relative higher activity than the N-terminal fragment in ISCs. The cleavage might induce faster degradation of Brm N- and C-terminus, since it was difficult to detect N- or C-fragments of Brm by Western blot analysis without MG132 treatment. It reveals that the degradation events of Brm including both ubiquitination and cleavage at D718 site can be important for Brm functional regulation under different conditions. To this end, the intrinsic signaling(s) may balance the activity of Brm complex through degradation of some important components, such as Brm, to maintain tissue homeostasis. Of note, the cleavage of Brm at D718 is occurred at a novel DATD sequence that is not conserved in human Brm. It has been reported that Cathepsin G, not caspase, cut hBrm during apoptosis, suggesting that the cleavage regulatory mechanism of Brm is relatively conserved between Drosophila and mammals (Jin, 2013).

This study provides evidence that the Brm complex plays an important role in Drosophila ISC proliferation and differentiation and is regulated by multi-levels of Hpo signaling. The findings indicate that Hpo signaling not only exhibits regulatory roles in organ size control during development but also directly regulates epigenetics through a control of the protein level of epigenetic regulatory component Brm. In mammals, it is known that Hpo signaling and SWI/SNF complex-mediated chromatin remodeling processes play critical roles in tissue development. Malfunction of the Hpo signaling pathway and aberrant expressions of SWI/SNF chromatin-remodeling proteins BRM and BRG1 have been documented in a wide variety of human cancers including colorectal carcinoma. Thus, this study that has implicated a functional link between Hpo signaling pathway and SWI/SNF activity may provide new strategies to develop biomarkers or therapeutic targets (Jin, 2013).

CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo

During development eukaryotic gene expression is coordinated by dynamic changes in chromatin structure. Measurements of accessible chromatin are used extensively to identify genomic regulatory elements. Whilst chromatin landscapes of pluripotent stem cells are well characterised, chromatin accessibility changes in the development of somatic lineages are not well defined. This study shows that cell-specific chromatin accessibility data can be produced via ectopic expression of E. coli Dam methylase in vivo, without the requirement for cell-sorting (CATaDa). Chromatin accessibility was profiled in individual cell-types of Drosophila neural and midgut lineages. Functional cell-type-specific enhancers were identified, as well as novel motifs enriched at different stages of development. Finally, global changes were shown in the accessibility of chromatin between stem-cells and their differentiated progeny. These results demonstrate the dynamic nature of chromatin accessibility in somatic tissues during stem cell differentiation and provide a novel approach to understanding gene regulatory mechanisms underlying development (Aughey, 2018).

The conserved Misshapen-Warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila

Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. This study shows that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP (Li, 2014).

Previous studies have shown that endothelial cells (ECs) produce regulatory factors in response to infection and damage and function as part of the niche to regulate intestinal stem cell (ISC)-mediated regeneration. Meanwhile, recent reports show that enteroblasts (EBs) can also produce growth factors including EGF receptor ligands, Wingless and Upd3, although the pathways that regulate their production are not known. The current results demonstrate that differentiating EBs also function as an important part of the niche to regulate ISC division via the Msn pathway. EB-specific knockdown of msn leads to highly increased Upd3 expression and midgut proliferation. A previous report suggests that undifferentiated EBs if remain in contact with the mother ISC can inhibit proliferation. Although the hyperproliferating midguts after loss of Msn contain many EBs, these EBs do go into normal differentiation and express high level of Upd3, which may overcome any inhibitory effect of undifferentiated EBs on ISC proliferation (Li, 2014).

Msn is known to regulate a number of biological processes. During embryonic dorsal closure the MAP kinase pathway Slipper-Hemipterous-JNK is downstream of Msn, and Slipper is able to bind to Msn in vitro. In the adult midgut, JNK is a mediator of aging-related intestinal dysplasia and is a stress-activated kinase in ECs to positively regulate ISC division. While the current RNAi experiments show that JNK has a function in EBs to negatively regulate ISC proliferation, this phenotype is not dependent on Upd3 or Yki. No change of JNK phosphorylation was detected after loss of Msn. Mammalian MAP4K4 has also been shown to function independently of JNK in some biological contexts. Therefore, Msn and JNK probably have independent functions in the midgut (Li, 2014).

This study has instead uncovered an interaction of Msn with Wts and subsequently regulation of Yki. Hpo-Wts-Yki has been demonstrated to have a function in ECs for stress and damage-induced response. Gal4 driven experiments have many caveats including cell-type specificity, differences in promoter strengths, and knockdown efficiency in different cell types. Nonetheless, the results of many parallel experiments that this study conducted strongly suggest that Msn and Hpo independently regulate Wts-Yki in EBs and ECs, respectively. How the Msn and Hpo pathways in the two cell types are coordinately regulated to produce an appropriate amount of Upd3 to achieve desirable intestinal growth under different circumstances remains an important question to be answered (Li, 2014).

Previous experiments in developing discs suggest that Wts and Yki but not Hpo act downstream of cytoskeleton regulators. Similarly, the mammalian Hpo homologs MST1/2 appear not to be involved in LATS regulation after cytoskeletal perturbation in some cell types. In vivo assay in midgut suggests a function for Msn, Yki and Upd3 downstream of actin capping proteins in EBs. Similarly, the Latrunculin B effect on MEFs suggests that MAP4K4 is required for cytoskeleton-regulated LATS and YAP phosphorylation. The situation in mammalian cells may be more complicated because the Msn/MAP4K4 subfamily also includes two other closely related kinases TNIK and MINK1. Proper regulation of Wts by the cytoskeleton may require both positive and negative regulators, because recent work in flies identified the LIM-domain protein Jub as a negative regulator of Wts in response to cytoskeletal tension. It will be interesting in future studies to determine how positive and negative regulators of Wts act in a coordinated manner to regulate cell fate and proliferation in response to cytoskeletal tension (Li, 2014).

Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence

Adult stem cells vary widely in their rates of proliferation. Some stem cells are constitutively active, while others divide only in response to injury. The mechanism controlling this differential proliferative set point is not well understood. The anterior-posterior (A/P) axis of the adult Drosophila midgut has a segmental organization, displaying physiological compartmentalization and region-specific epithelia. These distinct midgut regions are maintained by defined stem cell populations with unique division schedules, providing an excellent experimental model with which to investigate this question. This study has focused on the quiescent gastric stem cells (GSSCs) of the acidic copper cell region (CCR), which exhibit the greatest period of latency between divisions of all characterized gut stem cells, to define the molecular basis of differential stem cell activity. Molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. Under baseline conditions, when GSSCs are largely quiescent, the lowest levels of EGF ligands in the midgut are found in the CCR. However, acute epithelial injury by enteric pathogens leads to an increase in EGF ligand expression, specifically Spitz and Vein, in the CCR and rapid expansion of the GSSC lineage. Thus, the unique proliferative set points for gut stem cells residing in physiologically distinct compartments are governed by regional control of niche signals along the A/P axis (Strand, 2013).

The CCR epithelium is the exclusive site of large acid-secreting copper cells responsible for generating a low pH compartment in the midgut. Gastric stem cells in the CCR are normally quiescent but are robustly stimulated to replenish the unique differentiated cells of the gastric epithelium in response to injury by enteric pathogens or heat stress. This study resolvse outstanding issues related to the GSSC lineage, demonstrating the presence of tripotent GSSC lineages in the CCR. In addition, wa central role is demonstrated for the conserved EGF signaling pathway in controlling the emergence of gastric stem cells from quiescence. Taken together, two key differences between GSSCs and intestinal stem cells (ISCs) are now evident: the unique region specific cell lineages that they support (copper, interstitial, enteroendocrine vs. enterocyte and enteroendocrine) and their activity levels (quiescent vs. active). Thus, maintenance of physiologically and functionally distinct compartments of the adult midgut depends upon the activity of distinct stem cell lineages (Strand, 2013).

What is the nature of the unique molecular program that governs the observed differences in GSSC and ISC proliferative behavior? This study indicates that regional differences in gut stem cell proliferation are controlled by regional differences in EGF ligand availability. First, reporters of EGF pathway activity are normally very low in the CCR under baseline conditions, when GSSCs are quiescent. However, damage to the gastric epithelium by enteric infection increases local EGF ligand expression and Erk phosphorylation. This EGF activation directly correlates with an observed increase in proliferating GSSCs. Second, ectopic activation of the EGF pathway is sufficient to cell-autonomously promote GSSC proliferation in the absence of environmental challenge. Finally, functional EGF signaling is required for GSSC proliferation following enteric infection and for GSSC lineage expansion. Importantly, these studies of GSSCs in the CCR are similar to previous studies demonstrating that EGF signals are an essential part of the core niche program controlling the ISC lineage. Thus, regional control of EGF ligands, and perhaps other regulators of EGF pathway activity, are essential in generating gastrointestinal stem cell niches with distinct proliferative set points (Strand, 2013).

In this light, it is worth noting that over-expression of epidermal growth factors and their receptors are associated with human gastric cancer, the second leading cause of cancer-related deaths worldwide. In addition, Ménétrier’s disease is a hyperproliferative disorder of the stomach caused by over-expression of the EGF ligand TGF-α. Over production of TGF-α and increased EGF signaling is associated with an expansion of surface mucous cells and a reduction in parietal and chief cells. Gastric stem cells are the proposed cell-of-origin in Ménétrier’s disease, but this has not been directly tested due to a lack of gastric stem cell specific markers in the murine system. Advances in understanding how EGF ligand availability controls activity of the acid-secreting gastric stem cell lineage in Drosophila raises the possibility that hyperplastic conditions associated with the human stomach might arise when ectopic EGF ligands draw resident stem cells out of their quiescent state (Strand, 2013).

EGF signaling appears to be only one aspect of the region specific program controlling gastric stem cells in the adult copper cell region. Previous studies have shown that a Delta-lacZ enhancer trap line was not present in GSSCs under baseline conditions. In the course of this study, it was observed that Pseudomonas entomophila challenge also leads to an increase in Delta ligand expression in dividing cells, suggesting a role for Delta/Notch signaling in the GSSC lineage. In addition, elegant studies of GSSCs under baseline conditions have recently shown that the secreted BMP/Dpp signaling pathway is both necessary and sufficient to specify copper cells in the adult midgut and acts via the labial transcription factor. Interestingly, while the highest levels of Dpp pathway reporters are detected in the CCR, manipulation of the BMP/Dpp pathway did not affect GSSC proliferation. Thus, the GSSC lineage is influenced by secreted niche factors, which independently control both GSSC proliferation and cell fate specification (Strand, 2013).

In conclusion, understanding GI regionality and homeostatic diversity along the A/P axis is important for several reasons. It is now possible to gain insight into how the modification of a core GI niche program, which adapts each stem cell to its compartment specific physiology, leads to difference in lineage output. Second, disruption of regional identity along the GI tract is associated with a class of precancerous conditions called metaplasias, in which one region of the GI tract takes on the attributes of another. Finally, both the establishment and maintenance of tumorigenic lineages exhibit marked preferences along the A/P axis of the gut. The striking similarities between vertebrate and invertebrate GI biology, suggest that delving deeper into the mechanisms underlying Drosophila midgut regionalization will continue to provide important insights into these fundamental biological problems (Strand, 2013).

EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors

In holometabolous insects, the adult appendages and internal organs form anew from larval progenitor cells during metamorphosis. The adult Drosophila midgut, including intestinal stem cells (ISCs), develops from adult midgut progenitor cells (AMPs) that proliferate during larval development in two phases. Dividing AMPs, as visualized using esgGal4-driven GFP expression, first disperse, but later proliferate within distinct islands, forming large cell clusters that eventually fuse during metamorphosis to make the adult midgut epithelium. Signaling through the EGFR/RAS/MAPK pathway is necessary and limiting for AMP proliferation. Midgut visceral muscle produces a weak EGFR ligand, Vein, which is required for early AMP proliferation. Two stronger EGFR ligands, Spitz and Keren, are expressed by the AMPs themselves and provide an additional, autocrine mitogenic stimulus to the AMPs during late larval stages (Jiang, 2009).

Drosophila AMPs were previously thought to be relatively quiescent during larval development, dividing just once or twice, and not initiating rapid proliferation until the onset of metamorphosis. This is the case for several other larval progenitor/imaginal cell types, such as the abdominal histoblasts and cells in the salivary gland, foregut and hindgut imaginal rings. Studies have suggested that AMP proliferation might precede the onset of metamorphosis. However, the extensive proliferation of the AMPs that is seen in this study has not been reported and the early larval proliferative phase when the AMPs divide and disperse has not been reported. The extensive proliferation of the AMPs is similar to that of the larval imaginal disc cells, which also proliferate throughout larval development, dividing about ten times (Jiang, 2009).

AMPs occurs in two distinct phases. In early larvae, the AMPs divide and disperse throughout the midgut to form individual islets. During later larval development, the AMPs continue to divide but do so within these islets, forming large cell clusters. It is speculated that in the early larva, secretion of Vn from the midgut visceral muscle (VM) cells results in low-level activation of EGFR signaling in the AMPs, which is sufficient for their proliferation and might also promote their dispersal. No proliferation defects were seen in AMPs defective in shot function, suggesting that the mechanism of EGFR activation used by tendon cells during muscle/tendon development is probably not the same as in the larval midgut. Specifically, it is unlikely that the Shot-mediated concentration of Vn on AMPs activates EGFR signaling in the AMPs during early larval development. Consistent with this, dpERK staining is only seen in AMP clusters and not in the isolated AMPs present at early larval stages (Jiang, 2009).

The mechanisms that regulate the transition between these two proliferation phases remain unclear. Fewer AMP clusters are seen when sSpi, sKrn, lambdaTOP (activated Egfr) or Ras^V12 were induced in the AMPs starting from early larval stages, suggesting that EGFR signaling, in addition to its crucial role as an AMP mitogen, might also play a role in AMP cluster formation. In the late larval midgut (96-120 hours AED), high-level EGFR activation, resulting from expression of spi and Krn in the AMPs themselves, might not only promote AMP proliferation, but might also suppress AMP dispersal and thus promote formation of the AMP clusters. How the timing and location of Spi- or Krn-mediated EGFR activation are regulated during larval development is also unclear. It is noted, however, that the pro-ligand form of Krn acted similarly to sKrn, and that no functions were uncovered for the Rho-like gene products that regulate Spi and Krn function by proteolytic cleavage in other tissues. This suggests that the localized expression of these ligands in the AMP clusters might be the critical parameter that controls their effects. Consistent with this, Rho-independent cleavage and function of Krn have been documented (Reich, 2002; Jiang, 2009).

In the developing Drosophila wing, EGFR/RAS/MAPK signaling promotes the expression and controls the localization of the cell adhesion molecule Shotgun (Shg, Drosophila DE-cadherin). Ras^V12-expressing clones generated in the wing imaginal disc are round, much like the AMP clusters described in this study, owing to increased adhesive junctions. In developing Drosophila trachea, EGFR activity upregulates shg expression to maintain epithelial integrity in the elongating tracheal tubes. In the eye, EGFR activity leads to increased levels of Shg and adhesion between photoreceptors. Given these precedents, it seems reasonable to suggest that high-level EGFR activity in the AMP islets upregulates Shg and promotes the homotypic adhesion of the AMPs. Alternatively, changes in the differentiated cells of the midgut epithelium might promote AMP clustering. In either case, the dispersal of early AMPs and subsequent formation of late AMP clusters facilitate the formation of the adult midgut epithelium during metamorphosis (Jiang, 2009).

This study confirms previous reports that Drosophila AMPs replace larval midgut epithelial cells to form the adult midgut epithelium during metamorphosis. Furthermore, it was shown that the majority of AMPs lose esgGal4-driven GFP expression as they differentiate to form the new adult midgut epithelium. These cells lacked Prospero, which marks enteroendocrine cells in both the larval and adult midgut. They went through several rounds of endoreplication during late pupal development, and thus probably all differentiated into adult enterocytes (ECs). During early metamorphosis, some cells in the new midgut epithelium remained small and diploid and maintained strong esgGal4 expression. For several reasons, it is thought that these esg-positive cells are the future adult intestinal stem cells (ISCs). (1) esgGal4 expression marks AMPs, including adult ISCs and enteroblasts. (2) Mitoses in the adult midgut are only observed in ISCs, and this study observed mitoses only in the esg-positive cells during metamorphosis. (3) esg-positive cells migrated to the basal side of the midgut epithelium, the location of adult ISCs. (4) AMP clones generated during early larval development contained just a few esg-positive cells when the new adult midgut first formed (24 hours APF), but when such clones were scored in newly eclosed adults, they contained large numbers of ECs, as well as cells positive for the enteroendocrine marker Prospero and the ISC marker Delta. This suggests that a small fraction of AMPs differentiate into adult ISCs. However, esg-positive cells in the new pupal midgut lacked Delta expression until eclosion, suggesting that they are probably not mature adult ISCs (Jiang, 2009).

How a small fraction of AMPs are selected to become adult ISCs in the newly formed pupal midgut epithelium is not known. One possibility is that the adult ISCs are determined during larval development, long before the formation of the adult midgut. Another is that they are specified during early metamorphosis. This second hypothesis is preferred for several reasons. First, in the lineage analysis, it was found that all AMP clones induced during early larval stages formed multiple clusters. This suggests that there are no quiescent AMPs in the larval midgut. Second, when AMP clones were induced at mid-third instar, the mosaic clusters always contained multiple GFP-positive cells, suggesting that all AMPs in the mid-third instar midgut remain equally proliferative. Third, during larval development, differentiation of the AMPs were never observed, as judged by their ploidy (diploid) and lack of expression of the enteroendocrine marker Prospero. Fourth, all AMPs appeared to express esgGal4 throughout larval development. Given the crucial role that Notch signaling plays in regulating AMPs during embryonic midgut development and ISCs in adult midgut homeostasis, it is edexpect that Notch might also function to specify adult ISCs during metamorphosis (Jiang, 2009).

EGFR signaling is both required and sufficient to promote AMP proliferation. Hyperactivation of EGFR signaling, such as by expression of activated Ras (Ras^V12), promoted massive AMP overproliferation and generated hyperplastic midguts that were clearly dysfunctional. In contrast, inhibiting EGFR/RAS/MAPK signaling dramatically reduced AMP proliferation. Furthermore, the ability of EGFR signaling to induce ectopic AMP proliferation is almost unique. With the exception of larval hemocytes, activated EGFR signaling does not promote cell proliferation in the imaginal discs, salivary gland imaginal rings, abdominal histoblasts, foregut and hindgut imaginal rings. This suggests that the regulation of AMP proliferation is different from that in other imaginal cells (Jiang, 2009).

Despite the obvious differences between adult ISCs and their larval progenitors, the AMPs, there are also similarities. (1) When the new adult midgut epithelium forms, larval AMPs give rise to the new adult midgut including the adult ISCs. Many genes, such as esg, that are specifically expressed in the larval AMPs are also expressed in the adult ISCs. (2) The structure of the midgut epithelium with basal AMPs or ISCs is similar in larval and adult stages. (3) vn expression in larval VM persists in the adult midgut, suggesting that Vn from the adult VM might also regulate the ISCs (Jiang, 2009).

In two Drosophila stem cell models, the testis and ovary, stem cells reside in special niches comprising other supporting cell types. These niches maintain the stem cells and provide them with proliferative cues. For example, in the testis, germ stem cells attach to the niche that comprises cap cells. The cap cells release Jak/Stat and BMP ligands [Upd (Os) and Gbb/Dpp], which maintain the stem cells and induce their proliferation. Whether Drosophila ISCs utilize supporting cells that constitute a niche remains unclear. This study shows that multiple EGFR ligands are involved in the regulation of Drosophila AMP proliferation. During early larval development, the midgut VM expresses the EGFR ligand vn, which is required for AMP proliferation. Thus, the early AMPs might be considered to require a niche comprising non-epithelial VM. Later in larval development, however, the AMPs express two other EGFR ligands, spi and Krn, which are capable of autonomously promoting their proliferation and may render vn dispensable. This study found, however, that depleting spi and Krn in the AMPs did not affect AMP proliferation, suggesting that vn or another trigger of EGFR/RAS/MAPK activity might complement spi and Krn in late-stage larvae (Jiang, 2009).

Bunched and Madm function downstream of Tuberous Sclerosis Complex to regulate the growth of intestinal stem cells in Drosophila

The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. This study shows that the leucine zipper protein Bunched and the adaptor protein MLF1-adaptor molecule (Madm) are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, these data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells (Nie, 2015).

Homeostasis and regeneration of an adult tissue is normally supported by resident stem cells. Elucidation of the mechanisms that regulate stem cell-mediated homeostasis is important for the development of therapeutics for various diseases. The intestine with fast cell turnover rate supported by actively proliferating stem cells is a robust system to study tissue homeostasis. In the mouse intestine, two interconverting intestinal stem cell (ISC) populations marked by Bmi1 and Lgr5 located near the crypt base can replenish cells of various lineages along the crypt-villus axis Furthermore, recent data suggest that Lgr5+ cells are the main stem cell population and that immediate progeny destined for the secretory lineage can revert to Lgr5+ stem cells under certain conditions [6, 7]. Together, the results suggest previously unexpected plasticity in stem cell maintenance and differentiation in the adult mammalian intestine (Nie, 2015).

In the adult Drosophila midgut, which is equivalent to the mammalian stomach and small intestine, ISCs are distributed evenly along the basal side of the monolayered epithelium to support repair. The maintenance and regulation of Drosophila midgut ISCs depend on both intrinsic and extrinsic factors. When a midgut ISC divides, it generates a renewed ISC and an enteroblast (EB) that ceases to divide and starts to differentiate. The ISC-EB asymmetry is established by the Delta-Notch signaling, with Delta in the renewed ISC activating Notch signaling in the newly formed neighboring EB . Growth factors such as Wingless/ Wnt, insulin-like peptides, Decapentaplegic/BMP, Hedgehog and ligands for the EGF receptor and JAK-STAT pathways are secreted from surrounding cells and constitute the niche signals that regulate both ISC division and EB differentiation. ISC-intrinsic factors including Myc, Target of Rapamycin (TOR) and Tuberous Sclerosis Complex act to coordinate the growth and division of ISCs. Furthermore, chromatin modifiers such as Osa, Brahma and Scrawny function within ISCs to regulate Delta expression or ISC proliferation (Nie, 2015).

This study reports the identification of the leucine zipper protein Bunched (Bun) and the adaptor protein myeloid leukemia factor 1 adaptor molecule (Madm) as intrinsic factors for ISC proliferation. A single bun genomic locus generates multiple predicted transcripts that encode 4 long isoforms, BunA, F, G and P, and 5 short isoforms, BunB, C, D, E, H and O. The first identified mammalian homolog of Bun is TGF-β1 stimulated clone-22 (TSC-22). In the mouse genome four different TSC- 22 domain genes also encode multiple short and long isoforms. All isoforms of Bun and TSC-22 contain an approximately 200 amino acids C-terminal domain where the conserved TSC-box and leucine zippers are located. The originally identified TSC-22 is a short isoform and various assays suggest that it suppresses cancer cell proliferation and may function as a transcriptional regulator. Meanwhile, in Drosophila, the long Bun isoforms positively regulate growth, while the short isoforms may antagonize the function of long isoforms. Transgenic fly assays also demonstrate that the long TSC-22 can rescue the bun mutant phenotypes, whereas short isoforms cannot. These results suggest an alternative model that the long Bun isoforms positively regulate proliferation, while the short isoforms may dimerize with and inhibit the functions of long isoforms (Nie, 2015).

Madm also can promote growth. The long isoform BunA binds to Madm via a conserved motif located in the N- terminus that is not present in the short Bun isoforms. The molecular function of this novel BunA- Madm complex, nonetheless, remains to be elucidated. The results in this report demonstrate that Bun and Madm modulate the Tuberous Sclerosis Complex-target of Rapamycin (TOR)-eIF4E binding protein (4EBP) pathway to regulate the growth and division of ISCs in the adult midgut (Nie, 2015).

This report shows that Bun and Madm are intrinsically required for ISC growth and division. The results suggest a model that Bun and Madm form a complex in the cytoplasm to promote cellular growth and proliferation. The evidence that support this model includes the observation that transgenic expressed Bun localizes in the cytoplasm of midgut precursor cells, similar to the results from transfection in S2 cells and immune-staining in eye discs. Bun physically and functionally interacts with Madm, which has also been proposed as a cytoplasmic adaptor protein. Adding a nuclear localization signal to Bun reduced the growth promoting ability of Bun. Although there is a possibility this signal peptide changes the functionality in an unpredicted way, the interpretation is favored that Bun normally acts in the cytoplasm and with Madm to regulate the proliferation of ISCs. This is in contrast to mammalian TSC-22, which was reported to function in the nucleus (Nie, 2015).

The results seem to contradict a previous publication reporting that TSC-22 arrests proliferation during human colon epithelial cell differentiation. However, this apparent contradiction is resolved when the growing evidence for distinct functions for large and small Bun/ TSC-22 isoforms is considered. The Bun/TSC-22 proteins have short and long isoforms that contain the conserved TSC-box and leucine zippers in the C-terminal domain. The prototypical TSC-22 protein, TSC22D1-001, may act as a transcriptional regulator and repress cancer cell proliferation, particularly for blood lineages. Another recent model suggests that in Drosophila the long Bun isoforms interact with Madm and have a growth promoting activity, which is inhibited by the short Bun isoforms. Similarly, the long isoform, TSC22D1-002, enhances proliferation in mouse mammary glands, whereas the short isoform promotes apoptosis. Unpublished result that transgenic expression of BunB also has lower function than BunA in fly intestinal progenitor cells is consistent with this model where large isoforms have a distinct function, namely in growth promotion (Nie, 2015).

Loss of either Bun or Madm can potently suppress all the growth stimulation by multiple pathways in the midgut as shown in this report. These results are intrepeted to indicate that Bun and Madm do not act specifically in one of the signaling pathways tested but instead function in a fundamental process required for cell growth, such as protein synthesis or protein turnover. It is therefore speculated that Bun and Madm may regulate the TOR pathway. In support of this idea, it was shown that bunRNAi or MadmRNAi efficiently suppresses the Tuberous Sclerosis Complex 2RNAi-induced cell growth and p4EBP phenotypes. A recent study of genetic suppression of TOR complex 1-S6K function in S2 cells also suggests that Bun and Madm can interact with this pathway. Furthermore, proteomic analyses of Bun and Madm interacting proteins in S2 cells have shown interactions with ribosomal proteins and translation initiation factors. Therefore, a model is proposed that Bun and Madm function in the Tuberous Sclerosis Complex-TOR- 4EBP pathway to regulate protein synthesis in ISCs for their growth, which is a prerequisite for ISC proliferation. Suppression of Tuberous Sclerosis Complex mutant cell growth phenotype by bun or Madm RNAi was substantial but not complete. Earlier papers demonstrated that Bun also interacts with Notch and EGF pathway in ovary follicle cells. Therefore by definition Bun and Madm are neither 100% essential nor restricted to the TOR pathway. The genetic data suggest that Bun and Madm work downstream of Tuberous Sclerosis Complex and upstream of 4EBP, but they could also work in parallel to the TOR pathway components (Nie, 2015).

ISCs with loss of Tuberous Sclerosis Complex function have substantial cell size increase. Meanwhile, the Bun/ Madm overexpression caused increased ISC division but not cell hypertrophy. Both loss of Tuberous Sclerosis Complex and overexpression of Bun/Madm should promote cell growth but the phenotypes at the end are different. It is speculated that the reason is the Bun/Madm overexpressing ISCs are still capable of mitosis, while the Tuberous Sclerosis Complex mutant ISCs do not divide anymore thereby resulting in the very big cells. In Bun and Madm overexpressing mid- guts, the p-H3+ and GFP+ cell count showed a significant increase, indicating increased mitosis. Therefore, an explanation is that Bun and Madm overexpression may increase cell size/cell growth, but when they grow to certain size they divide, resulting in rather normal cell size (Nie, 2015). The knockout of the Madm mammalian homolog, NRBP1, can cause accumulation of the short isoform TSC22D2. Up-regulation of Madm/NRBP1 has been associated with poor clinical outcome and increased growth of prostate cancer. Further analysis based on this model may reveal whether high ratio of long Bun/TSC22 isoforms over short isoforms may associate with high Madm activity and poor clinical outcomes (Nie, 2015).

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. This study reports a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. These findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homoeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease (Perea, 2017).

These findings in Drosophila indicate that Ret is expressed not only by enteric neurons, but also by the adult somatic stem cells of the intestinal epithelium. In contrast to known Ret functions in other progenitor cell types -- for example, in spermatogonia or the hematopoietic system -- Ret is not required for the survival of adult somatic stem cells in the intestine, but sustains both their homeostatic and regenerative proliferative capacity. Gain- and loss-of-function experiments point to the existence of positive feedback between Ret and Wg signalling. Despite abundant genetic evidence that Wg signalling promotes stem cell proliferation in flies, the source of Wg has remained unclear. Using new, improved tools to visualise Wg expression, the current findings lend further support to recent data (Tian, 2016) indicating that the source of Wg ligand is not the stem cells themselves, despite the striking Ret-driven upregulation of Wg on their surface. How might Ret signalling in adult intestinal progenitors lead to Wg protein upregulation in these cells without affecting its transcript? Two possible ways in which it might do so is by upregulating the expression of Wg receptor(s) on their surface, and/or by promoting signalling from stem cells to the Wg-producing cells at the intestinal boundaries and/or the visceral muscles (Buchon, 2013; Tian, 2016) to increase Wg release/trafficking (Perea, 2017).

Epithelial Ret is not a peculiarity of the fly intestine; Ret is also expressed in the developing intestinal epithelium of mice, prior to the maturation of enteroendocrine or Lgr5-positive stem cells. Although immunohistochemical analyses have not revealed a specific Ret-positive cell population at this stage, ex vivo experiments using epithelial cultures devoid of enteric neuron or mesenchyme point to an intrinsic role for Ret at this stage in promoting epithelial maturation. The mechanism underlying the maturation-promoting effects of Ret may involve positive feedback between Ret and Wnt signalling similar to those found in flies. Indeed, the Wnt pathway target Axin2 is reduced in epithelial cultures derived from Ret51 mice and upregulated when wild-type FEnS are treated with the Ret ligand GDNF: a treatment that also promotes their branching. These data are consistent with the previous finding that elevated Wnt signalling promotes FEnS to organoid maturation and is reminiscent of the Wnt11/Ret autoregulatory loop promoting ureteric branching during kidney development. The relevant source of Wnt driving tissue maturation is currently unknown and is most likely not epithelial. The Drosophila finding that Wg ligand upregulation is not transcriptional underscores the importance of considering tissue crosstalk and non-autonomous signalling in any future studies addressing Wnt contributions to epithelial maturation in mice (Perea, 2017).

At first sight, the Ret effects on developmental maturation in mice appear to be different from its homeostatic role in flies. However, this study found that Ret continues to be expressed in the adult small intestine, where Ret expression is prominent in a subset of enteroendocrine cells positive for the secretory marker chromogranin-A. Based on their position, these cells may correspond to enterochromaffin cells: intriguing cells that contribute 90% of the serotonin in circulation, control gastrointestinal motility and secretions and have recently been shown to be chemosensory. A very recent study has blurred the distinction between enteroendocrine cells and their precursors by revealing expression overlaps between markers of enteroendocrine precursor identity and differentiated fate (including chromogranin-A), and by suggesting that differentiated enteroendocrine cells can have stem cell-like properties (Yan, 2017). This is exciting because it suggests that, whilst the cellular classification of Ret-positive cells based on known markers may differ between flies (ISCs) and mice (enteroendocrine), Ret-enabled stem cell functionality may contribute to regeneration in both epithelia. Intriguingly, endocrine tumours derived from the small intestine, ileal carcinoids, secrete serotonin and have been reported to express high levels of Ret. Conditional deletion of Ret in adult intestinal epithelium will, in future, determine its contribution to enteroendocrine fate and will help establish possible enteroendocrine contributions to intestinal homeostasis and tumour formation (Perea, 2017).

Consistent with ex vivo transfection studies in mammalian cells, pointing to physical association between Ret and c-Src, this study found that Src kinase Src42A is required downstream of Ret to activate Wg signalling. These findings therefore strengthen the link between two pathways previously known to control ISC proliferation in flies-Src and Wg, and may provide a physiological context for the previously reported, Src-dependent mitogenic effects of mutated, oncogenic Ret in the Drosophila developing retina. The finding that, in the Drosophila intestine, Src kinases control expression of a mitogenic module consisting of String/Cdc25 and cyclin E provides a simple link between Ret activation and its pro-proliferative effects. Overactive Src kinases do, however, lead to intestinal tumours so mechanisms must be in place to limit the positive Ret/Wg feedback loop so that it sustains homeostatic proliferation, but does not result in tumour formation. Availability of Wg ligand may be an extrinsic mechanism, but the focal adhesion kinase Fak may provide a cell-intrinsic break downstream of Ret/Src activation. Consistent with this idea, Ret expression leads to both Src42A and Fak phosphorylation, but this study found that the two kinases have opposing effects on proliferation: Src42A promotes proliferation downstream of Ret, whereas Fak blocks it. Hence, despite the fact that blocking Fak function may represent a therapeutic opportunity in some cancers, the current findings are more aligned with a previous study (Macagno, 2014) that suggested that, at least in the context of Ret-driven tumorigenesis, Fak can act as a tumour suppressor. In future, it will also be of interest to explore how the Ras/Raf/Erk pathway, activated by Ret in other contexts and previously shown to affect ISC proliferation in flies, intersects with Src/Fak/Wg signalling in response to Ret activation (Perea, 2017).

Both Wnt and Src pathways can have strong effects on proliferation, differentiation and/or tumorigenesis in the murine intestine. Src is required for mouse intestinal tumourigenesis following upregulation of Wnt signalling. Based on functional findings in Drosophila and expression data in mice, a possible contribution of the Ret- and CgA-positive cells to this process deserves further investigation. It will also be of interest to investigate how Src/Fak kinase signalling contributes to the maturation of foetal intestinal epithelial cells and whether this is important in the development of intestinal disorders (Perea, 2017).

Ret expression is one of the defining features of enteric neurons. This study has found another evolutionarily conserved and physiologically significant site of Ret expression: the intestinal epithelium. The presence of Ret in these two gastrointestinal cell types of very different developmental origin raises the possibility that the development and/or physiology of enteric neurons, intestinal epithelial progenitors and, in mammals, Ret-expressing intestinal lymphoid cells is coordinated. Such coordination may, for example, help ensure a match between the size of the intestinal epithelium, the number of innervating neurons during development and the transition from an immature foetal epithelium into a functional epithelium involved in nutrient uptake and interorgan signalling. In mammals, Ret ligands of the glial cell line derived neurotrophic factor (Gdnf) family may orchestrate Ret signalling in these three tissues. In flies (which lack these Ret ligands), integrins have been shown to interact with Ret in sensory neurons. Interfering with integrin expression in the Drosophila intestine can have different effects on intestinal progenitor proliferation, survival and/or orientation depending on whether the integrins are removed from the progenitors or their niche-the visceral muscles. Intriguingly, integrin downregulation in adult intestinal progenitors reduces their normal proliferation and can suppress their overproliferation in response to overactive Wingless signalling: phenotypes strikingly similar to those resulting from Ret downregulation. In the light of the known links between integrins and Fak/Src signalling in both normal and cancer cells and the effects that this study has found for Src and Fak downstream of Ret activation, Ret could provide a new route for the integrin activation of the Src/Fak complex. Alternatively, the recent finding that GDF15, a divergent member of the TGF-β superfamily, signals through a GDNF family receptor α-like in a Ret-dependent way also raises the intriguing possibility that TGF-β-like ligands modulate Ret signalling in the intestinal epithelium, potentially linking intestinal regeneration with the known GDF15 roles in food intake/body weight (Perea, 2017).

The crucial requirement for Ret in enteric nervous system development is underscored by disorders such as HSCR, in which Ret loss of function leads to almost complete absence of enteric innervation in varying lengths of the distal gut. Whilst the contribution of enteric aganglionosis to HSCR is unquestionable, the current findings raise the possibility that, if the epithelial expression of Ret is conserved in humans, dysregulation of epithelial signalling may contribute to disorders that, like HSCR, result from Ret mutation. Epithelial Ret signalling might also contribute to other aspects of gastrointestinal physiology previously shown to be affected by reduced Ret function, such as intestinal motility, gut-microbiota interactions and the compensatory response to massive small bowel resection. Interestingly, HSCR is typically diagnosed around birth due to defects in gastrointestinal functions. This coincides with the first demands on intestinal function, which could reflect not only neuronal defects related to peristalsis, but also defects associated with the transition from a foetal into a functional adult epithelium. Given that many of the pathways that drive tissue expansion and the maintenance of non-differentiated progenitor populations during foetal development are deregulated in cancer, a possible contribution of Ret signalling to colorectal tumours also deserves further investigation (Perea, 2017).

Accumulation of differentiating intestinal stem cell progenies drives tumorigenesis

Stem cell self-renewal and differentiation are coordinated to maintain tissue homeostasis and prevent cancer. Mutations causing stem cell proliferation are traditionally the focus of cancer studies. However, the contribution of the differentiating stem cell progenies in tumorigenesis is poorly characterized. This study reports that loss of the SOX transcription factor, Sox21a, blocks the differentiation programme of enteroblast (EB), the intestinal stem cell progeny in the adult Drosophila midgut. This results in EB accumulation and formation of tumours. Sox21a tumour initiation and growth involve stem cell proliferation induced by the Unpaired 2 mitogen released from accumulating EBs generating a feed-forward loop. EBs found in the tumours are heterogeneous and grow towards the intestinal lumen. Sox21a tumours modulate their environment by secreting matrix metalloproteinase and reactive oxygen species. Enterocytes surrounding the tumours are eliminated through delamination allowing tumour progression, a process requiring JNK activation. These data highlight the tumorigenic properties of transit differentiating cells (Zhai, 2015).

Sox100B regulates progenitor-specific gene expression and cell differentiation in the adult Drosophila intestine

Robust production of terminally differentiated cells from self-renewing resident stem cells is essential to maintain proper tissue architecture and physiological functions, especially in high-turnover tissues. However, the transcriptional networks that precisely regulate cell transition and differentiation are poorly understood in most tissues. This study identified Sox100B, a Drosophila Sox E family transcription factor, as a critical regulator of adult intestinal stem cell differentiation. Sox100B is expressed in stem and progenitor cells and required for differentiation of enteroblast progenitors into absorptive enterocytes. Mechanistically, Sox100B regulates the expression of another critical stem cell differentiation factor, Sox21a. Supporting a direct control of Sox21a by Sox100B, a Sox21a intronic enhancer was identified that is active in all intestinal progenitors and directly regulated by Sox100B. Taken together, these results demonstrate that the activity and regulation of two Sox transcription factors are essential to coordinate stem cell differentiation and proliferation and maintain intestinal tissue homeostasis (Meng, 2020).

The proper maintenance of tissue homeostasis is essential for their normal architecture and physiological functions, especially in high-turnover tissues, such as intestinal epithelium. In most tissues, this is achieved by their resident stem cells, which are capable of self-renewing and differentiating into a variety of cell types within tissues. To answer the fundamental question of how tissue homeostasis is properly maintained, it is critical to identify the genetic networks that control stem cell proliferation and differentiation. Although proliferation has been extensively studied over the decades, mechanisms by which progressive and robust differentiation is achieved in vivo remain less understood in many lineages (Meng, 2020).

The adult Drosophila intestinal epithelium provides a genetically tractable experimental system to examine molecular mechanisms regulating stem cell activities. The adult midgut epithelium is actively maintained by multipotent intestinal stem cells (ISCs), which self-renew to maintain a stable stem cell population and give rise to post-mitotic progenitors committed to one of two distinct cell lineages: diploid secretary enteroendocrine cells (EEs) and polyploid absorptive enterocytes (ECs). In the EC lineage, ISCs turn on the Notch signaling in the daughter cells termed enteroblasts (EBs) that are committed to differentiation into the absorptive fate. EBs then go through several rounds of endo-replication and finally differentiate into Pdm1-positive ECs. To maintain the secretory lineage, ISCs give rise to Prospero-positive pre-EE daughter cells. A number of signaling pathways and transcription factors have been implicated in regulating ISC differentiation, including Delta/Notch, JAK/STAT92E, escargot, Sox21a. However, understanding of the transcriptional network involved in the control of EB differentiation remains incomplete (Meng, 2020).

Sox (Sry-related HMG Box) family transcription factors are important regulators of cell fate specification and cell differentiation during development and in multiple adult stem cell populations. Sox21a, a Drosophila Sox B gene, is specifically expressed in ISCs and EBs and plays important roles in regulating ISC proliferation and EB differentiation into EC, both at homeostasis and under stress conditions (Chen, 2016, Meng, 2015, Zhai, 2015, Zhai, 2017). However, how ISC- and EB-specific Sox21a expression pattern is established remains unknown. This study investigated the expression and function of another Sox family transcription factor, the Sox E factor Sox100B, and found that it is required for ISC differentiation into the EC lineage. Sox100B is shown to be required for both Sox21a protein expression and the activity of a transcriptional enhancer located in the first intron of the Sox21a gene. This identification of Sox100B binding sites in this intronic enhancer strongly supports the notion that Sox21a is a direct Sox100B target gene. The results identify an essential player in the transcriptional network that regulates the complex process of stem cell differentiation in the adult Drosophila intestine (Meng, 2020).

This work shows that the Sox E family transcription factor Sox100B is expressed in ISCs and EBs in the adult Drosophila intestine, consistent with a recent report using a GFP-tagged genomic BAC construct for Sox100B (Doupe, 2018). Recently, another Sox family transcription factor Sox21a has already been shown to be expressed specifically in ISCs and EBs. These two Sox transcription factors do not simply share an overlapping expression pattern, as demonstrated by this work, but Sox100B is required for proper Sox21a expression in both cell types. These data altogether have led to a proposal of a transcriptional cascade in which Sox21a is a Sox100B target gene. The overlapping expression pattern is not surprising, given the fact that Sox factors are commonly co-expressed in several tissues. For example, Drosophila B Group Sox factors SoxNeuro and Dichaete are co-expressed in part of the neuroectoderm during early development of the CNS. However, this study presents evidence showing a direct transcriptional regulation between different Sox factors in the somatic stem cell lineage in Drosophila (Meng, 2020).

Previous studies have shown that transcription factors, such as Stat92E and AP-1 factor Fos are involved in regulating Sox21a expression at basal and stress conditions (Chen, 2016, Meng, 2015, Zhai, 2015). At basal condition, Stat92E has been implicated in regulating Sox21a expression, and the second intron of Sox21a alone is sufficient to drive gene expression in ISCs and EBs (Zhai, 2015); however, whether this intronic enhancer is directly regulated by Stat92E has not been addressed. This study has identified that the first intron of Sox21a is also sufficient to direct endogenous Sox21a expression pattern. Interestingly, this intronic enhancer is not dependent on Stat92E, suggesting that parallel signal inputs from both Sox21a introns act together to robustly control the ISC/EB-specific Sox21a expression pattern. In support of this model, this study found that the first intronic enhancer is specifically responsive to Sox100B. This model is consistent with the observation that Sox21a expression is strongly reduced but not absent in Sox100B mutant clones, suggesting that parallel inputs mediated by the second intron via other factors, such as Stat92E contribute to Sox21a expression even in the absence of Sox100B. This model also partially accounts for the notion that depleting Sox100B does not inhibit ISC proliferation while depleting Sox21a strongly inhibits ISC proliferation, and it is likely that residual Sox21a expression in Sox100B loss-of-function conditions allows ISC proliferation. In response to stress, Sox21a expression is strongly induced which involves multiple stress-sensing signaling pathways and factors, such as JNK, EGFR, AP-1 factor Fos, and Stat92E. This study has shown that Sox100B is required for stress-, JNK-, and RasV12-induced Sox21a expression but that Sox100B overexpression in ISCs and EBs is not sufficient to induce Sox21a expression. The current data suggest a model where Sox100B provides cell-type specificity and allows Sox21a expression in intestinal progenitors, ISCs and EBs, while other pathways control Sox21a induction during the differentiation process or in response to stresses. This raises an interesting question of whether Sox100B directly interacts with stress-sensing and differentiation pathways to ensure that Sox21a expression is stress inducible and gradually increases during differentiation. As an example of such a mechanism, mammalian Sox9 has been shown to physically interact with AP-1 factors to co-activate target gene expression in developing chondrocytes. It is anticipated that the identification of Sox100B-interacting cofactors will provide a better view regarding the mechanism(s) by which Sox100B cooperates with stress-sensing signaling and other differentiation pathways to precisely adapt stem cell activities to tissue demands (Meng, 2020).

The last decade has witnessed significant advances in understanding the mechanisms by which ISC activities are regulated in response to infection, tissue injury, and during aging, with a strong focus on ISC proliferation. In contrast, a well-defined progressive differentiation process is still inadequately understood. Sox100B has been recently implicated in regulating acute gut regeneration in response to pathogenic bacteria Pseudomonas entomophila (Lan, 2018); however, the exact role of Sox100B in this process has not been characterized. This study found that Sox100B is functionally required for robust stem cell differentiation, as a strong reduction in the EC lineage and a reduction to a lesser extent in the EE lineage were observed in the Sox100B mutant clones. This study further showed that the expression of a critical differentiation regulator Sox21a is reduced in Sox100B mutants, establishing a transcriptional cascade during the ISC differentiation process. However, in preliminary experiments, Sox21a overexpression alone is not sufficient to rescue the differentiation defects of the Sox100B mutants, suggesting that other Sox100B targets, in addition to Sox21a, are required for proper differentiation. Further identification of Sox100B transcriptional target genes is needed to fully decipher the role of Sox100B during the differentiation process (Meng, 2020).

Interestingly, Sox9, the mammalian counterpart of Drosophila Sox100B, is expressed in ISCs and differentiated Paneth cells at the bottom of the crypts. In Sox9 knockout intestine, Paneth cells are found missing and crypt hyperplasia is widely observed. This study also observed a mild but significant increase of ISC proliferation in Sox100B loss-of-function conditions, which could be due to disruption of normal differentiation process, since it has been well documented that blocking differentiation process by genetically manipulating Notch and Sox21a causes strong pro-mitotic feedback regulation on ISC proliferation. In addition to Sox9, several other Sox family factors are expressed in the mammalian intestine, and their expression pattern and function remain elusive. The transcriptional regulatory pattern between different Sox factors in the intestine could be conserved from flies to mammals, and such possibility needs to be further examined (Meng, 2020).

The Drosophila ortholog of mammalian transcription factor Sox9 regulates intestinal homeostasis and regeneration at an appropriate level

Balanced stem cell self-renewal and differentiation is essential for maintaining tissue homeostasis, but the underlying mechanisms are poorly understood. This study identified the transcription factor SRY-related HMG-box (Sox) 100B, which is orthologous to mammalian Sox8/9/10, as a common target and central mediator of the EGFR/Ras and JAK/STAT signaling pathways that coordinates intestinal stem cell (ISC) proliferation and differentiation during both normal epithelial homeostasis and stress-induced intestinal repair in Drosophila. The two stress-responsive pathways directly regulate Sox100B transcription via two separate enhancers. Interestingly, an appropriate level of Sox100B is critical for its function, as its depletion inhibits ISC proliferation via cell cycle arrest, while its overexpression also inhibits ISC proliferation by directly suppressing EGFR expression and additionally promotes ISC differentiation by activating a differentiation-promoting regulatory circuitry composed of Sox100B, Sox21a, and Pdm1. Thus, this study reveals a Sox family transcription factor that functions as a stress-responsive signaling nexus that ultimately controls tissue homeostasis and regeneration (Jin, 2020).

Homeostatic renewal of many adult tissues requires balanced stem cell proliferation and differentiation, a process that is commonly compromised in cancer and in tissue degenerative diseases. The intestinal epithelium in adult Drosophila midgut provides a genetically tractable system for understanding the underlying mechanisms of tissue homeostasis and regeneration driven by resident stem cells. The intestinal stem cells (ISCs) of the Drosophila midgut normally divide to renew themselves and give rise to two different types of progenitor cells that respectively differentiate into enterocyte cells (ECs) and enteroendocrine cells (EEs). Normally, ISCs divide occasionally and thereby maintain the ongoing renewal of the epithelium, a slow process that takes approximately 2-4 weeks. However, upon damage or infection, ISCs are able to rapidly divide to facilitate accelerated epithelial repair in as fast as two days (Jin, 2020).

Extensive studies have implicated the JAK/STAT and the EGFR/Ras/mitogen-activated protein kinase (MAPK) as the two major signaling pathways that regulate ISC proliferation and differentiation during both normal epithelial homeostasis and stress-induced intestinal repair. The EGFR signaling is considered to play a predominant role in the regulation of ISC proliferation because it is required for the JAK/STAT signaling activation-induced ISC proliferation, whereas the JAK/STAT signaling is not essential for EGFR/Ras signaling activation-induced ISC proliferation. The EGFR signaling is also important for remodeling of the differentiated cells, including the exclusion of damaged/aged ECs and incorporation of new cells. The JAK/STAT pathway is also essential for ISC differentiation. ISCs with compromised JAK/STAT activity generate progenitor cells that are incapable of further differentiation. Despite the importance of the two signaling pathways in controlling intestinal homeostasis, their downstream targets-which integrate pathway activities to coordinate ISC proliferation and differentiation-remain elusive (Jin, 2020).

Sox (SRY-related HMG-box) family transcription factors (TFs) are known to have diverse roles in cell-fate specification and differentiation in multicellular organisms. In mouse-small intestine, Sox9, a SoxE subfamily member, is expressed in ISCs to regulate ISC proliferation and differentiation, but whether it acts as an oncogene or a tumor suppressor is still in debate. In Drosophila midgut, Sox21a, a SoxB2 subfamily member, is specifically expressed in ISCs and transient progenitor cells, and is essential for progenitor cell differentiation into mature cells. This study identified Sox100B, the Drosophila ortholog of Sox9, as a common downstream gene target for both the JAK/STAT and the EGFR signaling in regulating ISC proliferation and differentiation. This study also revealed that an appropriate level of Sox100B is critical for its function in regulating ISC proliferation, in that it may allow it to serve as an important mediator for a balanced process of ISC proliferation and differentiation, thereby maintaining intestinal homeostasis (Jin, 2020).

Although it has been well established that in the Drosophila midgut, the stress-responsive JAK/STAT signaling and EGFR/Ras/MAPK signaling are the two major signaling pathways that regulate ISC proliferation and differentiation, the downstream signaling targets that coordinate ISC proliferation and differentiation for intestinal regeneration are still yet to be identified. Sox100B identified in this study may represent such a key target. First, the expression of Sox100B is regulated by both JAK/STAT- and EGFR-signaling pathways. Normally Sox100B is expressed specifically in ISCs and EBs, where JAK/STAT- and EGFR/Ras/MAPK-pathway activities are high, and its expression is highly dependent on the activity of JAK/STAT- and EGFR/Ras/MAPK-signaling activities. Second, similar to the functions of JAK/STAT and EGFR signaling, Sox100B is critically required for both ISC proliferation and differentiation. The sustained EGFR/Ras/MAPK activity in EBs is important for the initiation of DNA endoreplication during the process of EC differentiation, and the sustained JAK/STAT signaling activity in EBs is essential for terminal differentiation toward both EC and EE lineage. Depletion of Sox100B causes ISC quiescence, similar to that caused by the disruption of EGFR signaling, as well as arrest of EB differentiation, similar to that caused by the disruption of JAK/STAT signaling. Third, an appropriate level of Sox100B expression appears to be critical for intestinal homeostasis. This effect by the expression level, as well as its responsiveness to JAK/STAT, EGFR, and potentially other stress-induced signaling activities (not shown), such as Wnt and Hippo signaling, may position Sox100B as a central mediator that coordinates ISC proliferation and differentiation during intestinal homeostasis and regeneration in Drosophila (Jin, 2020).

Sox100B is a Sox family group-E transcription factor, homolog of mammalian Sox8/9/10. In mouse small intestine, Sox9 is expressed in stem cells and progenitor cells at the base of crypts, and loss of Sox9 in the intestinal epithelium causes ISC hyperplasia and failure of Paneth cell differentiation. Interestingly, in the stem cell zone, Sox9 is expressed at low levels in ISCs and high levels in the quiescent or reserved stem cells that are also considered as the secretory progenitors. A possible explanation for these observations is that a low level of Sox9 sustains actively dividing ISCs, while an increase of SOX9 converts these proliferating ISCs into quiescent ISCs that will eventually differentiate into Paneth cells. Similarly, Sox9 is also implicated in regulating colorectal cancer cells, but there are conflicting data regarding whether Sox9 functions as an oncogene or a tumor suppressor. These seemingly contradictory results can be reconciled with a proposed model that Sox9 functions at an appropriate level, with a critical dose of Sox9 that exhibits proliferation-promoting activity, while increasing or decreasing this dose both result in proliferation-inhibitory activity. It is worthy to note that the differentiation-promoting function of Sox9 could potentially further complicate the interpretation of the mutant phenotype. It has been shown in Drosophila gut that defects in differentiation can induce a stressed microenvironment that promotes cell proliferation and propels tumor development (Jin, 2020).

The results of this study suggest many aspects of functional conservation of this Sox E subfamily gene in ISCs from Drosophila to mammals. Sox100B regulates both ISC proliferation and differentiation in the Drosophila intestine, and in terms of regulating ISC proliferation, Sox100B also requires an appropriate expression level. This study has demonstrated that this modulation of Sox100B expression is largely due to a negative feedback mechanism, in which increased Sox100B caused by elevated EGFR/Ras/MAPK signaling in turn suppresses the expression of EGFR, thereby leading to damped EGFR-signaling activity. Of note, contradictory data were recently reported on the roles of Sox100B and Sox21a in regulating ISC proliferation: both a proliferation-promoting role and a tumor-suppressive role for Sox21a in ISCs have been reported; as for the role of Sox100B, a previous study showed in an RNAi genetic screen that Sox100B is required for P.e.-induced ISC proliferation, whereas another study showed that depletion of Sox100B by RNAi causes increased ISC proliferation. Consideration of the effects caused by different levels of Sox100B expression that was observed in the present study may help resolve understanding of apparently disparate functions for these genes as central coordinators of both ISC proliferation and differentiation. It is proposed that, normally, a low level of Sox protein expression sustains ISC proliferation. A transient increase of Sox protein may not only promote cell cycle exit but also activate programs for terminal differentiation, thereby leading to a coordinated ISC proliferation and differentiation and, consequently, a coherent process of epithelial renewal (Jin, 2020).

This study demonstrates that Sox100B directly regulates Sox21a to promote differentiation. One important downstream target of Sox100B and Sox21a appears to be Pdm1, a known EC-fate-promoting factor. Interestingly, overexpression of Pdm1 in progenitor cells rapidly shuts down both Sox100B and Sox21a expression, indicating a negative feedback mechanism. Therefore, the induced Sox100B-Sox21a-Pdm1 axis in the differentiating ECs not only promotes cell differentiation, but also acts in a feedback mechanism to turn down EGFR and JAK/STAT signaling activities, thereby allowing ECs to terminally differentiate. This differentiation-promoting axis might also have a role in turning down ISC-specific programs, which are independently regulated by EGFR or JAK/STAT signaling pathways. For example, downregulation of the stem-cell-factor Esg is required for EB differentiation, and ectopic expression of Pdm1 is able to antagonize Esg expression in progenitor cells. These kinds of feedback regulation could be a common strategy used for initiation and finalization of a cell-differentiation program (Jin, 2020).

In summary, this study identified the transcription factor Sox100B as a major effector downstream of JAK/STAT and EGFR pathways that acts at an appropriate level to coordinate ISC proliferation and differentiation during both normal intestinal homeostasis and during damage- and infection-induced intestinal regeneration in Drosophila. With the 'just-right' effect endowed by a feedback mechanism, Sox100B behaves as a homeostatic sensor in the intestinal epithelium that coordinates stem cell proliferation with stem cell differentiation under various environmental conditions. It is proposed that this expressional and functional modulation associated with Sox family transcription factors may be a general mechanism for maintaining tissue homeostasis and regeneration in many organs, including those in mammals, and that deregulation of this mechanism may lead to tissue degeneration or cancer development (Jin, 2020).

Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila

Adult stem cells or residential progenitor cells are critical to maintain the structure and function of adult tissues (homeostasis) throughout the lifetime of an individual. Mis-regulation of stem cell proliferation and differentiation often leads to diseases including cancer, however, how wildtype adult stem cells and cancer cells respond to cellular damages remains unclear. This study found that in the adult Drosophila midgut, intestinal stem cells (ISCs), unlike tumor intestinal cells, are resistant to various cellular damages. Tumor intestinal cells, unlike wildtype ISCs, are easily eliminated by apoptosis. Further, their proliferation is inhibited upon autophagy induction, and autophagy-mediated tumor inhibition is independent of caspase-dependent apoptosis. Interestingly, inhibition of tumorigenesis by autophagy is likely through the sequestration and degradation of mitochondria, as compromising mitochondria activity in these tumor models mimics the induction of autophagy and increasing the production of mitochondria alleviates the tumor-suppression capacity of autophagy. Together, these data demonstrate that wildtype adult stem cells and tumor cells show dramatic differences in sensitivity to cellular damages, thus providing potential therapeutic implications targeting tumorigenesis (Ma, 2016).

Personalized Therapeutics Approach Using an In Silico Drosophila Patient Model Reveals Optimal Chemo- and Targeted Therapy Combinations for Colorectal Cancer

In silico models of biomolecular regulation in cancer, annotated with patient-specific gene expression data, can aid in the development of novel personalized cancer therapeutic strategies. Drosophila melanogaster is a well-established animal model that is increasingly being employed to evaluate such preclinical personalized cancer therapies. This study reports five Boolean network models of biomolecular regulation in cells lining the Drosophila midgut epithelium and annotate them with colorectal cancer patient-specific mutation data to develop an in silico Drosophila Patient Model (DPM). Cell-type-specific RNA-seq gene expression data from the FlyGut-seq database were employed to annotate and then validate these networks. Next, three literature-based colorectal cancer case studies were used to evaluate cell fate outcomes from the model. Results obtained from analyses of the proposed DPM help: (i) elucidate cell fate evolution in colorectal tumorigenesis, (ii) validate cytotoxicity of nine FDA-approved CRC drugs, and (iii) devise optimal personalized treatment combinations. The personalized network models helped identify synergistic combinations of paclitaxel-regorafenib, paclitaxel-bortezomib, docetaxel-bortezomib, and paclitaxel-imatinib for treating different colorectal cancer patients. Follow-on therapeutic screening of six colorectal cancer patients from cBioPortal using this drug combination demonstrated a 100% increase in apoptosis and a 100% decrease in proliferation. In conclusion, this work outlines a novel roadmap for decoding colorectal tumorigenesis along with the development of personalized combinatorial therapeutics for preclinical translational studies (Gondal, 2021)

Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells

Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). Loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1 (Korzelis, 2014).

Stem cell identity is controlled by both extrinsic cues from the niche and cell-intrinsic transcriptional programs. Thus far, most studies of the Drosophila midgut have focused on the niche-derived signals that control midgut stem cell self-renewal. This study demonstrates a cell-intrinsic role for the Snail family transcription factor, Escargot, in controlling ISC self-renewal and differentiation. Loss of Esg leads to a rapid loss of all stem/progenitor cells in the midgut, due to their differentiation, whereas Esg overexpression keeps these cells permanently in an undifferentiated state. The dramatic effects of manipulating Esg levels support a central role for this Snail family member in controlling stem cell identity in the fly intestine (Korzelis, 2014).

A transcriptomics analysis indicated that Esg acts as a transcriptional repressor of a large diverse set of differentiation genes. These targets include transcription factors specific to ECs and EEs (Pdm1, Prospero) and genes used in digestion, immunity and cytoarchitectural specialization. Interestingly, one of these transcription factors, Pdm1, plays an important role in EC differentiation: ectopic expression of Pdm1 in progenitor cells was sufficient to trigger EC differentiation, partially mimicking the esg loss of function phenotype. The rapid loss of the Esg-expressing cell population upon Pdm1 overexpression suggests that Pdm1 might repress Esg expression, perhaps directly. In this case, Esg and Pdm1 together would constitute a negative feedback switch that governs EC differentiation (Korzelis, 2014).

Expression analysis also raised the possibility that Esg activates progenitor cell-specific genes in ISCs and EBs. These include the EGF signaling components Cbl, spitz, argos and Egfr as well as the Jak/Stat receptor domeless. Both EGFR and Jak/STAT pathways are crucial for ISC growth and maintenance, and receptivity to these signals is downregulated in differentiated ECs and EEs. While Snail family members are best understood as repressors, the Esg paralog Snail has been reported to function as a context-dependent transcriptional activator (Rembold, 2014), suggesting that an activating role for Esg is also plausible. The function of Esg as either an activator or repressor is likely determined by co-factors and/or other transcription factors acting on the same promoters that are expressed in the ISC and EB population. In the Drosophila embryo, Snail cooperates with Twist at distinct promoters to activate EMT gene expression during mesoderm formation (Rembold, 2014). Snail2 can bind to Sox9 to activate expression from its own promoter during chick neural crest formation. In its role as a repressor, Esg binds the co-repressor CtBP to maintain somatic Cyst stem cells and hub cells in the Drosophila male testis. Future work to unravel the complete transcriptional network within which Esg functions to maintain the stem/progenitor state should prove to be very interesting (Korzelis, 2014).

The data support a model in which Esg acts in a circuit with Delta-Notch signaling to control the switch from stem/progenitor identity to differentiated cell identities. In its simplest form, this circuit might be a bistable switch in which Esg and Notch mutually inhibited each other, with Esg being 'on' and dominant in progenitor cells and Notch signaling 'on' and dominant in their differentiated progeny, the enterocytes. However, the constant presence of a substantial population of intermediate progenitor cells, the enteroblasts (EBs), which express both Esg and Notch reporter genes, indicates that a simple bistable switch is not an accurate conception. Indeed, EBs, defined here as cells positive for both Esg and the Notch reporter Su(H)GBE-LacZ, can persist for many days in the absence of ISC division. Thus, the EB transition state is metastable. In this transition state, Notch is apparently active, but secondary downstream targets that directly affect differentiation, such as Pdm1, brush border Myosin and smooth septate junction proteins, remain repressed. Since these genes are rapidly activated following depletion of Esg, it is suggested that their repression is most likely mediated by Esg binding (Korzelis, 2014).

Two potential explanations are provided for the longevity of the EB transition state. First, it is suggested that the repression of esg transcription by Notch is indirect and that this delays esg silencing. Silencing of Esg is not likely to be mediated by the Notch-regulated transcription factor Su(H) (a transcriptional activator) but by downstream repressors that act only after enterocyte or endocrine differentiation has begun. Pdm1 in ECs and Prospero in EEs are presently the most obvious candidates. Both are specifically induced coincident with Esg silencing, in ECs and EEs, respectively, and Dam-ID assays suggest that Pros has binding sites in the esg locus. The finding that overexpression of Pdm1 caused the rapid differentiation of Esg+ stem/progenitor cells supports the notion that Pdm1 could directly repress Esg expression to control EC differentiation. Furthermore, nubbin/Pdm1 was found to restrict expression of Notch target genes in the Drosophila larval wing disc. Hence, Pdm1 likely triggers EC differentiation by downregulating both Esg and the expression of Notch target genes in the EB. Therefore, Notch is only transiently active in EBs but fully off in mature ECs with high levels of Pdm1 (Korzelis, 2014).

While a delay circuit that controls the silencing of Esg is likely, theoretically it cannot explain how Esg+ EBs can persist for such long periods during times of low gut epithelial turnover and then rapidly differentiate during gut regeneration. Hence, it is speculated that a second input signal acts in combination with Notch-dependent factor(s) to silence Esg. This second signal is likely to be a downstream effector of the growth factor signaling network that also drives ISC division and gut epithelial renewal. Of the transcriptional effectors involved in maintaining gut homeostasis, the most obvious candidate as an indirect mediator of esg repression is Stat92E, which is activated by the highly stress-dependent cytokines, Upd2 and Upd3. Tellingly, the cytokine receptor, Dome, Janus Kinase (hop) and Stat92E are all required for EB maturation into ECs. If the silencing of esg was dependent upon both Notch and Stat92E, and Delta-Notch signaling was irreversible once resolved; then, the Notch+ Esg+ EB transition state should in principle be stable in conditions of low Jak/Stat signaling, as is observed during periods of midgut quiescence. It needs to be noted, however, that ISCs and EBs maintain appreciable levels of Stat-reporter gene expression even during relative quiescence, and so, in this model, it would be Stat activity above some threshold that would combine with Notch signaling to trigger differentiation. Since Jak/Stat signaling also triggers ISC division, a surge in cytokine signaling could coordinately trigger both the differentiation of older EBs and the production of new ones in this model , thus explaining how a significant EB population is maintained even as stem cell activity waxes and wanes (Korzelis, 2014).

Snail family transcription factors have been described as regulators of epithelial-to-mesenchyme transitions (EMT) that occur during development, wound healing and cancer metastasis. In some contexts, notably metastasis, EMT is believed to accompany the acquisition of stem-like properties. Although Esg itself has not been reported to regulate EMT, its paralog in flies (Sna) and homologs in mammals (Snai1, Snai2) do promote EMT. Interestingly, RNA-seq experiments showed that not only Esg, but Snail, Worniu and the Zeb family members Zfh1 and Zfh2 were all expressed in intestinal stem cells and downregulated in ECs and EEs. Thus, these EMT-linked transcription factors may work together to affect different aspects of midgut homeostasis and ISC differentiation. Indeed, Esg-positive ISCs and EBs are morphologically more similar to mesenchymal cells than they are epithelial, whereas Esg-negative EEs and ECs have the pronounced apical-basal polarity typical of epithelial cells. Esg+ cells often make striking lateral projections, suggestive of dynamic behavior, and they have the capacity to multilayer when their differentiation is blocked or they are forced to overproliferate. Furthermore, a number of epithelial-class genes are repressed in Esg+ progenitors and activated upon EC and/or EE differentiation. These include genes encoding the apico-lateral cortical Lgl-Dlg-Scrib-Crb complex, septate junction proteins (e.g., Ssk, Cora, Mesh) and polarity factors including Par3 and Par6. Strikingly, Scrib and Ssk both have Esg-binding sites in their promoters, and their expression is highly regulated by Esg. However, some gene targets that are central to EMT in mammalian cells show opposite trends in the fly's ISC lineage. For instance, Esg+ progenitors express significant levels of integrins, and E-cadherin-typically lost during EMT-is highly upregulated specifically in ISCs and EBs. Thus, the Esg-regulated differentiation of Drosophila ISCs only partially resembles a mesenchymal-to-epithelial transition (MET) (Korzelis, 2014).

Esg's role in ISC maintenance nicely parallels the functions of other Snail family members in Drosophila and mammals. For instance, in Drosophila neuroblasts (neural stem cells), the Snail family member Worniu promotes self-renewal and represses neuronal differentiation. In mice, Snail family members have been associated with the regulation of the stem cell state in both normal and pathological conditions. For instance, mammary stem cells require the Snail family member Slug to retain their MaSC identity. Mouse Snai1 also represses the transition from the stem cell-like mitotically cycling trophoblast precursor cell to the endoreplicating trophoblast giant cell during rodent placental development. This process, which also requires a mitotic-to-endocycle switch upon differentiation, is strikingly similar to the role describe in this study for Esg in EC differentiation and its role during imaginal disc development (Korzelis, 2014).

More interesting yet, mouse Snai1 is specifically expressed and required for stem cell maintenance in the crypts of the mouse intestine and expands the stem cell population when overexpressed. However, few studies highlight the target genes responsible for the function of Snail family members in stem cell maintenance. One example is from mouse muscle progenitors (myoblasts), where Snai1 and Snai2 repress expression from MyoD target promoters and this is required to maintain their progenitor state. The work presented in this study shows that Esg affects many aspects of the differentiation process and that it can form a transcriptional switch with one of the targets it represses (Pdm1) to balance self-renewal and differentiation in this stem cell lineage. Together, these studies suggest that the function of Snail family transcription factors as repressors of differentiation genes is ancient and widespread and may be an essential component in balancing self-renewal with differentiation in diverse animal stem cell lineages (Korzelis, 2014).

Regulation of Drosophila intestinal stem cell proliferation by enterocyte mitochondrial pyruvate metabolism

Multiple signaling pathways in the adult Drosophila enterocyte sense cellular damage or stress and signal to intestinal stem cells (ISCs) to undergo proliferation and differentiation, thereby maintaining intestinal homeostasis. This study shows that misregulation of mitochondrial pyruvate metabolism in enterocytes can stimulate ISC proliferation and differentiation. These studies focus on the Mitochondrial Pyruvate Carrier (MPC), which is an evolutionarily-conserved protein complex that resides in the inner mitochondrial membrane and transports cytoplasmic pyruvate into the mitochondrial matrix. Loss of MPC function in enterocytes induces Unpaired cytokine expression, which activates the JAK/STAT pathway in ISCs, promoting their proliferation. Upd3 and JNK signaling are required in enterocytes for ISC proliferation, indicating that this reflects a canonical non-cell autonomous damage response. Disruption of lactate dehydrogenase in enterocytes has no effect on ISC proliferation but it suppresses the proliferative response to a loss of enterocyte MPC function, suggesting that lactate contributes to this pathway. These studies define an important role for cellular pyruvate metabolism in differentiated enterocytes to maintain stem cell proliferation rates (Wisidagama, 2019).

The TORC1 inhibitor Nprl2 protects age-related digestive function in Drosophila

Aging and age-related diseases occur in almost all organisms. Recently, it was discovered that the inhibition of target of rapamycin complex 1 (TORC1), a conserved complex that mediates nutrient status and cell metabolism, can extend an individual's lifespan and inhibit age-related diseases in many model organisms. However, the mechanism whereby TORC1 affects aging remains elusive. This study used a loss-of-function mutation in nprl2, a component of GATOR1 that mediates amino acid levels and inhibits TORC1 activity, to investigate the effect of increased TORC1 activity on the occurrence of age-related digestive dysfunction in Drosophila. The nprl2 mutation decreased Drosophila lifespan. Furthermore, the nprl2 mutant had a distended crop, with food accumulation at an early age. Interestingly, the inappropriate food distribution and digestion along with decreased crop contraction in nprl2 mutant can be rescued by decreasing TORC1 activity. In addition, nprl2-mutant flies exhibited age-related phenotypes in the midgut, including short gut length, a high rate of intestinal stem cell proliferation, and metabolic dysfunction, which could be rescued by inhibiting TORC1 activity. These findings showed that the gastrointestinal tract aging process is accelerated in nprl2-mutant flies, owing to high TORC1 activity, which suggested that TORC1 promotes digestive tract senescence (Xi, 2019).

The septate junction protein Tsp2A restricts intestinal stem cell activity via endocytic regulation of aPKC and Hippo signaling

Hippo signaling and the activity of its transcriptional coactivator, Yorkie (Yki), are conserved and crucial regulators of tissue homeostasis. In the Drosophila midgut, after tissue damage, Yki activity increases to stimulate stem cell proliferation, but how Yki activity is turned off once the tissue is repaired is unknown. From an RNAi screen, the septate junction (SJ) protein tetraspanin 2A (Tsp2A) was identified as a tumor suppressor. Tsp2A undergoes internalization to facilitate the endocytic degradation of atypical protein kinase C (aPKC), a negative regulator of Hippo signaling. In the Drosophila midgut epithelium, adherens junctions (AJs) and SJs are prominent in intestinal stem cells or enteroblasts (ISCs or EBs) and enterocytes (ECs), respectively. When ISCs differentiate toward ECs, Tsp2A is produced, participates in SJ assembly, and turns off aPKC and Yki-JAK-Stat activity. Altogether, this study uncovers a mechanism allowing the midgut to restore Hippo signaling and restrict proliferation once tissue repair is accomplished (Xu, 2019a).

An in vivo RNAi screen uncovers the role of AdoR signaling and adenosine deaminase in controlling intestinal stem cell activity

Metabolites are increasingly appreciated for their roles as signaling molecules. To dissect the roles of metabolites, it is essential to understand their signaling pathways and their enzymatic regulations. From an RNA interference (RNAi) screen for regulators of intestinal stem cell (ISC) activity in the Drosophila midgut, this study identified adenosine receptor (AdoR) as a top candidate gene required for ISC proliferation. Ras/MAPK and Protein Kinase A (PKA) signaling act downstream of AdoR and Ras/MAPK mediates the major effect of AdoR on ISC proliferation. Extracellular adenosine, the ligand for AdoR, is a small metabolite that can be released by various cell types and degraded in the extracellular space by secreted adenosine deaminase. Interestingly, down-regulation of adenosine deaminase-related growth factor A (Adgf-A) from enterocytes is necessary for extracellular adenosine to activate AdoR and induce ISC overproliferation. As Adgf-A expression and its enzymatic activity decrease following tissue damage, this study provides important insights into how the enzymatic regulation of extracellular adenosine levels under tissue-damage conditions facilitates ISC proliferation (Xu, 2019b).

The Drosophila midgut epithelium consists of multipotent intestinal stem cell (ISCs), their immediate progenies known as enteroblasts (EBs, which are progenitor cells primed for differentiation), and differentiated cells including enterocytes (ECs, which is the major cell type in number), and enteroendocrine cells (EEs). ISCs/EBs can adjust their proliferation and differentiation activities by deploying conserved core pathways such as JAK/Stat, Notch, Ras/MAPK, JNK, and Hippo. The dynamic responses of adult ISCs/EBs to different regenerative demands under physiological or pathological conditions depend on the machineries to detect microenvironment cues and modulate the activity of aforementioned core pathways, which have not been investigated in vivo systematically (Xu, 2019b). To understand how ISCs/EBs sense their microenvironment, an RNAi screen was performed to identify receptor-coding genes that regulate ISC activity, among which Adenosine Receptor (AdoR) emerged as a top candidate required for ISC self-renewal and proliferation. Characterization of the AdoR-signaling pathway revealed the role of AdoR downstream pathways in regulating different aspects of ISC activity. Importantly, this study demonstrated that the mitogenic activity of the AdoR ligand, adenosine, is inhibited by adenosine deaminase-related growth factor A (Adgf-A) from ECs and that Adgf-A activity decreases following tissue damage. Altogether, this study demonstrates how an EC-derived metabolic enzyme modulates ISC activity by restricting extracellular adenosine (Xu, 2019b).

An RNAi screen was performed for regulators of ISC activity and AdoR was identified as a gene required for Ras/MAPK and PKA signaling in the ISCs/EBs. Characterization of AdoR and its ligand revealed that, in the healthy midgut, EC-derived Adgf-A limits the bioavailability of extracellular adenosine and restricts AdoR signaling in ISCs/EBs to a baseline level that supports ISC maintenance. However, the damaged midgut lacks sufficient levels of Adgf-A to restrict extracellular adenosine, thus allowing the activation of AdoR and its downstream pathways to stimulate the regenerative activity of ISCs (Xu, 2019b).

Purines not only are required for nucleic acid synthesis and the cellular energy supply, but also represent the most primitive and common extracellular chemical messengers. Extracellular adenosine acts on P1-type purinergic receptors, i.e., AdoRs. The effects of AdoR signaling on cell growth are context-dependent. For example, adenosine inhibits the growth of imaginal disk cells, and Adgf-A was initially identified as a growth factor that stimulates the proliferation of Drosophila imaginal disk and embryonic cells in vitro (Zurovec, 2002). In contrast, in both larval lymph gland (Mondal, 2011) and adult midgut, AdoR supports proliferation and differentiation in the stem/progenitor cells whereas AdgfA from a nonautonomous source suppresses AdoR activity. Despite the remarkably similar roles of AdoR in controlling behaviors of 2 different types of stem/progenitor cells, AdoR activation leads to hemapoietic progenitor exhaustion but ISC expansion. Furthermore, Ras/MAPK activity, rather than PKA (as in the hemapoietic progenitors), functions as a necessary and sufficient downstream component mediating AdoR-induced ISC overproliferation (Xu, 2019b).

Identification of AdoR as an ISC regulator led to a dissection of the function of its downstream pathways, i.e., PKA and Ras/MAPK. Although earlier studies reported that EC-like differentiation in Caco2 colorectal cancer cells correlates with PKA activation and that pharmacological induction of cAMP/PKA suppresses the migration of mammalian intestinal or colorectal cancer cells, this study implicates PKA signaling in controlling ISC behaviors in vivo. This study found that PKA activation in ISCs/EBs induces ISC-EC differentiation and EB membrane elongation, whereas PKA activation in ECs nonautonomously stimulates ISC proliferation. PKA regulates cytoskeletal organizing proteins such as Rac, Cdc42, Rho, and PAK. Interestingly, PKA antagonizes Rac to induce morphological changes in neurons. A similar mechanism might explain how PKA affects EB morphology (Xu, 2019b).

Ras/MAPK activity in the ISCs/EBs is responsive to a wide spectrum of inputs, including the EGFR pathway, the PDGF- and VEGF-receptor-related pathway, and cytosolic Ca2+ levels. This study confirmed AdoR as another upstream signal that can affect Ca2+ and Ras/MAPK activity. Since earlier studies suggested that GPCRs might affect intracellular Ca2+ levels, whereas high levels of cytosolic Ca2+ levels can induce Ras/MAPK activity in ISCs/EBs, it is likely that the detailed mechanism for AdoR to activate Ras/MAPK implicates the regulation of Ca2+ levels (Xu, 2019b).

Following AdoR activation, both Ras/MAPK and PKA signaling are induced to facilitate ISC overproliferation and accelerated production of ECs, whereas the perdurance of PKA activity in a massive number of newly produced ECs has a synergistic effect with Ras/MAPK activity in ISCs/EBs in accelerating proliferation. Since human AdoRs are often highly expressed in carcinomas, a similar paradigm of PKA and Ras/MAPK synergy might fuel oncogenic growth in epithelial tissues (Xu, 2019b).

Mammalian AdoRs and human ADA2 have been extensively studied in the hematopoietic and immune systems where ADA2 is produced by differentiating monocytes to stimulate T cell and macrophage proliferation (Hasco, 2008; Zavialov, 2010). Although mammalian AdoRs are expressed in human digestive epithelial cells, their functions remain elusive. Different groups have reported contradictory results suggesting either a protective or a pathological role of AdoR signaling during tissue damage in the mouse intestine, which could be due to the differences in mouse culture conditions, genetic backgrounds, damage models, or inflammation responses. Therefore, this study in Drosophila might help clarify the function of AdoR signaling in the digestive epithelium and in epithelial stem cells (Xu, 2019b).

In carcinomas, ADA2 is focally and frequently deleted, based on copy number analysis. Deleterious ADA2 mutations have been identified in colorectal cancers in The Cancer Genome Atlas (TCGA) and Catalogue of Somatic Mutations in Cancer projects. Moreover, ADA2 expression is significantly down-regulated in colorectal cancers, according to microarray studies and RNA-seq datasets from TCGA. Further, anti-ADA2 stainings were detected in the normal digestive epithelium but not in colorectal cancers. Therefore, the down-regulation of ADA2 in colorectal carcinomas has been observed at DNA, RNA, and protein levels. Unfortunately, ADA2 cannot be studied in a mouse model because of a rodent-specific gene loss event during evolution. Moreover, murine developmental and physiological programs have adapted to the loss of ADA2, as transgenic expression of human ADA2 in mice results in abnormal development and embryonic/neonatal lethality. Therefore, these findings describe a striking case in which flies are uniquely suited for understanding the function and regulation of an important disease-related gene (Xu, 2019b).

Enteroendocrine cells support intestinal stem-cell-mediated homeostasis in Drosophila

Intestinal stem cells in the adult Drosophila midgut are regulated by growth factors produced from the surrounding niche cells including enterocytes and visceral muscle. The role of the other major cell type, the secretory enteroendocrine cells, in regulating intestinal stem cells remains unclear. This study shows that newly eclosed scute loss-of-function mutant flies are completely devoid of enteroendocrine cells. These enteroendocrine cell-less flies have normal ingestion and fecundity but shorter lifespan. Moreover, in these newly eclosed mutant flies, the diet-stimulated midgut growth that depends on the insulin-like peptide 3 expression in the surrounding muscle is defective. The depletion of Tachykinin-producing enteroendocrine cells or knockdown of Tachykinin leads to a similar although less severe phenotype. These results establish that enteroendocrine cells serve as an important link between diet and visceral muscle expression of an insulin-like growth factor to stimulate intestinal stem cell proliferation and tissue growth (Amcheslavsky, 2014).

Previous evidence shows that adult midgut mutant clones that have all the AS-C genes deleted are defective in EE formation while overexpression of scute (sc) or asense (ase) is sufficient to increase EE formation. Moreover, the Notch pathway with a downstream requirement of ase also regulates EE differentiation. To study the requirement of EEs in midgut homeostasis, attempts were made to delete all EEs by knocking down each of the AS-C transcripts using the ISC/EB driver esg-Gal4. The results show that sc RNAi was the only one that caused the loss of all EEs in the adult midgut. The esg-Gal4 driver is expressed in both larval and adult midguts, but the esg > sc RNAi larvae were normal while the newly eclosed adults had no EEs. Therefore, sc is likely required for all EE formation during metamorphosis when the adult midgut epithelium is reformed from precursors/stem cells (Amcheslavsky, 2014).

The sc6/sc10-1 hemizygous mutant adults were also completely devoid of midgut EEs, while other hemizygous combinations including sc1, sc3B, and sc5 were normal in terms of EE number. Df(1)sc10-1 is a small deficiency that has both ac and sc uncovered. sc1 and sc3B each contain a gypsy insertion in far-upstream regions of sc, while sc5 and sc6 are 1.3 and 17.4 kb deletions, respectively, in the sc 3' regulatory region. The sc6/sc10-1 combination may affect sc expression during midgut metamorphosis and thus the formation of all adult EEs (Amcheslavsky, 2014).

The atonal homolog 1 (Atoh1) is required for all secretory cell differentiation in mouse. However, esg-Gal4-driven atona; (ato) RNAi and the amorphic combination ato1/Df(3R)p13 showed normal EE formation. Nonetheless, older ato1/Df(3R)p13 flies exhibited a significantly lower increase of EE number, suggesting a role of ato in EE differentiation in adult flies (Amcheslavsky, 2014).

In sc RNAi guts, the mRNA expression of allatostatin (Ast), allatostatin C (AstC), Tachykinin (Tk), diuretic hormone (DH31), and neuropeptide F (NPF) was almost abolished, consistent with the absence of all EEs. On the other hand, the mRNA expression of the same peptide genes in heads showed no significant change. Even though the EEs and regulatory peptides were absent from the midgut, the flies were viable and showed no apparent morphological defects. There was no significant difference in the number of eggs laid and the number of pupae formed from control and sc RNAi flies, suggesting that the flies probably have sufficient nutrient uptake to support the major physiological task of reproduction. However, when the longevity of these animals was examined, the EE-less flies after sc RNAi showed significantly shorter lifespan. In addition, when the number of EEs was increased in adult flies by esgGal4;tubGal80ts (esg^ts)-driven sc overexpression, an even shorter lifespan was observed. These results suggest that a balanced number of EEs is essential for the long-term health of the animal. Moreover, there may be important physiological changes in these EE-less flies that are yet to be uncovered, such as reduced intestinal growth described in detail below (Amcheslavsky, 2014).

One of the phenotypic changes found for the sc RNAi/EE-less flies was that under normal feeding conditions, their midguts had a significantly narrower diameter than that of control midguts. When reared in poor nutrition of 1% sucrose, both wild-type (WT) and EE-less flies had thinner midguts. When reared in normal food, WT flies had substantially bigger midgut diameter, while EE-less flies had grown significantly less. The cross-section area of enterocytes in the EE-less midguts was smaller, suggesting that there is also a growth defect at the individual cell level (Amcheslavsky, 2014).

A series of experiments showed that ingestion of food dye by the sc RNAi/EE-less flies was not lower than control flies. The measurement of food intake by optical density (OD) of gut dye contents also showed similar ingestion. The measurement of excretion by counting colored deposits and visual examination of dye clearing from guts showed that there was no significant change in food passage. The normal fecundity also suggested that the mutant flies likely had absorbed sufficient nutrient for reproduction. Nonetheless, another phenotype that was detected was a substantial reduction of intestinal digestive enzyme activities including trypsin, chymotrypsin, aminopeptidase, and acetate esterase. These enzyme activities exhibit strong reduction after starvation of WT flies. The EE-less flies therefore have a physiological response as if they experience starvation although they are provided with a normal diet (Amcheslavsky, 2014).

A previous report has established that newly eclosed flies respond to nutrient availability by increasing ISC division that leads to a jump start of intestinal growth. When newly eclosed flies were fed on the poor diet of 1% sucrose, both WT and sc RNAi/EE-less guts had a very low number of p-H3-positive cells, which represent mitotic ISCs because ISCs are the only dividing cells in the adult midgut. When fed on normal diet, the WT guts had significantly higher p-H3 counts, but the sc RNAi/EE-less guts were consistently lower at all the time points. The sc6/sc10-1 hemizygous mutant combination exhibited a similarly lower mitotic activity on the normal diet (Amcheslavsky, 2014).

When possible signaling defects were investigated in the EE-less flies,in addition to other gut peptide mRNAs, the level of Dilp3 mRNA was also found to be highly decreased in these guts while the head Dilp3 was normal. This is somewhat surprising, because Dilp3 is expressed not in the epithelium or EEs but in the surrounding muscle. Dilp3 promoter-Gal4-driven upstream activating sequence (UAS)-GFP expression (Dilp3 > GFP) was used to visualize the expression in muscle. Both control and sc RNAi under this driver showed normal muscle GFP expression, demonstrating that sc does not function within the smooth muscle to regulate Dilp3 expression. The esg-Gal4 and Dilp3-Gal4, and the control UAS-GFP samples showed the expected expression in both midgut precursors and surrounding muscles. When these combined Gal4 drivers were used to drive sc RNAi, the smooth muscle GFP signal was clearly reduced. These guts also exhibited no Prospero staining and overall fewer cells with small sizes as expected from esg > sc RNAi (Amcheslavsky, 2014).

A previous report showed an increase of Dilp3 expression from the surrounding muscle in newly eclosed flies under a well-fed diet. This muscle Dilp3 expression precedes brain expression and is essential for the initial nutrient stimulated intestinal growth. The EE-less flies show similar growth and Dilp3 expression defects, suggesting that EE is a link between nutrient sensing and Dilp3 expression during this early growth phase (Amcheslavsky, 2014).

WT and AS-C deletion (scB57) mutant clones in adult midguts did not exhibit a difference in their cell numbers. Moreover, esg^ts > sc RNAi in adult flies for 3 days but did not undergo a decrease of mitotic count or EE number. Together, these results suggest that sc is not required directly in ISC for proliferation, and they imply that the ISC division defects observed in the sc mutant/EE-less flies is likely due to the loss of EEs. To investigate this idea further, the esg^ts > system to was used to up- and downshift the expression of sc at various time points, and the correlation of sc expression, EE number, and ISC mitotic activity were measured. The overexpression of sc after shifting to 29°C for a few days correlated with increased EE number, expression of gut peptides, and increased ISC activity. Then, flies were downshifted back to room temperature to allow the Gal80ts repressor to function again. The sc mRNA expression was quickly reduced within 2 days and remained low for 4 days. Although there was no working antibody to check the Sc protein stability, the expression of a probable downstream gene phyllopod showed the same up- and downregulation, revealing that Sc function returned to normal after the temperature downshift. Meanwhile, the number of Pros+ cells and p-H3 count remained higher after the downshift. Therefore, the number of EEs, but not sc mRNA or function, correlates with ISC mitotic activity (Amcheslavsky, 2014).

Another experiment that was independent of sc expression or expression in ISCs was performed. The antiapoptotic protein p35 was driven by the pros-Gal4 driver, which is expressed in a subset of EEs in the middle and posterior midgut. This resulted in a significant albeit smaller increase in EE number and a concomitant increase in mitotic activity, which was counted only in the middle and posterior midgut due to some EC expression of this driver in the anterior region. Therefore, the different approaches show consistent correlation between EE number and ISC division (Amcheslavsky, 2014).

Dilp3 expression was significantly although modestly increased in flies that had increased EE number after sc overexpression, similar to that observed in fed versus fasted flies. Whether Dilp3 was functionally important in this EE-driven mitotic activity was tested. Flies were generated that contained a ubiquitous driver with temperature controlled expression, i.e., tub-Gal80ts/UAS-sc; tub-Gal4/UAS-Dilp3RNAi. These fly guts showed a significantly lower number of p-H3+ cells than that in the tub-Gal80ts/UAS-sc; tub-Gal4/+ control flies. These results demonstrate that the EE-regulated ISC division is partly dependent on Dilp3. The expression of an activated insulin receptor by esg-Gal4 could highly increase midgut proliferation, and this effect was dominant over the loss of EEs after scRNAi, which is consistent with an important function of insulin signaling in the midgut (Amcheslavsky, 2014).

Normally hatched flies did not lower their EE number after esg^ts > sc RNAi, perhaps due to redundant function with other basic-helix-loop-helix proteins in adults. The expression of proapoptotic proteins by the prosts-Gal4 also could not reduce the EE number. Thus other drivers were screened and a Tk promoter Gal4 (Tk-Gal4) was identifed that had expression recapitulating the Tk staining pattern representing a subset of EEs. More importantly, when used to express the proapoptotic protein Reaper (Rpr), this driver caused a significant reduction in the EE number, Tk and Dilp3 mRNA, and mitotic count. The Tk-Gal4-driven expression of another proapoptotic protein, Hid, caused a less efficient killing of EEs and subsequently no reduction of p-H3 count. The knockdown of Tk itself by Tk-Gal4 also caused significant reduction of p-H3 count. A previous report revealed the expression by antibody staining of a Tk receptor (TkR86C) in visceral muscles, and the knockdown of TkR86C in smooth muscle by Dilp3-Gal4 or Mef2-Gal4 showed a modest but significant decrease in ISC proliferation. There was a concomitant reduction of Dilp3 mRNA in guts of all these experiments, while the head Dilp3 mRNA had no significant change in all these experiments. As a comparison, TkR99D or NPFR RNAi did not show the same consistent defect (Amcheslavsky, 2014).

In conclusion, this study has shown that among the AS-C genes, sc is the one essential for the formation of all adult midgut EEs and is probably required during metamorphosis when the midgut is reformed. In newly eclosed flies, EEs serve as a link between diet-stimulated Dilp3 expression in the visceral muscle and ISC proliferation. Depletion of Tk-expressing EEs caused similar Dilp3 expression and ISC proliferation defects, although the defects appeared to be less severe than that in the sc RNAi/EE-less guts. The results together suggest that Tk-expressing EEs are part of the EE population required for this regulatory circuit. The approach reported in this study has established the Drosophila midgut as a model to dissect the function of EEs in intestinal homeostasis and whole-animal physiology (Amcheslavsky, 2014).

The cellular diversity and transcription factor code of Drosophila enteroendocrine cells

Enteroendocrine cells (EEs) in the intestinal epithelium have important endocrine functions, yet this cell lineage exhibits great local and regional variations that have hampered detailed characterization of EE subtypes. Through single-cell RNA-sequencing analysis, combined with a collection of peptide hormone and receptor knockin strains, this study provides a comprehensive analysis of cellular diversity, spatial distribution, and transcription factor (TF) code of EEs in adult Drosophila midgut. Ten major EE subtypes were identified that totally produced approximately 14 different classes of hormone peptides. Each EE on average co-produces approximately 2-5 different classes of hormone peptides. Functional screen with subtype-enriched TFs suggests a combinatorial TF code that controls EE cell diversity; class-specific TFs Mirr and Ptx1 respectively define two major classes of EEs, and regional TFs such as Esg, Drm, Exex, and Fer1 further define regional EE identity. These single-cell data should greatly facilitate Drosophila modeling of EE differentiation and function (Guo, 2019).

Apart from the function in food digestion and absorption, the gastrointestinal tract is also considered as the largest endocrine organ due to the resident enteroendocrine cells (EEs). In mice and humans, EEs are scattered throughout the intestinal epithelium and take up only 1% of total intestinal cells, yet they produce more than 20 types of hormones that regulate a diverse of physiological processes, such as appetite, metabolism, and gut motility. There are at least 12 major subtypes of EEs based on hormones that they produce, and due to their great regional and local cellular diversity, the complete characterization of EE specification and diversification still remains as a challenge (Guo, 2019).

The adult Drosophila midgut has become an attractive model system for the understanding of EE cell diversity and their regulatory mechanisms. The EEs are scattered along the epithelium of the entire midgut, including anterior midgut (regions R1 and R2), middle midgut (the gastric region, R3) and posterior midgut (regions R4 and R5). They have important roles in regulating local stem cell division and lipid metabolism, as well as feeding and mating behaviors. Approximately 10 peptide hormone genes are found to be expressed in EEs, yielding more than 20 different peptide hormones. Studies using RNA in situ hybridization, antibody staining, and gene reporter tools have provided a glimpse of regional EE diversity in terms of peptide hormones that they produce. However, due to limited availability of antibodies against all these hormones and a limit in the number of hormones that can be simultaneously analyzed, the detailed characterization of EE subtypes and peptide profiles is still lacking (Guo, 2019).

As in mammals, EEs in the fly midgut are derived from multipotent intestinal stem cells (ISCs). The initial fate determination between absorptive enterocyte versus secretory EEs is controlled by Notch signaling and appears to be regulated by the antagonistic activities of E(spl)-C genes and achaete-scute complex genes. This is also analogous to the antagonistic activities between Hes1 (orthologous to E(spl)) and Math1 (paralogous to AS-C) in mammalian ISCs that control the initial cell fate decision. The committed EE progenitor cell usually divides one more time to yield a pair of EEs. Interestingly, the two EEs within each pair produce distinct hormone peptides as a result of differentially acquired Notch activity, suggesting that, at least in the posterior midgut, differential Notch signaling defines two major subtypes of EEs. The specification and commitment of EE fate requires the homeodomain transcription factor (TF) Prospero (Pros), and the maturation of peptide hormones in EEs requires a Neuro D family bHLH TF Dimmed (Dimm). Besides these general TFs that promote EE specification and function, little is known regarding the TFs that participate in EE subtype specification and regional EE identity (Guo, 2019).

Single-cell RNA-sequencing (scRNA-seq) has emerged as an efficient tool for revealing cell heterozygosity in different tissues and organisms. By using scRNA-seq and a collection of recently generated peptide and receptor knockin lines, this study provides a comprehensive analysis of EE cell diversity, peptide profiling, and regional distribution along the entire length of the fly midgut at single-cell resolution. In addition, TF enrichment analysis followed by genetic screen allowed thew identification of class and region EE regulators. These results suggest a TF code composed of class-specific and region-specific TFs generates EE cell diversity (Guo, 2019).

Using single-cell transcriptomics in combination with a collection of reporter lines, this study has provided a comprehensive characterization of the EE population in the entire midgut of adult Drosophila. In addition to the two major classes of EEs that respectively produce TK and AstC peptide hormones, a third class of EEs was identified that reside only in the anterior midgut (R2) and produce sNPF and CCHa2. Ten EE subtypes were identified that generally show region-specific distributions. In addition, functional screens with subtype-specific TFs have revealed class- and region-specific TFs that regulate subtype specification. These single-cell data should serve as an important resource for further understanding the differentiation, regulation, and function of EEs using Drosophila midgut as a genetic model system (Guo, 2019).

The single-cell data reveal 14 classes of peptide hormone genes that are expressed in EEs, compared to the previously known 10 classes. The midgut expression patterns of all these peptide hormones, including several peptide hormones whose gut expression patterns have not been clearly defined, such as Gbp5, ITP, and Nplp2 as well as sNPF, are also determined. As EEs perform their endocrine function by secreting various peptide hormones, the types of peptide hormones that they produced are usually used to classify EE subtypes in mammals. Indeed, the exclusive expression pattern of Tk and AstC is sufficient to distinguish between class I EEs and class II EEs. However, although different EE subtypes show distinct peptide hormone expression profiles, the types of peptide hormone expressed and the EE subtypes are not strictly correlated. In fact, the peptide hormone co-expression patterns are highly variable among individual EEs, even for EEs that belong to the same cluster or subtype. For example, for the II-m (C4) subtype, although they commonly produce Tk and NPF, their expression for Mip, Nplp2, and CCAP is highly variable. The external stimuli, such as stress and microbiota, may have an impact on the expression status of these variable peptide hormone genes. Alternatively, EEs could be plastic and change their peptide hormone expression profiles with age. Recent studies demonstrate that the mammalian EEs are plastic and can switch their hormone profiles as they differentiate and migrate upward along the crypt-villus axis (Guo, 2019).

One major limit associated with the scRNA-seq technology is that the spatial information of the cells is lost during tissue dissociation. In a way to overcome this limit, this study has developed a RSGE algorithm based on the region- and cell-type-specific transcriptome database from flygut-seq. As confirmed, for the various peptide hormone markers, including GAL4 knockin lines and antibodies, this algorithm has allowed generation of a reliable distribution map a for all the EE subtype clusters along the length of the midgut. The determination of the spatial distribution of EE subtypes should greatly facilitate the understanding of their regulation and function. For instance, DH31 and ITP expressing EEs are found be located in the posterior-most region of the midgut, and their location is clearly consistent with their known function: DH31 is known to regulate fluid secretion in Malpighian tubules, and ITP is known to regulate ion transport in hindgut. As regional difference for a common cell type is likely a general phenomenon in diverse tissues of many organisms, the algorithm in this study could provide an example of possible approaches for acquiring the lost spatial information of cells when conducting this type of single-cell analysis (Guo, 2019).

By analyzing the TF code for the EE subtypes followed by functional screen, this study has identified a number of TFs that participate in the specification of EE subtypes, including the class-I- and class-II-specific TFs Mirr and Ptx1 for the two major classes of EEs and region-specific TFs such as Esg, Drm, Fer1, and Sug that define regional EE identity. Previous studies in the posterior midgut have revealed that class I and II EEs are specified by differential Notch signaling. In this study, cell-type specific manipulating of Notch activity allows the conclusion that Notch must function transiently at the progenitor stage, between the two immediate daughters of an EEP, to define the two classes of EEs. As Mirr and Ptx1 are expressed only in differentiated EEs, the sequential activity of Notch and Mirr/Ptx1 indicates that these two TFs act downstream of Notch to specify class I versus class II EE type. The regional diversity of EEs is then further specified by region-specific TFs and possibly impacted by other environmental factors. It is proposed that EE cellular diversity is generated by a combination of class-specific and region-specific TFs, with class-specific TFs regulated by Notch signaling and region-specific TFs determined by anterior-posterior body planning during early development. The local EE diversity could also be regulated by environmental changes and age-related cell plasticity, possibilities that remain to be explored in the future (Guo, 2019).

Collectively, these single-cell data have provided a comprehensive characterization of EE cell diversity and their peptide hormone expression profiles. The TF code analysis also provides insights into EE diversity mechanisms. This data should greatly facilitate functional annotations of EE subtypes and gut peptide hormones under diverse physiological and pathological conditions, such as mating, starvation, bacterial infection, and so on. The Perrimon lab recently conducted single-cell transcriptomes for all types of midgut cells using the inDrop method. As EEs only represent a small fraction of total cells analyzed, their analysis primarily focused on progenitor cells and enterocytes (Hung, 2018). Therefore, the data and their scRNA-seq data should serve as complementary resources for understanding Drosophila gut cells. An online searchable database has been established to facilitate the use of these single-cell data (Guo, 2019).

The sugar-responsive enteroendocrine neuropeptide F regulates lipid metabolism through glucagon-like and insulin-like hormones in Drosophila melanogaster

The enteroendocrine cell (EEC)-derived incretins play a pivotal role in regulating the secretion of glucagon and insulins in mammals. Although glucagon-like and insulin-like hormones have been found across animal phyla, incretin-like EEC-derived hormones have not yet been characterised in invertebrates. This study shows that the midgut-derived hormone, neuropeptide F (NPF), acts as the sugar-responsive, incretin-like hormone in the fruit fly, Drosophila melanogaster. Secreted NPF is received by NPF receptor in the corpora cardiaca and in insulin-producing cells. NPF-NPFR signalling resulted in the suppression of the glucagon-like hormone production and the enhancement of the insulin-like peptide secretion, eventually promoting lipid anabolism. Similar to the loss of incretin function in mammals, loss of midgut NPF led to significant metabolic dysfunction, accompanied by lipodystrophy, hyperphagia, and hypoglycaemia. These results suggest that enteroendocrine hormones regulate sugar-dependent metabolism through glucagon-like and insulin-like hormones not only in mammals but also in insects (Yoshinari, 2021).

Haemocytes control stem cell activity in the Drosophila intestine

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes were recruited to the intestine and secreted the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switched their response to DPP by inducing expression of Thickveins, a second type I receptor that had previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promoted infection resistance, but also contributed to the development of intestinal dysplasia in ageing flies. The study proposes that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. It is proposed that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

The results extend the current model for the control of epithelial regeneration in the wake of acute infections in the Drosophila intestine. It is proposed that the control of ISC proliferation by haemocyte-derived DPP integrates with the previously described regulation of ISC proliferation by local signals from the epithelium and the visceral muscle, allowing precise temporal control of ISC proliferation in response to tissue damage, inflammation and infection (Ayyaz, 2015).

The association of haemocytes with the intestine is extensive, and can be dynamically increased on infection or damage. In this respect, the current observations parallel the invasion of subepithelial layers of the vertebrate intestine by blood cells that induce proliferative responses of crypt stem cells during infection. A role for macrophages and myeloid cells in promoting tissue repair and regeneration has been described in adult salamanders and in mammals, where TGFβ ligands secreted by these immune cells can inhibit ISC proliferation, but can also contribute to tumour progression. The results provide a conceptual framework for immune cell/stem cell interactions in these contexts (Ayyaz, 2015).

The observation that DPP/SAX/SMOX signalling is required for UPD-induced proliferation of ISCs suggests that SAX/SMOX signalling cooperates with JAK/STAT and EGFR signalling in the induction of ISC proliferation. Accordingly, while constitutive activation of EGFR/RAS or JAK/STAT signalling in ISCs is sufficient to promote ISC proliferation cell autonomously, this study found that this partially depends on Smox. Even in these gain-of-function conditions, ISC proliferation can thus be fully induced only in the presence of basal SMOX activity. As short-term overexpression of DPP in haemocytes does not induce ISC proliferation, it is further proposed that DPP/SAX/SMOX signalling can activate ISCs only when JAK/STAT and/or EGFR signalling are activated in parallel. However, long-term overexpression of DPP in haemocytes results in increased ISC proliferation, suggesting that chronic activation of immune cells disrupts normal signalling mechanisms and results in ISC activation even in the absence of tissue damage (Ayyaz, 2015).

BMP TGFβ signalling pathways are critical for metazoan growth and development and have been well characterized in flies. Multiple ligands, receptors and transcription factors with highly context-dependent interactions and function have been described. This complexity is reflected by the sometimes conflicting studies exploring DPP/TKV/SAX signalling in the adult intestine. These studies consistently highlight two important aspects of BMP signalling in the adult Drosophila gut: ISCs can undergo opposite proliferative responses to BMP signals; and there are various sources of DPP that differentially influence ISC function in specific conditions. By characterizing the temporal regulation of BMP signalling activity in ISCs, the results resolve some of these conflicts: it is proposed that early in the regenerative response, haemocyte-derived DPP triggers ISC proliferation by activating SAX/SMOX signalling, and ISC quiescence is re-established by muscle-derived DPP as soon as TKV becomes expressed. Of note, some of the conflicting conclusions described in the literature may have originated from problems with the genetic tools used in some studies. This study have used two independent RNAi lines (BL25782 and BL33618) that effectively decrease dpp mRNA levels in haemocytes when expressed using HmlΔ::Gal4 (Ayyaz, 2015).

The close association of haemocytes with the type IV collagen Viking suggests that the stimulation of ISC proliferation by haemocyte-derived DPP may also be controlled at the level of ligand availability, as suggested previously for DPP from other sources. The regulation of SAX/SMOX signalling by DPP observed in this study is surprising, but consistent with earlier reports showing that SAX can respond to DPP in certain contexts. Biochemical studies have suggested that heterotetrameric complexes between the type II receptor PUNT and the type I receptors SAX and TKV can bind DPP, and complexes with TKV/TKV homodimers preferentially bind DPP, and complexes with SAX/SAX homodimers preferentially bind GBB. In the absence of TKV, SAX has been proposed to sequester GBB, shaping the GBB activity gradient, but to fail to signal effectively. Expression of GBB in the midgut epithelium has recently been described, and ligand heterodimers between GBB and DPP are well established. Consistent with earlier reports, this study found that GBB knockdown in ECs significantly reduces ISC proliferation in response to infection. Complex interactions between haemocyte-derived DPP, epithelial GBB, and ISC-expressed SAX, PUNT and TKV thus probably shape the response of ISCs to damage, and will be an interesting area of further study (Ayyaz, 2015).

Similar complexities exist in the regulation of transcription factors by SAX and TKV. Canonically, SMOX is regulated by Activin ligands (Activin, Dawdle, Myoglianin and maybe more), and the type I receptor Baboon. This study has tested the role of Activin and Dawdle in ISC regulation, and, in contrast to DPP, this study could not detect a requirement for these factors in the induction of ISC proliferation after Ecc15 infection. Furthermore, the data establish a requirement for haemocyte-derived DPP as well as for SAX expression in ISCs in the nuclear translocation of SMOX after a challenge. This study thus indicates that in this context, SAX responds to DPP and regulates SMOX. Regulation of SMOX by SAX has been described before, yet SAX is also known to promote MAD phosphorylation, but only in the presence of TKV. Consistent with such observations, this study has detected MAD phosphorylation in ISCs only in the late recovery phase on bacterial infection, when TKV is simultaneously induced in ISCs. During this recovery phase, ISCs maintain high SAX expression, but SMOX nuclear localization is not detected anymore, suggesting that SAX cannot activate SMOX in the presence of TKV, and might actually divert signals towards MAD instead. The data also suggest that Medea (the Drosophila SMAD4 homologue) is not required for SMOX activity. Although surprising, this observation is consistent with recent reports that SMAD proteins in mammals can translocate into the nucleus and activate target genes in a SMAD4-independent manner. The specific signalling readouts in ISCs when these cells are exposed to various BMP ligands and are expressing different combinations of receptors are thus likely to be complex (Ayyaz, 2015).

The current findings demonstrate that the control of ISC proliferation by haemocyte-derived DPP is critical for tolerance against enteropathogens, but contributes to ageing-associated epithelial dysfunction, highlighting the importance of tightly controlled interactions between blood cells and stem cells in this tissue. Nevertheless, where haemocytes themselves are required for normal lifespan, loss of haemocyte-derived DPP does not impact lifespan. One interpretation of this finding is that beneficial (improved gut homeostasis) and deleterious (for example, reduced immune competence of the gut epithelium) consequences of reduced haemocyte-derived DPP cancel each other out over the lifespan of the animal. It will be interesting to test this hypothesis in future studies. Ageing is associated with systemic inflammation, and a role for immune cells in promoting inflammation in ageing vertebrates has been proposed. In humans, recruitment of immune cells to the gut is required for proper stem cell proliferation in response to luminal microbes, and prolonged inflammatory bowel disease further contributes to cancer development. It is thus anticipated that conserved macrophage/stem cell interactions influence the aetiology and progression of such diseases. The data confirm a role for haemocytes in age-related intestinal dysplasia in the fly intestine, and provide mechanistic insight into the causes for this deregulation. It can be anticipated that similar interactions between macrophages and intestinal stem cells may contribute to the development of IBDs, intestinal cancers, and general loss of homeostasis in the ageing human intestine (Ayyaz, 2015).

Drosophila Pez acts in Hippo signaling to restrict intestinal stem cell proliferation

The conserved Hippo signaling pathway acts in growth control and is fundamental to animal development and oncogenesis. Hippo signaling has also been implicated in adult midgut homeostasis in Drosophila. Regulated divisions of intestinal stem cells (ISCs), giving rise to an ISC and an enteroblast (EB) that differentiates into an enterocyte (EC) or an enteroendocrine (EE) cell, enable rapid tissue turnover in response to intestinal stress. The damage-related increase in ISC proliferation requires deactivation of the Hippo pathway and consequential activation of the transcriptional coactivator Yorkie (Yki) in both ECs and ISCs. This study identified Pez, an evolutionarily conserved FERM domain protein containing a protein tyrosine phosphatase (PTP) domain, as a novel binding partner of the upstream Hippo signaling component Kibra. Pez function (but not its PTP domain) is essential for Hippo pathway activity specifically in the fly midgut epithelium. Thus, Pez displays a tissue-specific requirement and functions as a negative upstream regulator of Yki in the regulation of ISC proliferation (Poernbacher, 2012).

The WW domain protein Kibra has recently been shown to function as a tumor suppressor in the Hippo pathway. Because Kibra is an adaptor molecule, attempts were made to identify physical binding partners of Kibra to further explore upstream Hippo signaling. Affinity purification-mass spectrometry (AP-MS) analysis with Kibra as bait identified Pez as a novel interaction partner of Kibra in Drosophila cultured cells. The same result was recently obtained in a large-scale proteomic study of Drosophila cultured cells. The binding between Pez and Kibra was confirmed by reciprocal coimmunoprecipitation (co-IP) experiments with epitope-tagged proteins. Furthermore, a yeast two-hybrid (Y2H) experiment revealed that the Kibra-Pez interaction is robust and direct (Poernbacher, 2012).

To address a possible function of Pez in the Hippo pathway, two loss-of-function alleles of Pez that were generated by different methods. Pez¹ is an EMS-induced allele resulting in an early premature translational stop codon. Pez² was generated by imprecise excision of the P element P{GawB}NP4748, removing most of the Pez coding sequence. Homozygotes for either Pez allele as well as heteroallelic Pez¹/Pez² flies are viable but smaller than controls. Combinations of the Pez alleles with the deficiency Df(2L)ED384 uncovering the Pez locus are also viable and cause a similar reduction in body size as the homozygous or heteroallelic combinations. One copy of a GFP-tagged Pez genomic rescue construct (gPez) restores normal body size. Therefore, both Pez¹ and Pez² are likely to represent strong or null alleles. For further experiments, heteroallelic Pez¹/Pez² flies were used as Pez mutant flies (Poernbacher, 2012).

In addition to their reduced body size, Pez mutant flies exhibit a developmental delay of 2 days and decreased fertility, all hallmarks of starvation. Pez mutant larvae are small and have decreased triglyceride (TAG) stores and increased expression of the starvation marker genes lipase-3 and 4E-BP. Clones of Pez mutant cells in larval fat bodies did not affect lipid droplets, thus excluding a fat body-autonomous requirement for Pez in lipid metabolism. Surprisingly, overexpression of Drosophila Pez in the developing eye or wing decreased the size of the adult organs, indicating that Pez restricts growth rather than promoting it. It is proposed that the starvation-like phenotype of Pez mutants is due to indirect effects on metabolism arising from a failure in nutrient utilization. Clones of Pez mutant cells in wing imaginal discs did not show growth defects in comparison to their corresponding wild-type sister clones. However, Pez mutant flies exhibit hyperplasia and extensive multilayering of the adult midgut epithelium. One copy of gPez restores normal tissue architecture. The structure of the larval midgut epithelium, as well as that of the other larval and adult epithelia, is not disturbed in Pez mutants. Thus, Pez specifically functions to restrict growth of the adult midgut epithelium (Poernbacher, 2012).

The Pez protein contains two conserved structural elements: an amino-terminal FERM domain (band 4.1-ezrin-radixin- moesin family of adhesion molecules) and a carboxyterminal protein tyrosine phosphatase (PTP) domain. A truncated version of the protein lacking the FERM domain (DFERM-Pez) or a phosphatase-dead protein (PezPD) still rescued the Pez mutant gut phenotype when overexpressed in ECs. However, overexpression of DFERM-Pez in the developing wing failed to decrease wing size, whereas overexpression of PezPD or of a truncated protein lacking the PTP domain (DPTP-Pez) caused a similar phenotype as overexpression of wild-type Pez, suggesting that the FERM domain is required for the growth-regulatory function of endogenous Pez but becomes dispensable when DFERM-Pez is overexpressed in ECs. In contrast, the potential phosphatase activity of Pez is clearly not needed for its function in growth control (Poernbacher, 2012).

Two other FERM domain proteins, Merlin (Mer) and Expanded (Ex), act in upstream Hippo signaling to control organ size in Drosophila. Together with the WW domain protein Kibra, Ex and Mer constitute the KEM complex that assembles at the apical junction of epithelial cells and regulates the core Hippo pathway kinase cassette. Overexpression of Kibra, Ex, or Mer in ECs of Pez mutant flies significantly suppressed the Pez gut phenotypes. Thus, Pez is not an essential mediator of Hippo signaling downstream of the KEM complex. Mer and Ex did not detectably coimmunoprecipitate with Pez in Drosophila S2 cells. However, Kibra and Pez coimmunoprecipitated and colocalized in S2 cells. This was dependent on the first WW domain of Kibra, whereas the FERM and PTP domains of Pez as well as two potential ligands of WW domains, a PPPY motif and a PPSGY motif, in the central linker region of Pez were dispensable. A fragment encompassing a proline-rich stretch of Pez (amino acids 368-627; PezPro) was sufficient for the binding to Kibra, whereas the remaining linker region (amino acids 622-967; PezLink) did not bind Kibra. Importantly, knockdown of Kibra via Myo1A-Gal4 caused mild overgrowth of the adult midgut epithelium, and overexpressed Kibra recruited gPez-GFP from the cell cortex of ECs into cytoplasmic punctae. The subcellular localizations of overexpressed Kibra, Ex, or Mer were not affected when Pez was absent (Poernbacher, 2012).

It is concluded that Pez and Kibra function together in a protein complex to regulate Hippo signaling in adult midgut ECs. The results establish that the Drosophila Pez protein acts as a component of upstream Hippo signaling, restricts transcriptional activity of Yki in epithelial cells of the adult midgut, and plays a crucial role in the control of ISC proliferation. Importantly, the involvement of Hippo signaling in intestinal regeneration is conserved in the mammalian system ] (Poernbacher, 2012).

The two mammalian homologs of Drosophila Pez are the widely expressed, cytosolic nonreceptor tyrosine phosphatases PTPD1/PTPN21 and PTPD2/PTP36/PTPN14/Pez. All three proteins share a similar domain structure including the well-conserved terminal FERM and PTP domains. The central region shows extensive sequence divergence but it contains several shorter regions of conservation that may function as adaptors in signal transduction. PTPD1 is a component of a cortical scaffold complex nucleated by focal adhesion kinase (FAK) and thus regulates a proliferative signaling pathway through a scaffolding function. PTPD2 has been implicated in the regulation of cell adhesion, as an inducer of TGF-β signaling, and in lymphatic development of mammals and choanal development of humans. Interestingly, PTPD2 is a potential tumor suppressor, based on sporadic mutations in breast cancer cells and colorectal cancer cells. It is tempting to speculate that mammalian PTPD2 shares the function of its fly homolog as a component of Hippo signaling that restrains the oncogenic potential of gut regeneration (Poernbacher, 2012).

Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies

The Drosophila Indy (I'm not dead yet) gene encodes a plasma membrane transporter of Krebs cycle intermediates, with robust expression in tissues associated with metabolism. Reduced INDY alters metabolism and extends longevity in a manner similar to caloric restriction (CR); however, little is known about the tissue specific physiological effects of INDY reduction. This study focused on the effects of INDY reduction in the Drosophila midgut due to the importance of intestinal tissue homeostasis in healthy aging and longevity. The expression of Indy mRNA in the midgut changes in response to aging and nutrition. Genetic reduction of Indy expression increases midgut expression of the mitochondrial regulator spargel/dPGC-1, which is accompanied by increased mitochondrial biogenesis and reduced reactive oxygen species (ROS). These physiological changes in the Indy mutant midgut preserve intestinal stem cell (ISC) homeostasis and are associated with healthy aging. Genetic studies confirm that dPGC-1 mediates the regulatory effects of INDY, as illustrated by lack of longevity extension and ISC homeostasis in flies with mutations in both Indy and dPGC1. These data suggest INDY may be a physiological regulator that modulates intermediary metabolism in response to changes in nutrient availability and organismal needs by modulating dPGC-1 (Rogers, 2014).

Caloric restriction (CR) extends lifespan in nearly all species and promotes organismal energy balance by affecting intermediary metabolism and mitochondrial biogenesis. Interventions that alter intermediary metabolism are though to extend longevity by preserving the balance between energy production and free radical production Indy (I'm Not Dead Yet) encodes a plasma membrane protein that transports Krebs' cycle intermediates across tissues associated with intermediary metabolism. Reduced Indy-mediated transport extends longevity in worms and flies by decreasing the uptake and utilization of nutrients and altering intermediate nutrient metabolism in a manner similar to CR. Furthermore, it was shown that caloric content of food directly affects Indy expression in fly heads and thoraces, suggesting a direct relationship between INDY and metabolism (Rogers, 2014 and references therein).

dPGC-1/spargel is the Drosophila homolog of mammalian PGC-1, a transcriptional co-activator that promotes mitochondrial biogenesis by increasing the expression of genes encoding mitochondrial proteins. Upregulation of dPGC-1 is a hallmark of CR-mediated longevity and is thought to represent a response mechanism to compensate for energetic deficits caused by limited nutrient availability. Increases in dPGC-1 preserve mitochondrial functional efficiency without consequential changes in ROS. Previous analyses of Indy mutant flies revealed upregulation of mitochondrial biogenesis mediated by increased levels of dPGC-1 in heads and thoraces (Rogers, 2014 and references therein).

Recently, dPGC-1 upregulation in stem and progenitor cells of the digestive tract was shown to preserve intestinal stem cell (ISC) proliferative homeostasis and extend lifespan. The Drosophila midgut is regenerated by multipotent ISCs, which replace damaged epithelial tissue in response to injury, infection or changes in redox environment. Low levels of reactive oxygen species (ROS) maintain stemness, self-renewal and multipotency in ISCs; whereas, age-associated ROS accumulation induces continuous activation marked by ISC hyper-proliferation and loss of intestinal integrity (Rogers, 2014 and references therein).

This study describes a role for Indy as a physiological regulator that modulates expression in response to changes in nutrient availability. This is illustrated by altered Indy expression in flies following changes in caloric content and at later ages suggesting that INDY-mediated transport is adjusted in an effort to meet energetic demands. Further, role was characterized for dPGC-1 in mediating the downstream regulatory effects of INDY reduction, such as the observed changes in Indy mutant mitochondrial physiology, oxidative stress resistance and reduction of ROS levels. Longevity studies support a role for dPGC-1 as a downstream effector of Indy mutations as shown by overlapping longevity pathways and absence of lifespan extension without wild-type levels of dPGC-1. These findings show that Indy mutations affect intermediary metabolism to preserve energy balance in response to altered nutrient availability, which by affecting the redox environment of the midgut promotes healthy aging (Rogers, 2014).

Reduction of Indy gene activity in fruit flies, and homologs in worms, extends lifespan by altering energy metabolism in a manner similar to caloric restriction (CR). Indy mutant flies on regular food share many characteristics with CR flies and do not have further longevity extension when aged on a CR diet. Furthermore, mINDY-/- mice on regular chow share 80% of the transcriptional changes observed in CR mice, supporting a conserved role for INDY in metabolic regulation that mimics CR and promotes healthy aging. This study shifted from systemic to the tissue specific effects of INDY reduction, focusing on the midgut due to the high levels of INDY protein expression in wild type flies and the importance of regulated intestinal homeostasis during aging. The evidence supports a role for INDY as a physiological regulator that senses changes in nutrient availability and alters mitochondrial physiology to sustain tissue-specific energetic requirements (Rogers, 2014).

The age-associated increase in midgut Indy mRNA levels that can be replicated by manipulations that accelerate aging such as increasing the caloric content of food or exposing flies to paraquat. Conversely, it was also shown that CR decreases Indy mRNA in control midgut tissues, which is consistent with previous findings in fly muscle and mouse liver. Diet-induced variation in midgut Indy expression suggests that INDY regulates intermediary metabolism by modifying citrate transport to meet tissue or cell-specific bioenergetic needs. Specifically, as a plasma membrane transporter INDY can regulate cytoplasmic citrate, thereby affecting fat metabolism, respiration, and via conversion to malate, the TCA cycle. Reduced INDY-mediated transport activity in the midgut could prevent age-related ISC-hyperproliferation by decreasing the available energy needed to initiate proliferation, thereby preserving tissue function during aging. This is supported by findings that nutrient availability affects ISC proliferation in adult flies and that CR can affect stem cell quiescence and activation (Rogers, 2014).

One of the hallmarks of CR-mediated longevity extension is increased mitochondrial biogenesis mediated by dPGC-1 (Spargel). Increased dPGC-1 levels and mitochondrial biogenesis have been described in the muscle of Indy mutant flies, the liver of mIndy-/- mice, and this study describes it in the midgut of Indy mutant flies. One possible mechanism for these effects can be attributed to the physiological effects of reduced INDY transport activity. Reduced INDY-mediated transport activity could lead to reduced mitochondrial substrates, an increase in the ADP/ATP ratio, activation of AMPK, and dPGC-1 synthesis. This is consistent with findings in CR flies and the livers of mINDY-/- mice. This study's analysis of mitochondrial physiology in the Indy mutant midgut shows upregulation of respiratory proteins, maintenance of mitochondrial potential and increased mitochondrial biogenesis, all of which are signs of enhanced mitochondrial health. The observed increase in dPGC-1 levels in Indy mutant midgut therefore appears to promote mitochondrial biogenesis and functional efficiency, representing a protective mechanism activated in response to reduced energy availability (Rogers, 2014).

Genetic interventions that conserve mitochondrial energetic capacity have been shown to maintain a favorable redox state and regenerative tissue homeostasis. This is particularly beneficial in the fly midgut, which facilitates nutrient uptake, waste removal and response to bacterial infection. Indy mutant flies have striking increases in the steady-state expression of the GstE1 and GstD5 ROS detoxification genes. As a result, any increase in ROS levels, whether from mitochondrial demise or exposure to external ROS sources can be readily metabolized to prevent accumulation of oxidative damage. Such conditions not only promote oxidative stress resistance, but also preserve ISC homeostasis as demonstrated by consistent proliferation rates throughout Indy mutant lifespan and preserved intestinal architecture in aged Indy mutant midguts. Thus, enhanced ROS detoxification mechanisms induced by Indy reduction and subsequent elevation of dPGC-1 contributes to preservation of ISC functional efficiency, and may be a contributing factor to the long-lived phenotype of Indy mutant flies (Rogers, 2014).

Several lines of evidence indicate that INDY and dPGC-1 are part of the same regulatory network in the midgut, in which dPGC-1 functions as a downstream effector of INDY. The similarity between dPGC-1 mRNA levels and survivorship of flies overexpressing dPGC-1 in esg-positive cells and Indy mutant flies suggests that Indy and dPGC-1 interact to extend lifespan. This is further supported by the lack of additional longevity extension when dPGC-1 is overexpressed in esg-positive cells of Indy mutant flies. Moreover, hypomorphic dPGC-1 flies in an Indy mutant background are similar to controls with respect to life span, declines in mitochondrial activity and ROS-detoxification. Together, these data suggest that dPGC-1 must be present to mediate the downstream physiological benefits and lifespan extension of Indy mutant flies (Rogers, 2014).

There are some physiological differences between the effects of Indy mutation and dPGC-1 overexpression in esg-positive cells. While Indy mutant flies are less resistant to starvation and more resistant to paraquat, a recent report showed that overexpressing dPGC-1 in esg-positive cells has no effect on resistance to starvation or oxidative stress. Additionally, mice lacking skeletal muscle PGC-1α were found to lack mitochondrial changes associated with CR but still showed other CR-mediated metabolic changes. In the fly INDY is predominantly expressed in the midgut, fat body and oenocytes, though there is also low level expression in the malpighian tubules, salivary glands, antenae, heart and female follicle cell membranes. Thus, the effects of INDY on intermediary metabolism and longevity could be partially independent from dPGC-1 or related to changes in tissues other than the midgut (Rogers, 2014).

This study suggests that INDY may function as a physiological regulator of mitochondrial function and related metabolic pathways, by modulating nutrient flux in response to nutrient availability and energetic demands. Given the localization of INDY in metabolic tissues, and importance of regulated tissue homeostasis during aging, these studies highlight INDY as a potential target to improved health and longevity. Reduced Indy expression causes similar physiological changes in flies, worms and mice indicating its regulatory role would be conserved. Further work should examine the interplay between Indy mutation and metabolic pathways, such as insulin signaling, which have been shown to promote stem cell maintenance and healthy aging in flies and mice. In doing so, the molecular mechanisms, which underlie Indy mutant longevity may provide insight for anti-aging therapies (Rogers, 2014).

Mitochondrial dynamics regulates Drosophila intestinal stem cell differentiation

Differentiation of stem/progenitor cells is associated with a substantial increase in mitochondrial mass and complexity. Mitochondrial dynamics, including the processes of fusion and fission, plays an important role for somatic cell reprogramming and pluripotency maintenance in induced pluripotent cells (iPSCs). However, the role of mitochondrial dynamics during stem/progenitor cell differentiation in vivo remains elusive. This study found differentiation of Drosophila intestinal stem cell is accompanied with continuous mitochondrial fusion. Mitochondrial fusion defective (opa1RNAi) ISCs contain less mitochondrial membrane potential, reduced ATP, and increased ROS level. Surprisingly, suppressing fusion also resulted in the failure of progenitor cells to differentiate. Cells did not switch on the expression of differentiation markers, and instead continued to show characteristics of progenitor cells. Meanwhile, proliferation or apoptosis was unaffected. The differentiation defect could be rescued by concomitant inhibition of Drp1, a mitochondrial fission molecule. Moreover, ROS scavenger also partially rescues opa1RNAi-associated differentiation defects via down-regulating JNK activity. It is proposed that mitochondrial fusion plays a pivotal role in controlling the developmental switch of stem cell fate (Deng, 2018).

Origin and dynamic lineage characteristics of the developing Drosophila midgut stem cells

Proliferating intestinal stem cells (ISCs) generate all cell types of the Drosophila midgut, including enterocytes, endocrine cells, and gland cells (e.g., copper cells), throughout the lifetime of the animal. Among the signaling mechanisms controlling the balance between ISC self-renewal and the production of different cell types, Notch (N) plays a pivotal role. This study investigates the emergence of ISCs during metamorphosis and the role of N in this process. Precursors of the Drosophila adult intestinal stem cells (pISCs) can be first detected within the pupal midgut during the first hours after onset of metamorphosis as motile mesenchymal cells. pISCs perform 2-3 rounds of parasynchronous divisions. The first mitosis yields only an increase in pISC number. During the following rounds of mitosis, dividing pISCs give rise to more pISCs, as well as the endocrine cells that populate the midgut of the eclosing fly. Enterocytes do not appear among the pISC progeny until around the time of eclosion. The "proendocrine" gene prospero (pros), expressed from mid-pupal stages onward in pISCs, is responsible to advance the endocrine fate in these cells; following removal of pros, pISCs continue to proliferate, but endocrine cells do not form. Conversely, the onset of N activity that occurs around the stage when pros comes on restricts pros expression among pISCs. Loss of N abrogates proliferation and switches on an endocrine fate among all pISCs. These results suggest that a switch depending on the activity of N and pros acts at the level of the pISC to decide between continued proliferation and endocrine differentiation (Takashima, 2016).

Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila

UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. This study shows that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. These data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans (Nagy, 2016).

UVRAG encodes a homolog of yeast Vps38 in metazoans. UVRAG/Vps38 and Atg14 are mutually exclusive subunits of two different Vps34 lipid kinase complexes, both of which contain Vps34, Vps15 and Atg6/Beclin 1. It is well established that Vps38 is required for endosome maturation and vacuolar and lysosomal protein sorting, whereas Atg14 is specific for autophagy in yeast. However, the function of UVRAG is much more controversial in mammalian cells. Although UVRAG was originally found to have dual roles in autophagy through promotion of autophagosome formation and fusion with lysosomes in various cultured cell lines based on, predominantly, overexpression experiments, recent reports have described that autophagosomes are normally generated and fused with lysosomes in the absence of UVRAG in cultured mammalian (HeLa) cells and in the Drosophila fat body (Nagy, 2016 and references therein).

The discoveries of UVRAG mutations in colorectal cancer cells, and that its overexpression increases autophagy and suppresses the proliferation of certain cancer cell lines, altogether suggest that this gene functions as an autophagic tumor suppressor. Such a role for UVRAG is thought to be related to its binding to Beclin 1, a haploinsufficient tumor suppressor gene required for autophagy. UVRAG appears to play roles similar to yeast Vps38 in the Drosophila fat body, and developing eye and wing: its loss leads to the accumulation of multiple endocytic receptors and ligands in an endosomal compartment, impaired trafficking of Lamp1 and Cathepsin L to the lysosome, and defects in the biogenesis of lysosome-related pigment granules. However, whether this gene is also required for the maintenance of intestinal homeostasis in Drosophila was unclear because the loss of UVRAG did not lead to uncontrolled cell proliferation in the developing eye or wing according to these reports. The current results showing that Uvrag deficiency causes intestinal dysplasia suggest that this gene is also important for the proper functioning of the adult gut in Drosophila (Nagy, 2016). A surprising aspect of this work is that UVRAG appears to function independently of autophagy in the intestine. There are other lines of evidence that also support that UVRAG has a more important role in endocytic maturation than in autophagy. First, it has been shown that truncating mutations in UVRAG that are associated with microsatellite-unstable colon cancer cell lines do not disrupt autophagy. Second, UVRAG depletion in HeLa cells does not prevent the formation or fusion of autophagosomes with lysosomes, but it does interfere with Egfr degradation. Third, a very recent paper has shown that overexpression of the colorectal-cancer-associated truncated form of UVRAG promotes tumorigenesis independently of autophagy status, that is, both in control and Atg5-knockout cells. That paper, again, relied on the overexpression of full-length or truncated forms of UVRAG, rather than the analysis of cancer-related mutations of the endogenous locus. Fourth, the endocytic function of UVRAG has been found to be required for developmental axon pruning that is independent of autophagy in Drosophila (Nagy, 2016 and references therein).

The results of this study indicate that UVRAG loss is accompanied with the sustained activation of JNK and STAT signaling in ISCs and EBs, and that these pathways are required for dysplasia in this setting. Sustained activation of these signaling routes is likely to be connected to the disruption of endocytic flux in the absence of UVRAG, because inhibiting endocytic uptake or degradation through dominant-negative dynamin expression or RNAi of Rab7, respectively, also leads to intestinal dysplasia. It is worth noting that the effects of inhibiting Shibire/dynamin function led to a much more severe hyperproliferation of ISCs and early death of animals. In line with this, the loss of early endocytic regulators, such as Rab5, in the developing eye causes overproliferation of cells and lethality during metamorphosis. Although eye development is not perturbed by the loss of the late endocytic regulators UVRAG or Rab7, these proteins are clearly important for controlling ISC proliferation and differentiation (Nagy, 2016).

A recent paper shows that hundreds of RNAi lines cause the expansion of the esg-GFP compartment in 1-week-old animals, which might be due to an unspecific ISC stress response in some cases. However, several lines of evidence support that impaired UVRAG-dependent endocytic degradation is specifically required to prevent intestinal dysplasia. First of all, activation of JNK stress signaling in esg-GFP-positive cells induces short-term ISC proliferatio, and almost all stem cells are lost through apoptosis by the 2- to 3-week age, the time when the Uvrag-mutant phenotype becomes obvious. In fact, UVRAG loss resembles an early-onset age-associated dysplasia that is normally observed in old (30-60 days) flies and involves the simultaneous activation of both JNK and STAT signaling. Second, UVRAG RNAi in ISCs and EBs leads to paracrine activation of the cytokine Unpaired3 in enterocytes, one of the hallmarks of niche appropriation by Notch-negative tumors. However, autocrine expression of the Unpaired proteins and JNK activation is observed in Uvrag-knockdown cells, unlike in Notch-negative tumors, and EBs with active Notch signaling accumulate in the absence of UVRAG, so the two phenotypes are clearly different. Third, it is the loss of autophagy that could be expected to mimic a stress response and perhaps induce stem cell tumors, but this does not seem to be the case – ISCs with Atg5 or Atg14 RNAi proliferate less in 3-week-old animals and an overall decrease of the esg-GFP compartment is seen, as opposed to the Uvrag-deletion phenotype (Nagy, 2016).

Taken together, this work indicates that endocytic maturation and degradation serves to prevent early-onset intestinal dysplasia in Drosophila, and its deregulation could be relevant for the development of colorectal cancer in humans (Nagy, 2016).

Activation of the Tor/Myc signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in Drosophila

The TOR (target of rapamycin) signaling pathway and the transcriptional factor Myc play important roles in growBh control. Myc acts, in part, as a downstream target of TOR to regulate the activity and functioning of stem cells. Tbis study explored the role of TOR-Myc axis in stem and progenitor cells in the regulation of lifespan, stress resistance and metabolism in Drosophila. Goth overexpression of rheb and myc-rheb in midgut stem and progenitor cells decreased the lifespan and starvation resistance of flies. TOR activation caused higher survival under malnutrition conditions. Furthermore, gut-specific activation of JAK/STAT and insulin signaling pathways were demonstrated to control gut integrity. Both genetic manipulations had an impact on carbohydrate metabolism and transcriptional levels of metabolic genes. These findings indicate that activation of the TOR-Myc axis in midgut stem and progenitor cells influences a variety of traits in Drosophila (Strilbytska, 2016).

Canonical nucleators are dispensable for stress granule assembly in Drosophila intestinal progenitors

Stressed cells downregulate translation initiation and assemble membrane-less foci termed stress granules (SGs). Extensively characterized in cultured cells, the existence of such structures in stressed adult stem cell pools remain poorly characterized. This study reports that Drosophila orthologs of mammalian SG components AGO1, ATX2, CAPRIN, eIF4E, FMRP, G3BP (Rasputin), LIN-28, PABP, and TIAR are enriched in adult intestinal progenitor cells where they accumulate in small cytoplasmic messenger ribonucleoprotein complexes (mRNPs). Treatment with sodium arsenite or rapamycin reorganized these mRNPs into large cytoplasmic granules. Formation of these intestinal progenitor stress granules (IPSGs) depended on polysome disassembly, led to translational downregulation, and was reversible. While canonical SG nucleators ATX2 and G3BP were sufficient for IPSG formation in the absence of stress, neither of them, nor TIAR, either individually or collectively, were required for stress-induced IPSG formation. This work therefore finds that IPSGs do not assemble via a canonical mechanism, raising the possibility that other stem cell populations employ a similar stress-response mechanism (Buddika, 2020).

This study characterized a population of SGs in Drosophila intestinal progenitor cells whose formation does not require the canonical stress granule nucleators needed in other cell types. A model is proposed describing IPSG formation that is based on three main observations: (1) IPSGs are bona fide SGs because they contain mRNAs and their formation can be blocked and reversed, (2) IPSGs are composed of at least nine conserved proteins that are highly expressed and distributed throughout the cytoplasm of progenitor cells prior to stress and that are known to associate with SGs in other cell types, and (3) IPSGs form even in the absence of three components, the Drosophila orthologs of mammalian ATX2, G3BP and TIA1, that are considered to be integral to SG formation in other cells. It is therefore proposed that following acute stresses, pre-existing mRNP particles aggregate together to form mature SGs, bypassing the role of these internally disorganized region (IDR)-rich proteins in nucleating stable cores needed during SG assembly in other cell types. This model indicates that the initial steps of SG assembly are variable and depend upon the cytoplasmic constituency of cells at resting state. Subsequent steps of IPSG assembly might follow the same progression proposed for SGs, including microtubule-dependent fusion of core mRNPs; super-resolution images of IPSGs indicate that they are not uniform but are rather likely composites of fused mRNPs. Since this study observed a complex and partially overlapping pattern of IPSG proteins in the absence of stress, it was hypothesize that mRNPs in unstressed cells are highly dynamic in nature. The pre-existence of such mRNPs may be an adaptation to the harsh intestinal environment and critical for proper epithelial homeostasis, allowing intestinal progenitor cells to rapidly respond to and recover from constant insult. The conservation of the proteins analyzed in this study raises the possibility that SGs in other cell types, including intestinal stem cells in other animals, may form via a pathway similar to Drosophila IPSGs (Buddika, 2020).

In contrast to IPSGs, other known SGs share a common need for three different IDR-containing proteins: ATX2, G3BP1/2 or TIA1/TIAR. These IDR-containing proteins play a critical role in establishing the core structures of nascent SGs that fuse during mature SG formation. Examples of the requirement of these proteins for SG formation include Drosophila G3BP in S2 cells, human G3BP1, either alone or in combination with its paralog G3BP2, in HEK293T, HeLa and U2OS cell types, Drosophila ATX2 and specifically its C-terminal IDR in S2 cells, and the prion-like domains of mammalian TIA1 in cultured COS7 cells. Despite this general requirement, there is also evidence suggesting possible redundancy between these proteins in some contexts. For example, while G3BP1/2 is required for arsenite-induced SG formation, it is not necessary for SG formation following osmotic stress (i.e. after treatment with NaCl or sorbitol). Furthermore, while loss of ATX2 or TIA1 severely compromises SG assembly in some mammalian and yeast cells, SGs are not completely eliminated. While these data suggest some redundancy between SG nucleators in some contexts, this possibility has not been previously investigated. Since it was observed that ATX2, RIN and ROX8 are co-expressed in intestinal progenitors, this study directly evaluated this potential functional redundancy using combinations of double and triple mutants of atx2, rin and rox8. This rigorous characterization showed that elimination of these integral SG nucleators, either alone or in combination, had little effect on either the size or number of SGs that form after either arsenite- or rapamycin-induced stress. While subtle defects cannot be completely ruled out, the grossly normal appearance of SGs in triple-mutant intestinal progenitors suggests the existence of a non-canonical mechanism for SG formation in these and potentially other cell types (Buddika, 2020).

This study also found that in a complex heterogenous tissue, such as the adult intestine, SGs selectively form in only a subset of cells. This observation may have been previously overlooked because much of the work on SG assembly has been conducted in homogenous cell culture systems rather than in intact tissues. Even the few previous studies on SG formation in Drosophila tissues has found that SG formation occurs uniformly by most cells throughout stressed tissues including, for example, heat-stressed ovarian follicular epithelia and larval imaginal discs, as well as mechanically stressed adult brain. Unlike the adult intestine, however, which is populated by both an active stem cell contingent as well as terminally differentiated cells, these tissues are all relatively homogenous with respect to the differentiation state of resident cell types. This analysis suggests that terminally differentiated intestinal cell types are refractory to SG formation because key RBPs are downregulated during their differentiation. Future studies investigating the molecular basis for this refractory state are medically relevant, since limiting SG formation could prevent the ectopic formation of pathogenic mRNP aggregates thought to underlie neurodegenerative disease (Buddika, 2020).

Embryonic and some somatic stem cell populations are known to maintain low levels of translational activity. This reduced translational activity helps these stem cells maintain an undifferentiated state, while increased translation drives differentiation. For instance, murine embryonic stem cells maintain global low translation during self-renewal, while differentiation proceeds with increased transcript abundance, ribosome loading, and protein synthesis and content. In addition, inhibition of translation by phosphorylation of the eukaryotic translation initiation factor 2α helps maintain low translation in mouse skeletal muscle stem cells; failure to maintain low translation result in loss of quiescence, initiation of the myogenic process and consequent differentiation. Using a microscopy-based OPP-staining approach, this study found that adult Drosophila intestinal progenitors, in contrast, display high levels of protein synthesis even under resting conditions. It is suggested that since increased translation in stem cells is a hallmark of differentiation, intestinal progenitor cells are in a state primed for differentiation that allows ISCs to rapidly proliferate and differentiate in order to replenish cells when needed. Since translation is an energy expensive process, stress responses may divert this energy to more immediate needs. It is proposed that SG formation provides this layer of regulation in intestinal progenitors during episodes of cellular stress. Furthermore, the characteristics of ISCs identified in this study may also be shared by stem cell populations that support other high-turnover adult tissues (Buddika, 2020).

While IPSGs were easily detectable in ex vivo-treated intestines, additional treatments in which adults were either fed chemical stressors or starved for various lengths of time with various vehicles (water, sugar-water or nothing) failed to induce SGs. It is hypothesized that ex vivo treatment induces IPSGs while feeding does not because orally fed chemicals are absorbed by enterocytes and fail to reach basally located and well protected progenitor cells. In addition, it is possible that endogenous IPSGs form transiently or require a dosage of proper duration that this study did not test. One other condition was found that induced small IPSG-like assemblies, namely heat shock. However, the induction was variable, and the treatments led to immediate death, precluding the ability to study them (Buddika, 2020).

There is considerable interest in identifying stem cell-specific factors, and this study shows that ten different RBPs are enriched in intestinal progenitors relative to surrounding differentiated cells. Previous transcriptional profiling analyses identified only one of these, lin-28, as being enriched in stem cells. Consistent with this, the other nine genes display relatively uniform transcript levels in non-differentiated versus differentiated cells. This apparent discrepancy between protein and transcript profiles in differentiated versus progenitor cells suggests active post-transcriptional regulatory mechanisms in intestinal cells. These mechanisms remain largely unexplored due to the lack of tools to profile the translatome relative to the transcriptome specifically in subsets of intestinal cells. Future work focused on developing such tools will likely identify post-transcriptional mechanisms that control stem cell behavior during resting and stressed conditions (Buddika, 2020).

Old and newly synthesized histones are asymmetrically distributed in Drosophila intestinal stem cell divisions

This study reports that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically partitioned during the division of Drosophila intestinal stem cells (ISCs). Furthermore, the inheritance patterns of old and new H3 and H4 in postmitotic cell pairs correlate with distinct expression patterns of Delta, an important cell fate gene. To understand the biological significance of this phenomenon, a mutant H3T3A was expressed to compromise asymmetric histone inheritance. Under this condition, an increase was observed in Delta-symmetric cell pairs and overpopulated ISC-like, Delta-positive cells. Single-cell RNA-seq assays further indicate that H3T3A expression compromises ISC differentiation. Together, these results indicate that asymmetric histone inheritance potentially contributes to establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings in Drosophila male germline stem cells (Zion, 2023).

In multicellular organisms, asymmetric cell division (ACD) of adult stem cells serves as an important mechanism for tissue homeostasis and regeneration. Disruption of this precisely regulated cell division mode can result in the dysregulation of stem cells, leading to cancer or tissue degeneration. Epigenetic mechanisms enable different cell types within a multicellular organism to establish distinct cellular identities while carrying the identical genetic information. Canonical histone proteins H3, H4, H2A, and H2B are synthesized and incorporated into DNA during replication as an octamer structure, forming the fundamental unit of chromatin, the nucleosome. It is well known that nucleosomes and chromatin structure can affect cell fate decisions; however, it remains largely unclear how epigenetic information is retained or altered during cell divisions to produce cells with different identities in multicellular organisms. Previous studies have shown that H3 and H4 histones are asymmetrically inherited during ACD of the Drosophila male germline stem cells (GSCs), where preexisting (old) histones are retained in the self‐renewed stem cell, while newly synthesized (new) histones are enriched in the differentiating daughter cell. In contrast, old and new H2A and H2B are inherited more symmetrically during ACD of male GSCs. It is hypothesized that the old H3 and H4 histones retain an epigenetic memory that is inherited by the self‐renewed stem cell, while the newly synthesized histones lacking this information can be used to establish a new gene expression program in the differentiating cell. Complementary to this hypothesis, previous studies have reported differences in post‐translational modifications between preexisting and newly synthesized histones. Additionally, it has been shown that nucleosomal density displays differences between old and new histone‐enriched sister chromatids, with the old histone side having higher overall nucleosomes than the new histone side. Nucleosome density and position have profound impacts on many cellular processes by modulating DNA accessibility to different regulators, including pioneer factors, transcription factors, and cell cycle regulators. One functional readout of the inheritance of asymmetric chromatin statuses in GSC divisions is the asymmetric recruitment of the DNA replication component Cdc6, which allows asynchronous cell cycle progression in the resulting daughter cells. Furthermore, when asymmetric H3 segregation is disrupted, progenitor germline tumors and germ cell loss phenotypes are both detected, suggesting that this process is required for both stem cell maintenance and proper germline differentiation. Recently, a new study revealed that proper interactions between homologous chromosomes at a critical 'stemness' gene, stat92E locus, depend on asymmetric H3 inheritance, without which stat92E gene expression becomes misregulated. The findings of asymmetric histone inheritance in Drosophila male GSCs set a precedent in studying epigenetic inheritance modes in multicellular organisms. The question remains, however, of whether this phenomenon is germ cell‐specific or if it serves as a more general mechanism. It also remains unclear whether asymmetrically inherited histones aid in defining distinct cell fates at a single‐cell level. Addressing these questions will not only greatly enhance our current understanding of how epigenetic inheritance modes dictate cell fates, but it will also help establish new methods to identify bona fide stem cells and asymmetrically dividing cells in vivo (Zion, 2023).

To investigate the generality of asymmetric histone inheritance, this study use the Drosophila intestinal stem cells (ISCs) in the midgut as a model system. One feature of ISCs is that they can alternate between ACD, which produces a self‐renewed ISC and a differentiating daughter of either an enteroblast (EB) or a pre‐enteroendocrine (pre‐ee) cell, and symmetric cell division (SCD), which results in two self‐renewed ISCs. As the Notch (N) signaling pathway is critical for cellular differentiation in the ISC lineage, the expression of Delta can be used as an ISC‐enriched cellular marker. It is proposed that the ISC lineage is a great system to study histone inheritance due to its well‐characterized lineage, clearly distinguishable ISC‐specific mitosis, and abundant ISCs in vivo. Indeed, previous studies have shown asymmetric inheritance of old versus new centromere‐specific histone H3 variant CENP‐A (i.e., CID in Drosophila) in the ISCs. Using this system this study reports that asymmetric canonical histone inheritance applies to this somatic stem cell lineage during ACD of ISCs and that misregulation of this process leads to midgut hyperplasia with ISC‐like cells. Collectively, these results demonstrate that asymmetric histone inheritance could be a more general feature for asymmetric stem cell divisions. This study also offers insight into how histone inheritance could influence the establishment or maintenance of cell identities and how misinheritance could lead to diseases such as cancer (Zion, 2023).

To study histone distribution and inheritance patterns during ISC divisions, a dual‐color histone labeling and tracking system was optimized in the ISCs, similar to what has been previously used. In this study, the expression of labeled histones was driven by a cell type‐specific escargot‐Gal4 (esg‐Gal4) to turn on the UAS‐histone transgene in the ISC lineage. After a heat‐shock‐induced switch from eGFP to mCherry‐labeled histone expression, ISCs were allowed to undergo at least a complete round of DNA replication after an approximately 18‐h recovery time, shown by robust incorporation of new replication‐dependent canonical histones genome‐wide. This time frame was determined through a time course experiment, where the expression and incorporation of new H3‐mCherry began around 12 h after heat shock. By 24 h, robust signals of both H3‐eGFP and H3‐mCherry could be detected. However, by 36 h old H3‐eGFP signals became almost undetectable. This time lapse required for new H3 incorporation is consistent with the replication‐dependent incorporation mode for canonical histones. Contrastingly, the incorporation of new histone variant H3.3‐mCherry which is independent of DNA replication showed robust signals 12 h after heat shock. Quantification showed minimal flipped‐out without heat shock but efficient flipped‐out at 18 h after heat shock, which is the time point at which most imaging‐based analysis was performed. These results are consistent with previous data using GFP‐tagged H3 versus GFP‐tagged H3.3 that show distinct incorporation modes between DNA replication‐dependent incorporation of the canonical H3 and DNA replication‐independent incorporation of the H3.3 histone variant in Drosophila. The ISCs co‐labeled with both eGFP (old histones) and mCherry (new histones) signals entered the subsequent mitosis, when sister chromatids are condensed and segregated equally into the daughter cells. The mitotic ISCs can be labeled using a mitotically enriched H3S10ph mark (phosphorylation at Serine 10 of H3). Using this mitotic mark, esg driver‐labeled ISCs can be distinguished in this histone tracer assay. Old versus new histone distribution patterns were studied at different mitotic stages of ISCs (Zion, 2023).

A long‐standing biological question is how distinct cell fates are established, maintained, and changed by epigenetic mechanisms at the single‐cell level in multicellular organisms, where cells have identical genomes. In this study, using the Drosophila ISC lineage as a model system, the inheritance of different canonical histones H3, H4, and H2A was studied during the ISC divisions. In postmitotic pairs of cells with an asymmetric Delta expression pattern, asymmetric histone inheritance patterns are detected, where the Delta‐high cell inherits more old histones and the Delta‐low cell inherits more new histones. In contrast, histones are distributed more symmetrically between the postmitotic pair of cells with similar Delta expression. It was hypothesized that the observed differences in Delta expression patterns are indicative of distinct cell identities resulting from different cell division modes. An asymmetric Delta expression pattern, with one Delta‐high cell and one Delta‐low cell, may indicate that these two cells result from an asymmetric ISC division, where the Delta‐high cell will maintain an ISC identity while the Delta‐low cell will take on differentiation. In contrast, a symmetric Delta expression pattern could result from a symmetric ISC division, where both Delta‐expressing cells will maintain the ISC identity. Interestingly, the percentages of asymmetric versus symmetric Delta expression patterns in the postmitotic pairs (79.6% versus 20.4%) are largely consistent with the ratios of asymmetric versus symmetric ISC divisions as reported in other studies, as well as the H3 inheritance modes in the mitotic ISCs observed in this study (75.0% versus 25.0%). These findings link the asymmetric histone inheritance mode with the establishment of distinct cell identities after one cell division. Furthermore, this asymmetric inheritance mode is specific to H3 and to the less extent to H4, as old and new H2A histones are almost always inherited symmetrically. This molecular specificity could be explained by the incorporation of old H3 and H4 into chromatin as a (H3‐H4)2 tetramer, while old H2A and H2B are incorporated as (H2A‐H2B) dimers. Since H3 and H4 carry most of the post‐translational modifications that influence gene expression, the asymmetric inheritance of old versus new (H3‐H4)2 serves as an elegant mechanism for establishing distinct epigenomes in the daughter cells that arise from stem cell ACD, possibly leading to differential gene expression programs and potentially other distinct cellular features (see Model for old (green) and new (red) histone inheritance patterns and their potential roles in the ISC system (Zion, 2023).

The biological significance of the asymmetric histone inheritance is further exemplified when this pattern is disrupted by the H3^T3A histone mutant, where an increase was detected in Delta‐symmetric postmitotic pairs of cells as well as the overpopulated ISC‐like cells. Interestingly, based on intestinal morphology, hyperplasia was observed in the H3T3A mutant more often in the posterior midgut. Previous work in the field has identified the posterior midgut as a region that is more sensitive to tumor formation, acting as a 'tumor hotspot'. In the current experiments, the posterior midgut appears to be more sensitized to the H3T3A mutant, consistent with these findings. These data connect misregulated histone inheritance with potential changes in cell fate determination. Recently, large‐scale analysis identified histone mutations in 3.8% of human tumor samples, a ratio similar to the mutations of known cancer‐associated genes such as BRCA2 and NOTCH1. In particular, mutations at the Thr3 residue of H3 have been found in a variety of human tumor samples, including lung, breast, skin, bladder, and liver cancers. However, the molecular mechanisms underlying these 'oncohistones' are not fully understood. The current findings on the oncohistone H3T3A illuminate how this mutation could lead to the loss of proper epigenetic inheritance and the onset of tumor formation (Zion, 2023).

The asymmetric inheritance mode of histones was first reported during the ACD of male Drosophila GSCs, which opened a new avenue of research; however, many essential questions remained, such as whether this asymmetric histone inheritance mode is specific to germ cells, stem cells, and/or asymmetrically dividing cells. The results reported in this paper provide a solid basis to addressing these questions. Despite significant differences in the niche structure, signaling cascades for regulating stem cell activity, and cellular differentiation pathways between the ISC and male GSC lineages, several key features of asymmetric histone inheritance are common between these two stem cell systems. First, the cellular specificity in the GSC lineage has been demonstrated by asymmetric histone inheritance mode in asymmetrically dividing GSCs but not in symmetrically dividing spermatogonial cells. In the ISC lineage, this cellular specificity is manifested by ISCs displaying an asymmetric histone inheritance mode in Delta‐asymmetric postmitotic pairs but not for Delta‐symmetric postmitotic pairs. Second, this asymmetry has the molecular specificity for H3 and H4 histones in both systems, emphasizing the importance of these two canonical histones in carrying and passing on or resetting an 'epigenetic memory.' Finally, expression of the mutant histone H3T3A abolishes asymmetric histone inheritances in both systems, resulting in stem cell or progenitor cell hyperplasia. Therefore, these results demonstrate that the asymmetric histone inheritance mode is not specific to either germ cells or stem cells, but likely contingent on the asymmetric mode of mitosis with the mission to generate two distinct daughter cells (Zion, 2023).

Zdditionally, asymmetric histone inheritance could occur at a local level, likely at critical genes that regulate stem cell fate or proper cellular differentiation. Similar local asymmetry of histone inheritance was shown in induced asymmetrically dividing mouse embryonic stem cells, with the H3K27me3 as a key histone modification displaying distinct distribution at the differentially expressed genes between the two daughter cells\. It will be intriguing to explore in the future what histone modifications are associated with old versus new histones (Zion, 2023).

Furthermore, asymmetric sister centromere in recognizing sister chromatids during ACD has also been reported in Drosophila male GSCs and female GSCs. Asymmetric inheritance of old versus new centromere‐specific histone variant CENP‐A/CID has also been reported in the Drosophila ISCs. More studies in the future are needed to understand how global (i.e., in fly male GSCs and ISCs) and local (i.e., in fly female GSCs and mESCs) canonical histone asymmetries are established, which is likely by developmentally programmed DNA replication. Furthermore, how canonical histone asymmetries on sister chromatids are recognized and differentially inherited is likely due to the differential attachment of the mitotic spindle to asymmetric sister centromeres. Finally, the distinct distribution of epigenetic information between the two daughter cells derived from ACD probably prepares them for distinct cellular behaviors, which is crucial to development, tissue homeostasis, and regeneration of multicellular organisms (Zion, 2023).

It has been debated whether the two cells resulting from ISC division are intrinsically asymmetric, or only become asymmetric through the extrinsic signaling cues after ISC division. Previous studies demonstrate that intrinsic polarity mechanisms result in the asymmetric distribution and inheritance of Par proteins to the apical daughter cell during ACD of ISCs, in order to promote differentiation. Furthermore, differential Notch activities due to the polarized Par complex induce distinct cellular differentiation pathways. Recent work has also shown that the spindle orientation in ISCs is tightly linked with cell fate, where planar orientation gives rise to two ISCs and angular orientation generates the ISC/EB pair of daughter cells\. Through analyzing different mitotic stages of ISCs, separable old versus new H3 distribution is detectable in prophase and prometaphase ISCs regardless of the ISC division modes, indicating that this asymmetry is likely intrinsically established prior to mitosis. Interestingly, old versus new H3T3A signals are still separable in prophase and prometaphase ISCs, similar to that of wild‐type H3, suggesting that this mutant histone does not interfere with differential histone incorporation before mitosis. However, increased symmetric segregation patterns in anaphase and telophase H3T3A‐expressing ISCs indicate that sister chromatids differentially enriched with old versus new H3T3A signals cannot be properly recognized and segregated. Because flies have two major autosomes (2nd and 3rd chromosomes) in addition to the sex chromosomes, even the randomized segregation of sister chromatids could lead to an asymmetric pattern at a low percentage, as shown previously. However, even though rare asymmetric histone inheritance occurs with randomized segregation pattern, the daughter cell enriched with old histone is still the cell expressing a higher level of Delta, indicative of ISC identity. Together, these findings indicate that ISC cell fate is likely specified by the intrinsic epigenetic information and the polarized extrinsic cues possibly act to ensure their differential segregation pattern. In summary, given the unique features of the ISC system, such as the ability to precisely label each derivative cell in the entire lineage, the large number of ISCs in their endogenous niche, and the sensitivity of ISC activity to environmental changes such as nutrition as well as aging, it will become a new in vivo model system to study the fundamental principles of different histone inheritance modes and relevant biological consequences under physiological and pathological conditions (Zion, 2023).

Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis and regeneration. In mammals, the progenies of intestinal stem cells (ISCs) can dedifferentiate to generate ISCs upon ablation of resident stem cells. However, whether and how mature tissue cells generate ISCs under physiological conditions remains unknown. This study shows that infection of the Drosophila melanogaster intestine with pathogenic bacteria induces entry of enteroblasts (EBs), which are ISC progenies, into the mitotic cycle through upregulation of epidermal growth factor receptor (EGFR)-Ras signaling. Ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive enteroblast mitosis cell autonomously. Furthermore, the dividing enteroblasts did not gain ISC identity as a prerequisite to divide, and the regenerative ISCs are produced through EB mitosis. Taken together, this work uncovers a new role for EGFR-Ras signaling in driving EB mitosis and replenishing the ISC pool during fly intestinal regeneration, which may have important implications for tissue homeostasis and tumorigenesis in vertebrates (Tian, 2022).

Wnt/beta-catenin signaling within multiple cell types dependent upon kramer regulates Drosophila intestinal stem cell proliferation

The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/beta-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, \the cellular determinants of ISC proliferation, harnessing kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. Wnt signaling within Prospero-positive cells supports ISC proliferation, and kramer regulates Wnt signaling in this context by antagonizing kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes kramer as a physiological regulator of Wnt/beta-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/β-catenin signaling (Sun, 2023).

Long non-coding RNA CR46040 is essential for injury-stimulated regeneration of intestinal stem cells in Drosophila

Long non-coding RNAs (lncRNAs) play important regulatory roles in stem cells self-renewal, pluripotency maintenance and differentiation. Till now, there is very limited knowledge about how lncRNAs regulate intestinal stem cells (ISCs), and lncRNAs mediating ISCs regeneration in Drosophila have yet been characterized. This study identify a lncRNA, CR46040, that is essential for the injury-induced ISCs regeneration in Drosophila. Loss of CR46040 greatly impairs ISCs proliferation in response to tissue damage caused by dextran sulfate sodium (DSS) treatment. This study demonstrates that CR46040 is a genuine lncRNA that has two isoforms transcribed from the same transcription start site, and works in trans to regulate intestinal stem cells. Mechanistically, CR46040 knock-out flies are failed to fully activate JNK, JAK/STAT and HIPPO signaling pathways after tissue damage, which are required for ISCs proliferation after intestinal injury. Moreover, CR46040 knock-out flies are highly susceptible to DSS treatment and enteropathogenic bacteria Erwinia carotovora ssp. carotovora 15 (Ecc15) infection. These findings characterize, for the first time, a lncRNA that mediates damage-induced ISCs proliferation in Drosophila, and provide new insights into the functional links among the long non-coding RNAs, ISCs proliferation and tissue homeostasis (Xu, 2023).

Sleep loss impairs intestinal stem cell function and gut homeostasis through the modulation of the GABA signalling pathway in Drosophila

Sleep is essential for maintaining health. Indeed, sleep loss is closely associated with multiple health problems, including gastrointestinal disorders. However, it is not yet clear whether sleep loss affects the function of intestinal stem cells (ISCs). Mechanical sleep deprivation and sss mutant flies were used to generate the sleep loss model. qRT-PCR was used to measure the relative mRNA expression. Gene knock-in flies were used to observe protein localization and expression patterns. Immunofluorescence staining was used to determine the intestinal phenotype. The shift in gut microbiota was observed using 16S rRNA sequencing and analysis. Sleep loss caused by mechanical sleep deprivation and sss mutants disturbs ISC proliferation and intestinal epithelial repair through the brain-gut axis. In addition, disruption of SSS causes gut microbiota dysbiosis in Drosophila. As regards the mechanism, gut microbiota and the GABA signalling pathway both partially played a role in the sss regulation of ISC proliferation and gut function. The research shows that sleep loss disturbed ISC proliferation, gut microbiota, and gut function. Therefore, the results offer a stem cell perspective on brain-gut communication, with details on the effect of the environment on ISCs (Zhou, 2023).

Drosophila Sulf1 is required for the termination of intestinal stem cell division during regeneration

Stem cell division is activated to trigger regeneration in response to tissue damage. The molecular mechanisms by which this stem cell mitotic activity is properly repressed at the end of regeneration are poorly understood. This study shows that a specific modification of heparan sulfate (HS) is critical in regulating Drosophila intestinal stem cell (ISC) division during normal midgut homeostasis and regeneration. Loss of the extracellular HS endosulfatase Sulf1 results in increased ISC division during normal homeostasis, which is caused by upregulation of mitogenic signaling including the JAK/STAT, EGFR, and Hedgehog pathways. Using a regeneration model, this study found that ISCs failed to properly halt division at the termination stage in Sulf1 mutants, showing that Sulf1 is required for terminating ISC division at the end of regeneration. It is proposed that post-transcriptional regulation of mitogen signaling by HS structural modifications provides a novel regulatory step for precise temporal control of stem cell activity during regeneration (Takemura, 2015).

Diversity of fate outcomes in cell pairs under lateral inhibition

Cell fate determination by lateral inhibition via Notch/Delta signalling has been extensively studied. Most formalised models consider Notch/Delta interactions in fields of cells, with parameters that typically lead to symmetry breaking of signalling states between neighbouring cells, commonly resulting in salt-and-pepper fate patterns. This study considers the case of signalling between isolated cell pairs. The bifurcation properties of a standard mathematical model of lateral inhibition was found to lead to stable symmetric signalling states. This model was applied to the adult intestinal stem cell (ISC) of Drosophila, whose fate is stochastic but dependent on the Notch/Delta pathway. A correlation was observed between signalling state in cell pairs and their contact area. This behaviour is intrepeted in terms of the properties of the model in the presence of population variability in contact areas, which affects the effective signalling threshold of individual cells. The results suggest that the dynamics of Notch/Delta signalling can contribute to explain stochasticity in stem cell fate decisions, and that the standard model for lateral inhibition can account for a wider range of developmental outcomes than previously considered (Guisoni, 2017).

Tis11 mediated mRNA decay promotes the reacquisition of Drosophila intestinal stem cell quiescence

Adult stem cell proliferation rates are precisely regulated to maintain long-term tissue homeostasis. Defects in the mechanisms controlling stem cell proliferation result in impaired regeneration and hyperproliferative diseases. Many stem cell populations increase proliferation in response to tissue damage and reacquire basal proliferation rates after tissue repair is completed. Although proliferative signals have been extensively studied, much less is known about the molecular mechanisms that restore stem cell quiescence. This study shows that Tis11, an Adenine-uridine Rich Element (ARE) binding protein that promotes mRNA degradation, is required to re-establish basal proliferation rates of adult Drosophila intestinal stem cells (ISC) after a regenerative episode. Tis11 limits ISC proliferation specifically after proliferation has been stimulated in response to heat stress or infection, and Tis11 expression and activity are increased in ISCs during tissue repair. Based on stem cell transcriptome analysis and RNA immunoprecipitation, it is proposed that Tis11 activation represents an integral part of a negative feedback mechanism that limits the expression of key components of several signaling pathways that control ISC function and proliferation. The results identify Tis11 mediated mRNA decay as an evolutionarily conserved mechanism of re-establishing basal proliferation rates of stem cells in regenerating tissues (McClelland, 2017).

Drosophila dyskerin is required for somatic stem cell homeostasis

Drosophila represents an excellent model to dissect the roles played by the evolutionary conserved family of eukaryotic dyskerins. These multifunctional proteins are involved in the formation of H/ACA snoRNP and telomerase complexes, both involved in essential cellular tasks. Since fly telomere integrity is guaranteed by a different mechanism, the specific role played by dyskerin in somatic stem cell maintenance was investigated. Focus was placed on Drosophila midgut, a hierarchically organized and well characterized model for stemness analysis. Surprisingly, the ubiquitous loss of the protein uniquely affects the formation of the larval stem cell niches, without altering other midgut cell types. The number of adult midgut precursor stem cells is dramatically reduced, and this effect is not caused by premature differentiation and is cell-autonomous. Moreover, only a few dispersed precursors found in the depleted midguts could maintain stem identity and the ability to divide asymmetrically, and show cell-growth defects or undergo apoptosis. Loss is mainly specifically dependent on defective amplification. These studies establish a strict link between dyskerin and somatic stem cell maintenance in a telomerase-lacking organism, indicating that loss of stemness can be regarded as a conserved, telomerase-independent effect of dyskerin dysfunction (Vicidomini, 2017).

Intestinal stem cell pool regulation in Drosophila

Intestinal epithelial renewal is mediated by intestinal stem cells (ISCs) that exist in a state of neutral drift, wherein individual ISC lineages are regularly lost and born but ISC numbers remain constant. To test whether an active mechanism maintains stem cell pools in the Drosophila midgut, partial ISC depletion was performed. In contrast to the mouse intestine, Drosophila ISCs failed to repopulate the gut after partial depletion. Even when the midgut was challenged to regenerate by infection, ISCs retained normal proportions of asymmetric division and ISC pools did not increase. The loss of differentiated midgut enterocytes (ECs), however, slows when ISC division is suppressed and accelerates when ISC division increases. This plasticity in rates of EC turnover appears to facilitate epithelial homeostasis even after stem cell pools are compromised. This study identifies unique behaviors of Drosophila midgut cells that maintain epithelial homeostasis (Jin, 2017).

To achieve homeostasis in a stem cell pool, stem cell divisions typically give rise to one new stem cell and one cell that is destined to differentiate. This lineage asymmetry can be determined cell-intrinsically, for instance by the asymmetric partitioning of determinants during division, or by localized niche factors. In the latter case, lineage asymmetry may be observed only in populations, rather than by following each and every stem cell division. Studies in mice and flies have documented this sort of population asymmetry in ISC pools and have demonstrated the phenomenon of neutral drift, whereby individual stem cell lineages are born and extinguished at equivalent rates as a result of divisions that either duplicate stem cells or fail at self-renewal. In addition, in mice, dedifferentiation of progenitor cells within the crypt has been observed as a mechanism for restoring lost stem cells. However, the precise response of the gut after stem cell pools are compromised is not well understood in either Drosophila or mice. Understanding this response has considerable practical value, since many anti-cancer chemotherapies deplete intestinal and other stem cells, and thereby give rise to debilitating side effects such as gastrointestinal mucositis (Jin, 2017).

In this study it was asked whether the fly intestinal stem cell pool is self-regulatory and capable of regeneration following the ablation of about 50% of the ISCs. Surprisingly, it was found that the fly's ISC population did not repopulate itself after ISC depletion. Instead, the remaining ISCs behaved essentially as in normal midguts: they divided at normal rates and responded normally to gut epithelial damage with increased division, but did not duplicate at higher frequencies or regenerate a normal-sized stem cell pool, even after long recovery periods. Nevertheless, midguts with about half the normal ISC number retained their normal size for many weeks, indicating that somehow homeostasis was maintained. In exploring this phenomenon it was found that the rate of stem cell division has a strong influence on the rate of loss of differentiated epithelial cells, both when ISC divisions were accelerated or retarded. Hence, it is suggested that homeostasis in ISC-depleted guts was made possible by a reduction in the rate of cell loss from the gut epithelium. One explanation for this may be that EC loss rates are substantially determined by ISC division rates, rather than directly by damage from digestive wear and tear and adverse interactions with the gut microbiota, as generally assumed. The demonstration that EC loss can be accelerated by promoting ISC proliferation is consistent with this view. It is speculated that, as between ECs and ISC tumors, competition between old and newborn ECs for attachment to the basement membrane may underlie these effects on EC lifespan. In support of this, midgut epithelial cell crowding induced by increased ISC proliferation due to stress was shown to be relieved by the loss of excess cells though apoptosis. The ability to alter the rate of epithelial replacement to match the capabilities of the stem cell pool represents an unexpected mechanism of homeostatic plasticity (Jin, 2017).

Experiments were performed in which ISCs were completely ablated, and no recovery of the ISC pool was observed over the lifespan of flies. This is consistent with another recent report in which ISCs were completely ablated. In both of these cases the esg-Gal4 driver was used for depletion, allowing the conclusion that there are no esg- ISC precursors in the adult fly. Interestingly, in contrast to a recent report that found that flies lacking ISCs had almost normal lifespans, this study found that complete ISC ablation reduced fly survival. The data suggest that while flies can survive partial ISC loss for at least 4 weeks, complete ISC depletion results in a loss of midgut homeostasis and reduced survival. (Jin, 2017).

The dynamics of ISC pool maintenance in the fly midgut have significant differences from those in the mammalian intestine. Symmetric ISC lineages are very often observed in the mouse intestine, while only 10% of ISC lineages are typically symmetric in the fly midgut. Strikingly, murine ISC pools readily recover after stem cell depletion whereas in the fly ISC depletion appears to be irreversible. Furthermore, in the murine intestine, selection of stem cells based on niche occupancy is important, and partially differentiated TA cells can revert into ISCs if they can access the niche. These phenomena are not observed in the fly midgut, which has a dispersed niche, no TA cells, and a fixed number of ISCs. The different behavior of ISCs in these two species could be due to differing requirements for stem cell capability. The mouse's lifespan is more than ten times longer than the fly's, so murine ISCs need to maintain gut homeostasis for much longer. Mammalian ISCs have to renew themselves many more times during the host's lifespan and also accumulate more genomic damage from DNA replication, which could alternatively drive cell death or transformation. Perhaps because of these pressures, the mammalian intestine evolved a more flexible system for stem cell pool control, which allows both better recovery from injury and the capability to select defective ISCs while maintaining a normal-sized stem cell pool (Jin, 2017).

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. This study sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells (Schell, 2017).

It was first observed almost 100 years ago that, unlike differentiated cells, cancer cells tend to avidly consume glucose, but not fully oxidize the pyruvate that is generated from glycolysis. This was originally proposed to be due to dysfunctional or absent mitochondria, but it has become increasingly clear that mitochondria remain functional and critical. Mitochondria are particularly important in proliferating cells because essential steps in the biosynthesis of amino acids, nucleotide and lipid occur therein. Most proliferating stem cell populations also exhibit a similar glycolytic metabolic program, which transitions to a program of mitochondrial carbohydrate oxidation during differentiation. The first distinct step in carbohydrate oxidation is import of pyruvate into the mitochondrial matrix, where it gains access to the pyruvate dehydrogenase complex (PDH) and enters the tricarboxylic acid (TCA) cycle as acetyl-CoA. The two proteins that assemble to form the mitochondrial pyruvate carrier (MPC) have been recently described. This complex is necessary and sufficient for mitochondrial pyruvate import in yeast, flies and mammals, and thereby serves as the junction between cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. Decreased expression and activity of the MPC underlies the glycolytic program in colon cancer cells in vitro, and forced re-expression of the MPC subunits increased carbohydrate oxidation and impaired the ability of these cells to form colonies in vitro and tumours in vivo. This impairment of tumorigenicity was coincident with a loss of key markers and phenotypes associated with stem cells. This has prompted an examination of whether glycolytic non-transformed stem cells might also exhibit low MPC expression and mitochondrial pyruvate oxidation, which must increase during differentiation (Schell, 2017).

The role of the MPC was studied in the ISCs of the fruit fly Drosophila, which share key aspects of their biology with mammalian ISCs. Both MPC1 and MPC2 are expressed in all four cell types of the intestine, with the lowest level of expression in the ISCs and the highest expression in the differentiated enteroendocrine cells. Confocal imaging of intestines dissected from dMPC1 mutants revealed that the epithelium exhibits multilayering unlike the normal single-cell layer seen in controls. This is a classic overgrowth phenotype that is associated with oncogene mutations in Drosophila. Accordingly, MARCM clonal analysis was used to determine whether a specific loss of the MPC in ISCs leads to an increase in their proliferation. On average, newly divided GFP-marked dMPC1 mutant clones are more than twofold larger than control clones, which were generated in parallel using a wild-type chromosome, indicating that the MPC is required in the ISC lineage to suppress proliferation. Because GFP-marked clones could include cells that differentiate into mature enterocytes or enteroendocrine cells, clonal analysis was conducted in the absence of Notch, thereby blocking ISC differentiation. Under these conditions, an approximately twofold increase was observed in the size of dMPC1 mutant ISC clones. To confirm and extend these results, MPC function was specifically disrupted in the ISCs by using the Dl-GAL4 driver in combination with UAS-GFP, which facilitates stem cell identification. Once again, approximately twofold more GFP-marked stem cells were observed relative to controls when either dMPC1 or dMPC2 expression was disrupted by RNA-mediated interference (RNAi) along with increased ISC proliferation as detected by staining for phosphorylated histone H3 (pHH3). Similar results were obtained when RNAi was targeted to the E1 or E2 subunits of PDH to specifically disrupt the next step in mitochondrial pyruvate oxidation. Importantly, an opposite phenotype was seen when Ldh was reduced by RNAi in the ISCs or progenitor cells. Ldh suppression is known to result in a significant increase in pyruvate levels, which can promote pyruvate oxidation. Taken together with the results with Pdh RNAi, these observations support the model that the MPC limits stem cell proliferation through promoting oxidative pyruvate metabolism in the mitochondria. It also appears to be sufficient as specific overexpression of MPC1 and MPC2 in ISCs or progenitors caused a reduction in stem cell proliferation, the opposite of the loss-of-function phenotype. This can be seen in either Pseudomonas-infected intestines, which undergo rapid stem cell proliferation, or under basal conditions in aged animals. Consistent with this, MPC overexpression under basal conditions had no effects on intestinal morphology, while the intestines from infected flies displayed a fully penetrant size reduction, which is probably due to the inability of ISCs to maintain tissue homeostasis. Taken together, these results demonstrate that mitochondrial pyruvate uptake and metabolism is both necessary and sufficient in a stem cell autonomous manner to regulate ISC proliferation and maintain intestinal homeostasis in Drosophila (Schell, 2017).

Studies in Drosophila, intestinal organoids and mice provide strong evidence that the MPC is necessary and sufficient in a cell autonomous manner to suppress stem cell proliferation. Consistently, this study has demonstrated that ISCs maintain low expression of the subunits that comprise the MPC, which enforces a mode of carbohydrate metabolism wherein glucose is metabolized in the cytosol to pyruvate and other biosynthetic intermediates. This glycolytic metabolic program appears to be sufficient to drive robust and continuous stem cell proliferation. High mitochondrial content was observed in ISCs, which must be geared primarily toward biosynthetic functions and/or oxidation of other substrates such as fatty acids. Increased fatty acids, the metabolism of which is enhanced in MPC-deficient and MPC-inhibited organoids, have been shown to promote ISC expansion and proliferation via enhanced beta-catenin signalling and increasing tumour-initiating capacity. MPC expression increases following differentiation, consistent with the shift in demand from macromolecule biosynthesis to ATP production in support of post-mitotic differentiated cell function. A similar switch in MPC expression can be seen following differentiation of embryonic stem cells, haematopoietic stem cells and trophoblast stem cells. Conversely, MPC expression is reduced after reprogramming fibroblasts to induced pluripotent stem cells. This suggests that the effects of altering pyruvate flux that wad observed in this study might not be restricted to ISCs, but instead be representative of similar effects on multiple stem cell populations. Interestingly, Myc is known to drive a metabolic program that is similar to that observed following MPC loss, characterized by increased glycolysis and reliance on glutamine and fatty acid oxidation with reduced glucose oxidation. This suggests that Myc may play a role in repressing the MPC in stem cells, possibly acting downstream of Wnt/beta-catenin signalling. Consistent with this, Myc and its repressive co-factors localize to the Mpc1 promoter and Myc expression is strongly anti-correlated with Mpc1 expression (Schell, 2017).

Taken together, these studies demonstrate that changes in the MPC and mitochondrial pyruvate metabolism are required to properly orchestrate the proliferation and homeostasis of intestinal stem cells. Importantly, this metabolic program, mediated at least partially by the MPC, appears to be instructive for cell fate, rather than a downstream consequence of cell fate. Future work will define the extent to which the results presented in this study relate to those showing that diet quality and quantity can modulate ISC behaviour. It is tempting to speculate that ISC metabolism is used as a signal for increased or decreased demand for intestinal epithelium. Perhaps of most importance will be to define the mechanisms whereby altered partitioning of pyruvate metabolism affects stem cell proliferation and fate. It is speculated that the robust changes that were observed in fatty acid oxidation and histone acetylation, which are probably downstream of altered metabolite utilization for acetyl-CoA production, play an important role. While the mechanisms are not as yet defined, these studies establish a paradigm wherein mitochondrial metabolism does not merely provide a permissive context for proliferation or differentiation, but rather plays a direct and instructive role in controlling stem cell fate (Schell, 2017).

Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut

Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, this study identified two regulators of cytosolic Ca2+ levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca2+ signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. These findings provide a link between redox regulation and Ca2+ signaling and reveal a novel mechanism by which ISCs detect stress signals (Xu, 2017).

This study found that the two cation channels TRPA1 and RyR are critical for cytosolic Ca2+ signaling and ISC proliferation. Under homeostatic conditions, the basal activities of TRPA1 and RyR are required for maintaining cytosolic Ca2+ in ISCs to ensure their self-renewal activities and normal tissue turnover. Agonists, including but not limited to low levels of ROS, could be responsible for the basal activities of TRPA1 and RyR. Under tissue damage conditions, increased ROS stimulates the channel activities of TRPA1 to mediate increases in cytosolic Ca2+ in ISCs. As for RyR, besides its potential to directly sense ROS, it is known to act synergistically with TRPA1 in a positive feedback mechanism to release more Ca2+ from the ER into the cytosol upon sensing the initial Ca2+ influx through TRPA1 (Xu, 2017).

Previously, Deng (2015) identified L-glutamate as a signal that can activate metabotropic glutamate receptor (mGluR) in ISCs, which in turn modulates the cytosolic Ca2+ oscillation pattern via phospholipase C (PLC) and inositol-1,4,5-trisphosphate (InsP3). Interestingly, L-glutamate and mGluR RNAi mainly affected the frequency of Ca2+ oscillation in ISCs, while their influence on cytosolic Ca2+ concentration was very weak. Strikingly, the number of mitotic cells induced by L-glutamate (i.e. an increase from a basal level of ~5 per midgut to ~10 per midgut) is far less than what has been observed in tissue damage conditions (depending on the severity of damage, the number varies from ~20 to more than 100 per midgut following damage). Consistent with this, in a screen for regulators of ISC proliferation in response to tissue damage, this study tested three RNAi lines targeting mGluR (BL25938, BL32872, and BL41668, which was used by Deng, 2015), and none blocked the damage response in ISCs, suggesting that L-glutamate and mGluR do not play a major role in damage repair of the gut epithelium (Xu, 2017).

This study found that ROS can trigger Ca2+ increases through the redox- sensitive cation channels TRPA1 and RyR under damage conditions. In particular, it was demonstrated using voltage-clamp experiments that the TRPA1-D isoform, which is expressed in the midgut, is sensitive to the oxidant agent paraquat. In addition, the results of previous studies have demonstrated the direct response of RyR to oxidants via single channel recording and showed that RyR could amplify TRPA1-mediated Ca2+ signaling through the Ca2+-induced Ca2+ release (CICR) mechanism. Interestingly, expression of oxidant- insensitive TRPA1-C isoform in the ISCs also exhibits a tendency to induce ISC proliferation, indicating that ROS may not be the only stimuli for TRPA1 and RyR under physiological conditions. Possible other activators in the midgut may be irritant chemicals, noxious thermal/mechanical stimuli, or G-protein-coupled receptors (Xu, 2017).

Altogether, the concentration of cytosolic Ca2+ in ISCs appears to be regulated by a number of mechanisms/inputs including mGluR and the ion channels TRPA1 and RyR. Although mGluR might make a moderate contribution to cytosolic Ca2+ in ISCs, TRPA1 and RyR have a much stronger influence on ISC Ca2+ levels. Thus, it appears that the extent to which different inputs affect cytosolic Ca2+ concentration correlates with the extent of ISC proliferation (Xu, 2017).

Although, as a universal intracellular signal, cytosolic Ca2+ controls a plethora of cellular processes, we were able to demonstrate that cytosolic Ca2+ levels regulate Ras/MAPK activity in ISCs. Specifically, we found that trpA1 RNAi or RyR RNAi block Ras/MAPK activation in stem cells, and that forced cytosolic Ca2+ influx by SERCA RNAi induces Ras/MAPK activity. Moreover, Ras/MAPK activation is an early event following increases in cytosolic Ca2+, since increased dpErk signal was observed in stem cells expressing SERCA RNAi before they undergo massive expansion, and when Yki RNAi was co-expressed to block proliferation. It should be noted that a more variable pattern of pErk activation was observed with prolonged increases of cytosolic Ca2+, suggesting complicated regulations via negative feedback, cross-activation, and cell communication at late stages of Ca2+ signaling. This might explain why Deng failed to detect pErk activation after 4 days induction of Ca2+ signaling (Deng, 2015). Previously, Ras/MAPK activity was reported to increase in ISCs, regulating proliferation rather than differentiation, in regenerating midguts, which is consistent with the findings about TRPA1 and RyR (Xu, 2017).

The Calcineurin A1/CREB-regulated transcription coactivator/CrebB pathway previously proposed to act downstream of mGluR-calcium signaling (Deng, 2015) is not likely to play a major role in high Ca2+-induced ISC proliferation, as multiple RNAi lines targeting CanA1 or CrebB were tested and none of them suppressed SERCA RNAi-induced ISC proliferation. In support of this model, it was also found that the active forms of CanA1/ CRTC/ CrebB cannot stimulate mitosis in ISCs when their cytosolic Ca2+ levels are restricted by trpA1 RNAi, whereas mitosis induced by the active forms of Ras or Raf is not suppressed by trpA1 RNAi (Xu, 2017).

Prior to this study, it has been shown that paracrine ligands such as Vn from the visceral muscle, and autocrine ligands such as Spi and Pvf ligands from the stem cells, can stimulate ISC proliferation via RTK-Ras/MAPK signaling. It study found that multiple RTK ligands in the midgut are down-regulated by trpA1 RNAi expression in the ISCs, including spi and pvf1 that can be induced by SERCA RNAi. Further, it was demonstrated that high Ca2+ fails to induce ISC proliferation in the absence of EGFR. As spi is induced by EGFR-Ras/MAPK signaling in Drosophila cells, and DNA binding mapping (DamID) analyses indicate that spi might be a direct target of transcriptional factors downstream of EGFR-Ras/MAPK in the ISCs, the autocrine ligand Spi might therefore act as a positive feedback mechanism for EGFR-Ras/MAPK signaling in ISCs (Xu, 2017).

In summary, this study identifies a mechanism by which ISCs sense microenvironment stress signals. The cation channels TRPA1 and RyR detect oxidative stress associated with tissue damage and mediate increases in cytosolic Ca2+ in ISCs to amplify and activate EGFR-Ras/MAPK signaling. In vertebrates, a number of cation channels, including TRPA1 and RyR, have been associated with tumor malignancy. The current findings, unraveling the relationship between redox-sensing, cytosolic Ca2+, and pro-mitosis Ras/MAPK activity in ISCs, could potentially help understand the roles of cation channels in stem cells and cancers, and inspire novel pharmacological interventions to improve stem cell activity for regeneration purposes and suppress tumorigenic growth of stem cells (Xu, 2017).

Loss of the mucosal barrier alters the progenitor cell niche via JAK/STAT signaling

The mucous barrier of the digestive tract is the first line of defense against pathogens and damage. Disruptions in this barrier are associated with diseases such as Crohn's disease, colitis and colon cancer, but mechanistic insights into these processes and diseases are limited. Loss of a conserved O-glycosyltransferase (PGANT4) in Drosophila has been shown to result in aberrant secretion of components of the peritrophic/mucous membrane in the larval digestive tract. This study shows that loss of pgant4 disrupts the mucosal barrier, resulting in epithelial expression of the IL-6-like cytokine Upd3, leading to activation of JAK/STAT signaling, differentiation of cells that form the progenitor cell niche and abnormal proliferation of progenitor cells. This niche disruption could be recapitulated by overexpressing upd3 and rescued by deleting upd3, highlighting a crucial role for this cytokine. Moreover, niche integrity and cell proliferation in pgant4-deficient animals could be rescued by overexpression of the conserved cargo receptor Tango1 and partially rescued by supplementation with exogenous mucins or treatment with antibiotics. These findings help elucidate the paracrine signaling events activated by a compromised mucosal barrier and provide a novel in vivo screening platform for mucin mimetics and other strategies to treat diseases of the oral mucosa and digestive tract (Zhang, L. 2017).

The mucous barrier that lines the respiratory and digestive tracts is the first line of defense against pathogens and provides hydration and lubrication. This unique membrane separates the delicate epithelia from factors present in the external environment. In mammals, the mucous lining of the intestine allows nutrient penetration while conferring protection from both bacteria and the mechanical damage associated with digestion of solid food. The principal components of mucous membranes are mucins, a diverse family of proteins that are expressed in tissue-specific fashions. Whereas secreted mucins vary greatly in sequence and size, they all share highly O-glycosylated serine- and threonine-rich regions that confer unique structural and rheological properties, allowing the formation of hydrated gels. Deletion of specific components of the mucous membranes present in the lung (Muc5b) or the intestinal tract (Muc2) resulted in defects in mucociliary clearance or an increased incidence colorectal cancer, respectively. Changes in mucus production or the glycosylation status of mucins have also been associated with oral pathology in Sjogren's syndrome, the development of colitis and intestinal tumors in mice, and the progression of ulcerative colitis and colon cancer in humans. However, the detailed mechanisms by which loss/alteration of this protective layer results in epithelial pathology remain unknown (Zhang, L. 2017).

Many components and factors that confer the unique properties of this protective lining, including the mucins and the enzymes that mediate their dense glycosylation, are conserved across species. Indeed, Drosophila melanogaster contains a similar protective lining known as the peritrophic membrane, consisting of a chitin scaffold that is bound by highly O-glycosylated mucins. During larval development, where animals ingest solid food and undergo massive growth in preparation for metamorphosis, specialized secretory cells (PR cells) of the anterior midgut produce this membrane, which is thought to protect epithelial cells of the digestive tract from mechanical and microbial damage. Previous work in Drosophila has shown that loss of one component of the peritrophic membrane resulted in a thinner and more permeable membrane, where adult flies were viable yet more susceptible to oral infections. However, the exact role of the entirety of this lining in the integrity and protection of cells of the digestive tract remains unknown (Zhang, L. 2017).

Extensive research elucidating the development and function of the many cell types that comprise the Drosophila digestive tract has provided insights into mammalian digestive system formation and function. The Drosophila larval midgut is composed of specialized epithelial cells (enterocytes [ECs] and enteroendocrine cells) for digestion and nutrient absorption, as well as progenitor cells that will eventually form the adult midgut epithelium. These adult midgut progenitor cells (AMPs) reside in a protected niche, formed by peripheral cells (PCs) that wrap and shield them from external signaling. PCs are characterized by a unique crescent shape, with long processes that surround AMPs, restricting proliferation and differentiation until metamorphosis. The larval digestive tract therefore represents an ideal system to interrogate the role of the mucous layer in protection of both the epithelium and the progenitor cell niche at a stage when the mechanical and microbial stresses associated with the ingestion of solid food are abundant (Zhang, L. 2017).

Previous work has shown that loss of a conserved UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase responsible for initiating O-linked glycosylation (PGANT4) resulted in aberrant secretion of components of the peritrophic membrane in the larval digestive tract (Tran, 2012). This study shows that pgant4 mutants are devoid of a peritrophic membrane, resulting in epithelial cell damage and expression of the IL-6-like inflammatory cytokine, unpaired 3 (Upd3). Upd3 expression resulted in increased JAK/STAT signaling in the progenitor cell niche, causing niche cell differentiation and aberrant progenitor cell proliferation. These effects were dependent on Upd3 and could be rescued by deleting upd3 or partially rescued by feeding animals antibiotics or exogenous mammalian intestinal mucins. Moreover, overexpression of the conserved extracellular matrix cargo receptor, Tango1 (transport and Golgi organization 1), in secretory cells of the digestive tract resulted in restoration of the peritrophic membrane and rescue of niche integrity. These results elucidate new mechanistic details regarding how a compromised mucous lining can influence epithelial integrity and the progenitor cell niche and provide an in vivo screening platform for compounds and strategies that could restore mucosal barrier function (Zhang, L. 2017).

This study shows the peritrophic membrane is essential to protect the integrity of the epithelial cell layer and maintain an appropriate environment for the progenitor cell niche. Moreover, a dynamic and specific response to the loss of this membrane occurs via the production of the IL-6-like cytokine Upd3 from epithelial cells, which in turn signals to niche cells in a paracrine fashion, causing differentiation and morphological changes. This demonstrates the multipotent nature of PCs, which can respond to specific cytokines to alter their fate. Once the PC morphology and fate were altered, JAK/STAT signaling was activated in AMPs exposed to Upd3, causing aberrant cell proliferation/DNA replication. This represents the first example where loss of the protective mucous lining activates signaling from epithelial cells to alter the fate of niche cells and change the behavior of progenitor cells. These studies highlight the importance of this membrane in both epithelial and progenitor cell biology and elucidate the paracrine signaling cascade that is specifically activated when this barrier is compromised (Zhang, L. 2017).

Interestingly, the mucinous peritrophic membrane could be restored by overexpression of the conserved cargo receptor Tango1. Tango1 is an essential protein that functions to package large extracellular matrix proteins, such as collagen and mucins, into secretory vesicles. Loss of the mammalian ortholog of Tango1 (Mia3) in a murine model resulted in lethality with global defects in collagen secretion and extracellular matrix composition. Alterations in Tango1/Mia3 expression have also been associated with colon and hepatocellular carcinomas in humans. Previous work in Drosophila demonstrated that PGANT4 glycosylates Tango1, protecting it from Dfur2-mediated proteolysis in the digestive tract. This study shows that Tango1 overexpression specifically in the secretory PR cells of the digestive tract can restore the mucinous membrane throughout the midgut to rescue epithelial viability and niche integrity, further demonstrating the crucial role of the peritrophic membrane in digestive system homeostasis and health. These results suggest the possibility of exogenous Tango1 expression as a potential strategy to restore secretion, mucous membranes, and/or extracellular matrix composition and confer epithelial protection (Zhang, L. 2017).

The larval digestive system offers unique opportunities to investigate the role of the individual components of the mucinous membrane and restorative strategies in epithelial biology. Unlike the adult stage, the larval portion of the life cycle is devoted to continuous feeding and digestion to orchestrate the massive growth of cells and tissues in preparation for metamorphosis. As such, larvae consume many types of solid food and will readily ingest various compounds. Indeed, oral supplementation with an intestinal mucin (Muc2) partially rescued JAK/STAT signaling, suggesting that this could serve as a strategy for epithelial protection. Muc2 is a major component of the protective mucous membrane that lines the small intestine and colon of mammals. Muc2 is thought to confer lubrication for food passage as well as to form a barrier between microbes and epithelial cells of the digestive tract. The current results suggest that Muc2 supplementation could be providing similar properties in the Drosophila digestive tract. Interestingly, supplementation with the gastric mucin (Muc5AC) dramatically exacerbated JAK/STAT signaling, suggesting that the different structural, rheological, or binding properties of each mucin are mediating distinct cellular responses in this system. Current work is focused on deciphering the specific functional regions of various secreted mucins and testing their ability to confer epithelial protection using this in vivo system. Human diseases of the digestive tract are associated with disrupted mucinous linings, and disease severity is often correlated with the severity of barrier disruption. Interestingly, these diseases are also characterized by increased levels of the mammalian ortholog of Upd3 (IL-6), increased JAK/STAT activation, and increased cell proliferation, similar to what is seen in Drosophila, suggesting conserved mechanisms for responding to mucosal disruption/injury. It is widely known that immune cells are one source of IL-6 in mammals, but recent studies have demonstrated that mechanically damaged epithelial and endothelial cells also produce IL-6. This study demonstrated that epithelial expression of Upd3 is both necessary and sufficient for the changes in PC fate and AMP proliferation, as disruption of the niche could be recapitulated by overexpression of upd3 from ECs and rescued by deletion of upd3. How peritrophic membrane loss is signaling to up-regulate upd3 expression in ECs is currently unknown. However, previous studies in the adult Drosophila digestive system have shown up-regulation of upd, upd2, and upd3 in response to enteric infection or damage-inducing agents, such as bleomycin or dextran sulfate sodium, suggesting roles for both microbial insults and physical/mechanical damage to epithelial cells. Indeed, the results also suggest roles for microbial and mechanical damage in the absence of the peritrophic membrane, as both antibiotics and mucin supplementation were able to reduce upd3 expression and cell proliferation. This study demonstrates that the larval midgut can serve as a model system to study how cells/tissues sense and respond to damage as well as to decipher how upd3 is specifically activated in epithelial cells under various conditions (Zhang, L. 2017).

As a mucous layer is present across most internal epithelial surfaces, understanding the mechanisms by which it confers protection and epithelial homeostasis will be informative in treating various diseases affecting the integrity of this layer. Mucosal healing has been proposed as a treatment option for inflammatory bowel disease and other diseases of the digestive tract that are characterized by destruction of the mucosa and epithelial surfaces. Likewise, mucins are a component in some oral treatments for dry mouth caused by head and neck irradiation or Sjogren's syndrome. Other therapeutics for various autoimmune and inflammatory diseases include JAK inhibitors (Jakinibs) and drugs directed against particular cytokines. This study has shown that genetic restoration of the peritrophic membrane can restore digestive system health and that antibiotic treatment or mucin supplementation can partially rescue damage-induced signaling cascades, suggesting that this Drosophila system may be a viable platform for testing compounds to remediate epithelial damage. Future studies will focus on testing newly emerging mucin mimetics (designed to confer epithelial protection and appropriate rheology/hydration), synthetic mucins (where the extent of glycosylation can be specifically modified), glycan-based hydrogels, and drugs that target conserved steps in the JAK/STAT signaling cascade. Lessons learned in Drosophila may inform future strategies for functional restoration of mucosal protection (Zhang, L. 2017).

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair

Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. This study found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. These findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation (Lechuga, 2022).

The septate junction component bark beetle is required for Drosophila intestinal barrier function and homeostasis

Age-related loss of intestinal barrier function has been documented across species, but the causes remain unknown. The intestinal barrier is maintained by tight junctions (TJs) in mammals and septate junctions (SJs) in insects. Specialized TJs/SJs, called tricellular junctions (TCJs), are located at the nexus of three adjacent cells, and this study have shown that aging results in changes to TCJs in intestines of adult Drosophila melanogaster. This study now demonstrates that localization of the TCJ protein bark beetle (Bark) decreases in aged flies. Depletion of bark from enterocytes in young flies led to hallmarks of intestinal aging and shortened lifespan, whereas depletion of bark in progenitor cells reduced Notch activity, biasing differentiation toward the secretory lineage. These data implicate Bark in EC maturation and maintenance of intestinal barrier integrity. Understanding the assembly and maintenance of TCJs to ensure barrier integrity may lead to strategies to improve tissue integrity when function is compromised (Hodge, 2023).

Gut barrier defects, increased intestinal innate immune response, and enhanced lipid catabolism drive lethality in N -glycanase 1 deficient Drosophila

Intestinal barrier dysfunction leads to inflammation and associated metabolic changes. However, the relative impact of infectious versus non-infectious mechanisms on animal health in the context of barrier dysfunction is not well understood. This study established that loss of Drosophila N -glycanase 1 (Pngl) leads to gut barrier defects, which cause starvation and increased JNK activity. These defects result in Foxo overactivation, which induces a hyperactive innate immune response and lipid catabolism, thereby contributing to lethality associated with loss of Pngl. Notably, germ-free rearing of Pngl mutants did not rescue lethality. In contrast, raising Pngl mutants on isocaloric, fat-rich diets improved animal survival in a dosage-dependent manner. These data indicate that Pngl functions in Drosophila larvae to establish the gut barrier, and that the immune and metabolic consequences of loss of Pngl are primarily mediated through non-infectious mechanisms (Pandey, 2023).

Nutritional control of stem cell division through S-Adenosylmethionine in Drosophila intestine

The intestine has direct contact with nutritional information. The mechanisms by which particular dietary molecules affect intestinal homeostasis are not fully understood. In this study, S-adenosylmethionine (SAM) was identified as a universal methyl donor synthesized from dietary methionine, as a critical molecule that regulates stem cell division in Drosophila midgut. Depletion of either dietary methionine or SAM synthesis reduces division rate of intestinal stem cells. Genetic screening for putative SAM-dependent methyltransferases has identified protein synthesis as a regulator of the stem cells, partially through a unique diphthamide modification on eukaryotic elongation factor 2. In contrast, SAM in nutrient-absorptive enterocytes controls the interleukin-6-like protein Unpaired 3, which is required for rapid division of the stem cells after refeeding. This study sheds light upon a link between diet and intestinal homeostasis and highlights the key metabolite SAM as a mediator of cell-type-specific starvation response (Obata, 2018).

Autophagy maintains stem cells and intestinal homeostasis in Drosophila

Intestinal homeostasis is maintained by tightly controlled proliferation and differentiation of tissue-resident multipotent stem cells during aging and regeneration, which ensures organismal adaptation. This study shows that autophagy is required in Drosophila intestinal stem cells to sustain proliferation, and preserves the stem cell pool. Autophagy-deficient stem cells show elevated DNA damage and cell cycle arrest during aging, and are frequently eliminated via JNK-mediated apoptosis. Interestingly, loss of Chk2, a DNA damage-activated kinase that arrests the cell cycle and promotes DNA repair and apoptosis, leads to uncontrolled proliferation of intestinal stem cells regardless of their autophagy status. Chk2 accumulates in the nuclei of autophagy-deficient stem cells, raising the possibility that its activation may contribute to the effects of autophagy inhibition in intestinal stem cells. This study reveals the crucial role of autophagy in preserving proper stem cell function for the continuous renewal of the intestinal epithelium in Drosophila (Nagy, 2018).

Spen limits intestinal stem cell self-renewal

Precise regulation of stem cell self-renewal and differentiation properties is essential for tissue homeostasis. Using the adult Drosophila intestine to study molecular mechanisms controlling stem cell properties, this study identified the gene split-ends (spen) in a genetic screen as a novel regulator of intestinal stem cell fate (ISC). Spen family genes encode conserved RNA recognition motif-containing proteins that are reported to have roles in RNA splicing and transcriptional regulation. This study demonstrates that spen acts at multiple points in the ISC lineage with an ISC-intrinsic function in controlling early commitment events of the stem cells and functions in terminally differentiated cells to further limit the proliferation of ISCs. Using two-color cell sorting of stem cells and their daughters, spen-dependent changes in RNA abundance and exon usage were identified and potential key regulators were found downstream of spen. This work identifies spen as an important regulator of adult stem cells in the Drosophila intestine, provides new insight to Spen-family protein functions, and may also shed light on Spen's mode of action in other developmental contexts (Andriatsilavo, 2018).

The mechanisms by which ISCs undergo asymmetric and symmetric divisions are still not completely understood. In a genetic screen, this study has identified spen as an essential regulator of adult stem cells in Drosophila. The data indicate that in the absence of spen activity, stem cells have aberrant high levels of Delta (Dl) protein and fail to properly commit into EB daughter cells resulting in large increase in numbers of ISC-like cells and that this activity is cell autonomous roles in the ISC. Furthermore, spen acts in EBs to limit ISC numbers, and in EBs, EEs and ECs cells to suppress ISC proliferation. Therefore, in spen mutant tissue, stem cell autonomous and non-autonomous mechanisms that act to drive ISC proliferation, increased ISC numbers, and aberrant accumulation of Dl protein in ISCs. While spen mutants are dependent on Akt and InR for their growth, they are not as sensitive to a reduction in EGFR activity as wild-type control clones are. These findings argue that Spen does not act as a general regulator of Notch signaling in all tissues, and is upstream of, or parallel to, Notch pathway activation to promote intestinal stem cell commitment. Through a transcriptomic analysis, this study has identified genes controlled by spen in ISCs and EBs, both at the transcript and exon level, providing candidate ISC regulators downstream of spen. Critically, this work shows that the RNA binding protein Spen is an important regulator of asymmetric fate outcomes of ISC division and its proliferation (Andriatsilavo, 2018).

It is interesting that spen has both stem cell-autonomous and non-autonomous activities to regulate stem cell numbers and proliferation. The knockdown of spen in ISCs and EBs can expand stem cell numbers, whereas the knockdown in EEs and ECs led to a dramatic increase in the number of dividing ISCs, but did not change the density of ISCs. It is believed, therefore, that the growth of spen mutant clones is influenced by both cell autonomous and non-autonomous processes: spen inactivation in the stem cell leads to more ISC symmetric self-renewing divisions, however, EB, EE and EC cells are still produced. The mutant EB, EE, and EC cells can further drive proliferation of the extra ISCs through non-autonomous regulation. It is possible that the non-autonomous regulation of ISC proliferation detected upon downregulation of spen in EEs and ECs could be due to stress induced in the tissue owing to disruption of numerous spen targets genes in these cells. Importantly, these findings highlight the complex process of stem cell deregulation arising in tumor contexts whereby inactivation of a tumor suppressor genes may have numerous functions in different cells within a tumor that collaborate to drive tumorigenesis (Andriatsilavo, 2018).

One intriguing effect of spen inactivation is the high levels of accumulation of the Notch ligand Dl at the plasma membrane and in intracellular vesicles. RNAseq data revealed that Dl mRNA levels or exon usage are not affected, suggesting a regulation at the protein level, perhaps during its endocytic trafficking steps. This phenotype of Dl protein accumulation is reminiscent of those occurring upon Dl trafficking perturbation, such as in neuralized mutants. A previous study has shown genetic interaction of spen and regulators of trafficking of Notch ligands. Indeed, in the developing Drosophila eye disc, spen genetically interacts with the endocytic adaptor Epsin/liquid facets (lqf), which promotes Dl trafficking and internalization facilitating Dl activation. Spen, however, is likely not a general regulator of protein trafficking as the localization of the membrane protein Sanpodo occurred normally in spen mutant clones in the intestine. Therefore, Spen likely regulates Dl trafficking in a tissue-specific manner and indirectly through transcriptional or post-transcriptional control of a downstream gene (Andriatsilavo, 2018).

In addition, the data indicated that the knockdown of Akt and InR activity could reduce the number of Dl+ ISCs produced in the spen mutant background. The InR pathway has previously been implicated in regulating symmetric cell divisions of the ISC during adaptive growth. Thus, it appears as if Akt and InR are also facilitating symmetric cell division in the spen mutant context. These data also show that spen mutant stem cells are insensitive to a reduction of EGFR signaling upon overexpression of EGFR-DN, suggesting that spen and EGFR pathway have opposing roles in regulating ISC proliferation. While a decrease in EGFR signaling reduces ISC proliferation, spen inactivation, in contrast, leads to an increase in proliferation. spen has been shown to genetically interact with the EGFR signaling pathway during Drosophila embryogenesis and during eye development. Nevertheless, in these contexts, spen potentiates EGFR signaling. Thus, spen may have a tissue specific effect regarding EGFR pathway regulation. The precise link between spen and EGFR signaling regulation is nevertheless still unclear. Indeed, while a constitutive active form of Ras can rescue the lethality caused by a spen dominant negative form in embryo, spen seems to act downstream or in parallel of Ras activation during photoreceptor specification. Additionally, spen loss of function in cone cells during pupal eye development affects the EGF receptor ligand Spitz level, which suggest a role at the level of the EGF receptor activation. Further studies will be required to better understand the link between spen and the EGFR signaling pathway in the regulation of intestinal stem cell regulation, as well as in other developmental contexts (Andriatsilavo, 2018).

In addition to describing a new function of spen in adult stem cell regulation, the data raise a number of interesting questions about the function of spen and its downstream target genes in stem cell regulation. Importantly, this study demonstrates that inactivation of a large SPEN family member also results in considerable alteration of exon usage, like the smaller family members (spenito in flies, RBM15 and RBM15b in mouse). Thus, spen activity is important for transcriptional and post-transcriptional regulation in Drosophila, although it cannot be excluded that some of these effects might be indirect. Interestingly, in absence of spen function, altered exon usage of spenito was detected in EBs, raising the possibility of feedback control between family members, reminiscent of the plant SPEN family member FPA (Andriatsilavo, 2018).

Little is known about the impact on intestinal stem cell self-renewal of RNA binding proteins. A recent study found that inactivation of Tis11, encoding a regulator of RNA stability, led to larger clones than controls. The Tis11 phenotype differs markedly from that of spen as it appears not to affect cell fate decisions since ISCs do not accumulate as in spen mutant clones. Importantly, this study provides a data set that opens new perspectives for future investigation on the relationship between Spen and RNA regulation at transcriptional and post-transcription levels. Interestingly, the data also reveals that Spen co-regulates several genes encoding proteins that are involved in similar biological processes, such as cytoplasmic ribosomal proteins, immune response pathway and Imd pathway genes, and genes involved in Chitin metabolism. This may explain previously reported spen phenotypes in other tissues (Andriatsilavo, 2018).

Recent studies have highlighted important, essential functions of Spen family proteins from X-inactivation in mammals to sex determination and fat metabolism in flies. This family of proteins is also frequently mutated in cancers, though mechanistically the role SPEN family proteins in cancers is not understood. This work demonstrates that spen inactivation causes the deregulation of numerous genes and results in alteration of the stem cell fate acquisition. In mammals, RBM15 has a critical function in promoting hematopoietic stem cell return to quiescence. These findings raise the possibility that conserved functions of SPEN family proteins may help restrict stem cell activity required for cancer prevention (Andriatsilavo, 2018).

RAL GTPases drive intestinal stem cell function and regeneration through internalization of WNT signalosomes

Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, this studyidentified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, this study has identified a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential (Johansson, 2019).

The WT1-like transcription factor Klumpfuss maintains lineage commitment of enterocyte progenitors in the Drosophila intestine

In adult epithelial stem cell lineages, the precise differentiation of daughter cells is critical to maintain tissue homeostasis. Notch signaling controls the choice between absorptive and entero-endocrine cell differentiation in both the mammalian small intestine and the Drosophila midgut, yet how Notch promotes lineage restriction remains unclear. This study describes a role for the transcription factor Klumpfuss (Klu) in restricting the fate of enteroblasts (EBs) in the Drosophila intestine. Klu is induced in Notch-positive EBs and its activity restricts cell fate towards the enterocyte (EC) lineage. Transcriptomics and DamID profiling show that Klu suppresses enteroendocrine (EE) fate by repressing the action of the proneural gene Scute, which is essential for EE differentiation. Loss of Klu results in differentiation of EBs into EE cells. These findings provide mechanistic insight into how lineage commitment in progenitor cell differentiation can be ensured downstream of initial specification cues (Korzelius, 2019).

Ecdysone-regulated genomic networks in Drosophila: Midgut gene expression during metamorphosis

During insect metamorphosis, each tissue displays a unique physiological and morphological response to the steroid hormone 20-hydroxyecdysone (ecdysone). Gene expression was assayed in five tissues during metamorphosis onset. Larval-specific tissues display major changes in genome-wide expression profiles, whereas tissues that survive into adulthood display few changes. In one larval tissue, the salivary gland, a computational approach was used to identify a regulatory motif and a cognate transcription factor involved in regulating a set of coexpressed genes. During the metamorphosis of another tissue, the midgut, genes encoding factors from the hedgehog, Notch, EGF, dpp, and wingless pathways are activated by the ecdysone regulatory network. Mutation of the ecdysone receptor abolishes their induction. Cell cycle genes are also activated during the initiation of midgut metamorphosis, and they are also dependent on ecdysone signaling. These results establish multiple new connections between the ecdysone regulatory network and other well-studied regulatory networks (Li, 2003).

Developmental patterns of gene expression were studied from five different tissues and organs: central nervous system (CNS), wing imaginal disc (WD), larval epidermis and attached connective tissue (ED), midgut (MG), and salivary gland (SG), during late larval and early prepupal development when ecdysone triggers metamorphosis. At these stages of development, the five tissues display very different morphological and physiological responses to ecdysone. The wing imaginal disc responds to the hormone by initiating evagination, or unfolding, as it changes from a compact epithelial bilayer to an extended appendage. The salivary glands secrete glue proteins that are used to immobilize the puparium during metamorphosis. The cuticle attached to the larval epidermis undergoes a process of hardening and tanning to form the pupal case. The central nervous system (CNS) displays little morphological change during the late third instar ecdysone pulse, but the animal displays changes in behavior and in neurosecretory status. The two major types of cells in the larval midgut, larval epidermal cells and adult epidermal progenitor cells (midgut imaginal islands), respond in opposite ways to ecdysone. The larval epidermal cells initiate the process of programmed cell death, while the imaginal cells proliferate and form the adult midgut (Li, 2003).

One tissue, the midgut, was selected to assay during its complete metamorphosis, which occurs from 18 hr before puparium formation (BPF) to 12 hr APF. During this 30 hr period, eleven time points were examined as the larval midgut is destroyed and replaced with the adult midgut. The two major cell types present in this organ are distinguishable by size. The larval epithelial cells are large, with decondensed polyploid nuclei, and undergo programmed cell death in response to ecdysone. Embedded among the larval cells are small diploid imaginal midgut cells, which proliferate in response to the hormone to form the adult epithelial cells. Additionally, the midgut contains relatively small numbers of muscle, tracheal, and endocrine cells (Li, 2003).

In total, transcripts from a surprisingly large fraction of the genome, >30%, changed significantly during the metamorphosis of the midgut (18 hr BPF to 12 hr APF). Broad classes of temporally separable gene expression patterns are evident. These classes include sets of transcripts that rapidly decrease coincident with onset of programmed cell death in the larval cells, sets that are induced during early or late metamorphosis, and sets of transcripts expressed at highest levels during the middle period of the time course when the larval cells are in the final stages of cell death and the adult cells are rearranging to form new tissue (Li, 2003).

Within these broad classes, specific sets of genes that have related functions and show parallel expression were identified, indicating that they make up gene batteries. Six such examples, included coregulated transcripts that encode proteins found in specific macromolecular complexes, biochemical pathways, organellar functions, and structural components of the cells that compose this tissue. Transcripts encoding proteasome components increase during the ecdysone pulse that triggers the onset of cell death in larval cells. Transcripts encoding glycolytic enzymes rapidly decrease during the initiation of metamorphosis, but gradually resume expression as the imaginal cells proliferate. Vacuolar ATPases shows a pattern similar to the glycolytic enzymes, whereas tubulin- and actin-encoding transcripts peak during the intense period of imaginal cell proliferation and migration as the adult midgut is formed. Transcripts encoding structural components of the peritrophic membrane of the mature larval gut gradually decrease during its replacement with adult tissue (Li, 2003).

The expression patterns were examined of regulatory genes known to be involved in the ecdysone transcriptional hierarchy predicted to control the gene batteries that were identified. Also examined was the expression of genes with known roles in programmed cell death or cell cycle control. The expression of known ecdysone-responsive regulatory genes was consistent with previous observations in midgut. Although the larval midgut is composed of cell types that undergo divergent responses to ecdysone -- apoptosis and cell proliferation -- it was nonetheless possible to detect significant changes in transcript levels from genes encoding proteins involved in both processes. The apoptosis activator gene ark was expressed at 4 hr BPF. E93 and reaper, which encode proteins that serve as critical control points in the commitment to programmed cell death, were expressed at PF, as was the initiator caspase dronc. These midgut expression profiles were compared to those reported for salivary glands at and after 10 hr APF, when a prepupal pulse of ecdysone triggers apoptosis in that tissue; almost the entire genetic cascade was found to be similarly activated in salivary glands and midgut albeit at two distinct periods of development. However, one notable difference was observed at the top level of the cascade. In the salivary gland, E93 is activated by βFTZ-F1, whereas in the midgut the βFTZ-F1 gene is not induced until 6-8 hr after E93 is induced. The regulation of E93 therefore does not depend on βFTZ-F1 in the midgut, but must rely on another as yet unidentified factor(s). During midgut metamorphosis, developmental modulation of transcript levels were also observed for genes encoding DNA polymerases, cyclins, CDCs, and other cell cycle regulators, as well as genes encoding DNA repair proteins such as Hus1, Rad23, and PCNA/Mus209 (Li, 2003).

Which of the genes that are differentially expressed at the onset of midgut metamorphosis require ecdysone signaling? Ecdysone-dependent transcriptional activity was removed using mutant Ecdysone Receptor (EcR) alleles, rescuing null EcR mutants to the third larval instar by using a heat shock-inducible EcR transgene. Gene expression was examined in mutant midguts that were isolated from mutant animals arrested at the end of the third larval stage (stage 2a mutants). 376 (76%) of the 495 genes that are significantly induced during the onset of midgut metamorphosis (18 hr BPF to 2 hr APF) required EcR function, whereas 296 (64%) of 460 transcripts that decline significantly in level during this time period require ecdysone signaling through EcR. Thus, a very large proportion of the genes that are developmentally regulated at the initiation of metamorphosis in this organ are under the control of the transcription factors that mediate the ecdysone signal. However, it does not appear that EcR function is a general requirement for transcription, because a significant fraction of differentially expressed genes are unaffected in EcR mutant tissue (Li, 2003).

Of the several different classes of genes expressed during midgut metamorphosis, the regulation of all genes in the proteasome, tubulin/actin, and lysozyme clusters requires EcR to exhibit their normal changes in developmental expression. However, many genes in the v-ATPase cluster and nearly half the genes in the peritrophin cluster did not require EcR. The downregulation of hexokinase A, 6-phosphofructokinase, and pyruvate kinase genes in the glycolysis pathway were affected in the EcR mutants, while many others in this pathway were not. Hexokinase A, 6-phosphofructokinase, and pyruvate kinase are rate-controlling enzymes in the glycolytic pathway, indicating that their ecdysone dependence is functionally significant. The expression of the numerous known ecdysone receptor target genes such as E75, E74, broad, E23, and DHR3 required EcR as expected. The induction dynamics for the E74 and DHR3 transcription factor genes was as expected, as was their dependence on EcR. In contrast to E74 and DHR3, DHR78 has previously been described to reside upstream of EcR at the top of the ecdysone regulatory hierarchy -- the expression of EcR is dependent on the wild-type function of DHR78. However, DHR78 can also be induced by ecdysone in organ culture. The results demonstrate that DHR78 wild-type induction is indeed dependent on EcR function. Taken together, these data indicate a positive feedback loop between EcR and DHR78 during the onset of metamorphosis in the midgut (Li, 2003).

Genes encoding factors involved in cell cycle and growth control, and in DNA repair, are also under the control of EcR. In spite of the role of ecdysone in stimulating cell proliferation during metamorphosis, no cell cycle genes have previously been linked to the ecdysone regulatory hierarchy. The induction of the cell cycle regulatory genes CyclinB, cdc2, and CyclinD were all observed to be dependent on EcR function. The rapid induction of cdc2 during the late third instar ecdysone pulse is similar to that observed for direct targets of EcR. The CyclinD gene is also induced at this time, but its maximal induction occurs several hours after that observed for cdc2. Cyclin D promotes cellular growth, whereas Cyclin B/Cdc2 controls G2/M transitions in proliferative cells. The dependence of these three genes on EcR function indicates that ecdysone may control cell proliferation, at least in part, through their regulation. Coordinate with the induction of CyclinB, cdc2, and CyclinD, the induction was observed of DNA polymerase-delta and DNA repair genes such as Rad23, and PCNA/mus209. The induction of these DNA repair and synthesis genes is also EcR dependent. The expression changes of these genes may be the result of the direct action of EcR, or due to the action of factors directly controlled by the ecdysone receptor complex. It is unlikely that the increase in expression of these genes is simply due to increased numbers of proliferative cells because the total number of divisions between 18 hr BPF and PF are few, and not all cell cycle or DNA repair genes showed an increase in expression at the initiation of metamorphosis. For example, the level of CyclinJ, which is known to be required during early embryonic division cycles, is actually reduced in expression from 18 hr BPF to PF. When the expression of cell death genes was examined in EcR mutant tissue, E93 induction was observed as well as induction of the Ark caspase activator and the dronc caspase gene required wild-type function of EcR (Li, 2003).

Factors in several well-studied signaling pathways are induced during midgut metamorphosis. These include Wnt (dishevelled, armadillo, and zeste white 3), TGFβ/BMP (sara, daughters against dpp, and glass bottom boat), EGFR (torpedo/egfr, rhomboid/veinlet, vein, and keren/spitz2), and Notch pathway genes (delta, kuzbanian, suppressor of hairless, E(spl)malpha, and E(spl)mβ). All of these pathways are used during embryonic midgut development, and these data indicate they are reused during midgut metamorphosis. Genes in the Hedgehog signaling pathway (hedgehog, smoothened, and cubitus interruptus) changed significantly as well (Li, 2003).

To determine whether any of the genes in these pathways are expressed as a consequence of ecdysone signaling, the EcR mutant expression data was examined for those genes that were induced during the late third instar ecdysone pulse. The induction of zeste white-3/shaggy, keren/spitz2, kuzbanian, and hedgehog are all dependent on the presence of functional EcR. The induction dynamics of the EGFR ligand gene keren/spitz2, the Notch proteolytic activation factor gene kuzbanian, and the shaggy/zeste white-3 kinase gene are similar to genes that are known direct targets of ecdysone signaling. The induction of hedgehog follows a secondary response pattern, as do genes from the E(spl) complex that are induced in response to Notch activation, although these induction kinetics are also consistent with these genes being partially activated directly by the ecdysone receptor and partially with other factors (i.e., they may be 'early-late' genes). These data show that the regulatory network controlled by ecdysone in midguts includes the activation of known components of the Wnt, EGFR, Hedgehog, and Notch pathways. Notably, ligand production for the EGF, Hedgehog, TGFβ/BMP, and Notch pathways is under control of ecdysone. The specific roles that each of these pathways plays during metamorphosis are currently unknown. These results nonetheless indicate new connections between ecdysone signaling and the activity of several other signaling pathways during the metamorphosis of this organ, either through direct targeting of the ecdysone receptor or through the actions of downstream factors (Li, 2003).

Ecdysone-induced receptor tyrosine phosphatase PTP52F regulates Drosophila midgut histolysis by enhancement of autophagy and apoptosis

The rapid removal of larval midgut is a critical developmental process directed by molting hormone ecdysone during Drosophila metamorphosis. To date, it remains unclear how the stepwise events can link the onset of ecdysone signaling to the destruction of larval midgut. This study investigated whether ecdysone-induced expression of receptor protein tyrosine phosphatase PTP52F regulates this process. The mutation of the Ptp52F gene caused significant delay in larval midgut degradation. Transitional endoplasmic reticulum ATPase (TER94), a regulator of ubiquitin proteasome system, was identified as a substrate and downstream effector of PTP52F in the ecdysone signaling. The inducible expression of PTP52F at the puparium formation stage resulted in dephosphorylation of TER94 on its Y800 residue, ensuring the rapid degradation of ubiquitylated proteins. One of the proteins targeted by dephosphorylated TER94 was found to be Drosophila inhibitor of apoptosis 1 (DIAP1), which was rapidly proteolyzed in cells with significant expression of PTP52F. Importantly, the reduced level of DIAP1 in response to inducible PTP52F was essential not only for the onset of apoptosis but also for the initiation of autophagy. This study demonstrates a novel function of PTP52F in regulating ecdysone-directed metamorphosis via enhancement of autophagic and apoptotic cell death in doomed Drosophila midguts (Santhanam, 2014).

This study shows that ecdysone-induced expression of PTP52F and the subsequent tyrosine dephosphorylation of TER94 coordinate to construct upstream signaling determinants for a precise time-dependent degradation of larval midgut. The transient expression of Ptp52F gene at the PF stage is regulated by the functional EcR. Immediately after the level of endogenous PTP52F protein is detectable in larval midgut, TER94 becomes dephosphorylated on its Y800 residue. This modification may be critical to the rapid degradation of ubiquitylated proteins through a TER94-dependent regulation of ubiquitin proteasome system (UPS). Although the exact mechanism remains elusive, recent studies have suggested that only the tyrosine-dephosphorylated form and not the tyrosine-phosphorylated form of VCP interacts with cofactors for processing ubiquitylated substrates of UPS. Because VCP and TER94 share some evolutionarily conserved features, it is proposed that the same phosphorylation- and dephosphorylation-dependent mechanism may be adopted by TER94. Ubiquitylated DIAP1, a potential substrate of UPS, was found to be targeted by the Y800 dephosphorylated form of TER94. DIAP1 was rapidly degraded in cells in which levels of PTP52F were increased, as illustrated by in vivo observations in Drosophila midgut during metamorphosis. Consequently, the proteolysis of DIAP1 in response to inducible expression of PTP52F terminates the inhibitory effect on autophagy, allowing the initiation of autophagic cell death accompanied by apoptotic cell death for the destruction of the larval midgut tissues. Since the regulatory role of all Drosophila homologs of caspases have been ruled out in the process of larval midgut histolysis, it is likely that DIAP1 degradation-induced autophagic signaling may activate a yet-unknown pathway leading to the onset of apoptotic cell death in dying midgut. Additional experiments are needed to identify downstream effectors of PTP52F that modulate the cross talk between autophagy and apoptosis in the context of midgut maturation (Santhanam, 2014).

Identification of TER94 as a substrate dephosphorylated by PTP52F in larval midgut is interesting and important. From the time of their original cloning and identification, Drosophila TER94 and its vertebrate ortholog VCP have been characterized as key mediators involved in ER-associated degradation (ERAD), a major quality control process in the protein secretary pathway. Additional investigations have demonstrated degradation of proteins with no obvious relationship to ERAD by a VCP-mediated process, suggesting that TER94 and VCP may perform general functions in the proteolysis of ubiquitylated proteins. However, it remains unknown how this process is regulated under physiological conditions. The current study presents evidence that TER94-dependent degradation of ubiquitylated proteins is enhanced by PTP52F-mediated dephosphorylation of the penultimate Y800 residue. It has been suggested that the penultimate tyrosine (Y805 in VCP and Y800 in TER94) must be in a dephosphorylated form in order to interact with substrate-processing cofactors, such as the peptides N-glycanase (PNGase) and Ufd3, during UPS-mediated proteolysis. In addition, tyrosine phosphorylation levels of VCP/TER94 determine how fast ubiquitylated proteins are degraded by the USP pathway. Clearly, the finding that PTP52F is responsible for dephosphorylation of the penultimate tyrosine residue is critical for uncovering the functional role of VCP/TER94 in the regulation of protein degradation under physiologically relevant conditions (Santhanam, 2014).

This study has demonstrated that the timely degradation of DIAP1 in doomed larval midgut of developing flies is regulated by ecdysone-induced PTP52F. DIAP1 was identified to ubiquitylate proapoptotic proteins in living cells, thereby suppressing cell death signaling. Interestingly, DIAP1 can be ubiquitylated for degradation itself. The proteolytic process of ubiquitylated DIAP1 remained unclear until a recent report suggesting that TER94-mediated UPS pathway is involved in this process. This study has further shown that it is the dephosphorylated form of TER94 that is responsible for rapid DIAP1 degradation. In addition, although a previous study suggested that DIAP1 might suppress Atg1-mediated PCD, it was not known whether degradation of ubiquitylated DIAP1 could promote autophagy in vivo. This study has explored the underlying mechanism through which autophagic cell death is initiated by degradation of DIAP1. The data show that the constitutively tyrosine-phosphorylated form of TER94 acts as a gatekeeper ensuring the death signaling downstream of DIAP1 in'switch-off' mode. Developmental stage-dependent dephosphorylation of TER94 by inducible expression of PTP52F converts the autophagic death signaling into 'switch-on' mode through degradation of DIAP1. These findings thus explain, at least in part, how the massive destruction of larval midgut is precisely controlled by autophagic cell death. In conclusion, this study shows a novel function of PTP52F involved in the onset of autophagy and apoptosis essential for the removal of obsolete midgut tissues. Reversible tyrosine phosphorylation signaling controlled by PTP52F plays an indispensable role in the process of cell death-directed midgut maturation. Therefore, these findings open a new avenue for understanding the previously unexplored function of R-PTPs linked to regulation of autophagic and apoptotic cell death (Santhanam, 2014).

Dpp regulates autophagy-dependent midgut removal and signals to block ecdysone production

Animal development and homeostasis require the programmed removal of cells. Autophagy-dependent cell deletion is a unique form of cell death often involved in bulk degradation of tissues. In Drosophila the steroid hormone ecdysone controls developmental transitions and triggers the autophagy-dependent removal of the obsolete larval midgut. The production of ecdysone is exquisitely coordinated with signals from numerous organ systems to mediate the correct timing of such developmental programs. This study shows that blocking Dpp signaling induces premature autophagy, rapid cell death, and midgut degradation, whereas sustained Dpp signaling inhibits autophagy induction. Furthermore, Dpp signaling in the midgut prevents the expression of ecdysone responsive genes and impairs ecdysone production in the prothoracic gland. It is proposed that Dpp has dual roles: one within the midgut to prevent improper tissue degradation, and one in inter-organ communication to coordinate ecdysone biosynthesis and developmental timing (Denton, 2018).

Apoptosis restores cellular density by eliminating a physiologically or genetically induced excess of enterocytes in the Drosophila midgut

Using pathogens or high levels of opportunistic bacteria to damage the gut, studies in Drosophila have identified many signaling pathways involved in gut regeneration. Dying cells emit signaling molecules that accelerate intestinal stem cell proliferation and progenitor differentiation to replace the dying cells quickly. This process has been named 'regenerative cell death'. This study, mimicking environmental conditions, showed that the ingestion of low levels of opportunistic bacteria was sufficient to launch an accelerated cellular renewal program despite the brief passage of bacteria in the gut and the absence of cell death and this is is due to the moderate induction of the JNK pathway that stimulates stem cell proliferation. Consequently, the addition of new differentiated cells to the gut epithelium, without preceding cell loss, leads to enterocyte overcrowding. Finally, it was shown that a couple of days later, the correct density of enterocytes is promptly restored by means of a wave of apoptosis involving Hippo signaling and preferential removal of old enterocytes (Loudhaief, 2017).

Cp1/cathepsin L is required for autolysosomal clearance in Drosophila

Macroautophagy/autophagy is a highly conserved lysosomal degradative pathway important for maintaining cellular homeostasis. Much of the current knowledge of autophagy is focused on the initiation steps in this process. Recently, an understanding of later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling, is becoming evident. Autophagy can function in both cell survival and cell death, however, the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remain to be established. Using proteomic analysis of Drosophila larval midguts during degradation, this study identified a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. Cp1/cathepsin L-deficient larval midgut cells accumulate aberrant autophagic vesicles due to a block in autophagic flux, yet later stages of midgut degradation are not compromised. The accumulation of large aberrant autolysosomes in the absence of Cp1 appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material, rather than a defect in autophagosome-lysosome fusion. Finally, this study found that other cathepsins may also contribute to proper autolysosomal degradation in Drosophila larval midgut cells. These findings provide evidence that cathepsins play an essential role in the autolysosome to maintain basal autophagy flux by balancing autophagosome production and turnover (Xu, 2020).

Suppression of insulin production and secretion by a Decretin hormone

Decretins, hormones induced by fasting that suppress insulin production and secretion, have been postulated from classical human metabolic studies. From genetic screens, this study identified Drosophila Limostatin (Lst), a peptide hormone that suppresses insulin secretion. Lst is induced by nutrient restriction in gut-associated endocrine cells. limostatin deficiency leads to hyperinsulinemia, hypoglycemia, and excess adiposity. A conserved 15-residue polypeptide encoded by limostatin suppresses secretion by insulin-producing cells. Targeted knockdown of CG9918, a Drosophila ortholog of mammalian Neuromedin U receptors (NMURs), in insulin-producing cells phenocopied limostatin deficiency and attenuated insulin suppression by purified Lst, suggesting CG9918 encodes an Lst receptor. Human NMUR1 is expressed in islet &beta; cells, and purified NMU suppressed insulin secretion from human islets. A human mutant NMU variant that co-segregates with familial early-onset obesity and hyperinsulinemia failed to suppress insulin secretion. The study proposes Lst as an index member of an ancient hormone class called decretins, which suppress insulin output. (Alfa, 2015).

The coupling of hormonal responses to nutrient availability is fundamental for metabolic control. In mammals, regulated secretion of insulin from pancreatic b cells is a principal hormonal response orchestrating metabolic homeostasis. Circulating insulin levels constitute a dynamic metabolic switch, signaling the fed state and nutrient storage (anabolic pathways) when elevated, or starvation and nutrient mobilization (catabolic path ways) when decreased. Thus, insulin secretion must be precisely tuned to the nutritional state of the animal. Increased circulating glucose stimulates b cell depolarization and insulin secretion. In concert with glucose, gut-derived incretin hormones amplify glucose-stimulated insulin secretion (GSIS) in response to ingested carbohydrates, thereby tuning insulin output to the feeding state of the host (Alfa, 2015).

While the incretin effect on insulin secretion during feeding is well-documented, counter-regulatory mechanisms that suppress insulin secretion during or after starvation are incompletely understood. Classical starvation experiments in humans and other mammals revealed that sustained fasting profoundly alters the dynamics of insulin production and secretion, resulting in impaired glucose tolerance, relative insulin deficits, and 'starvation diabetes'. Remarkably, starvation-induced suppression of GSIS was not reverted by normalizing circulating glucose levels, suggesting that the dampening effect of starvation on insulin secretion perdures and is uncoupled from blood glucose and macronutrient concentrations. Based on these observations, it has been postulated that hormonal signals induced by fasting may actively attenuate insulin secretion suggested that enteroendocrine 'decretin' hormones may constrain the release of insulin to prevent hypoglycemia. This concept is further supported by recent studies identifying a G protein that suppresses insulin secretion from pancreatic b cell. Thus, after nutrient restriction, decretin hormones could signal through G protein-coupled receptors (GPCRs) to attenuate GSIS from b cells (Alfa, 2015).

The discovery of hormonal pathways regulating metabolism in mammals presents a formidable challenge. However, progress has revealed conserved mechanisms of metabolic regulation by insulin and glucagon-like peptides in Drosophila, providing a powerful genetic model to address unresolved questions relevant to mammalian metabolism. Similar to mammals, secretion of Drosophila insulin-like peptides (Ilps) from neuroendocrine cells in the brain regulates glucose homeostasis and nutrient stores in the fly. Ilp secretion from insulin-producing cells (IPCs) is responsive to circulating glucose and macronutrients and is suppressed upon nutrient withdrawal. Notably, recent studies have identified hormonal and GPCR-linked mechanisms regulating the secretion of Ilps from IPCs, suggesting further conservation of pathways regulating insulin secretion in the fly (Alfa, 2015).

In mammals, the incretin hormones gastric inhibitory peptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by enteroendocrine cells following a meal and enhance glucose-stimulated insulin production and secretion from pancreatic b cells. Thus, It was postulated that a decretin hormone would have the 'opposite' hallmarks of incretins. Specifically, a decretin (1) derives from an enteroendocrine source that is sensitive to nutrient availability, (2) is responsive to fasting or carbohydrate deficiency, and (3) suppresses insulin production and secretion from insulin-producing cells. However, like incretins, the action of decretins on insulin secretion would be manifest during feeding, when a stimulus for secretion is present (Alfa, 2015).

This study identifed a secreted hormone, Limostatin (Lst), that suppresses insulin secretion following starvation in Drosophila. lst is regulated by starvation, and flies deficient for lst display phenotypes consistent with hyperinsulinemia. Lst production was shown to be localized to glucose-sensing, endocrine corpora cardiaca (CC) cells associated with the gut, and show that lst is suppressed by carbohydrate feeding. Using calcium imaging and in vitro insulin secretion assays, a 15-aa Lst peptide (Lst-15) was identified that is sufficient to suppress activity of IPCs and Ilp secretion. An orphan GPCR was identified in IPCs as a candidate Lst receptor. Moreover, Neuromedin U (NMU) is likely a functional mammalian ortholog of Lst that inhibits islet b cell insulin secretion. These results establish a decretin signaling pathway that suppresses insulin output in Drosophila (Alfa, 2015).

Limostatin is a peptide hormone induced by carbohydrate restriction from endocrine cells associated with the gut that suppresses insulin production and release by insulin-producing cells. Thus, Drosophila Lst fulfills the functional criteria for a decretin and serves as an index member of this hormone class in metazoans. Results here also show that Lst signaling from corpora cardica cells may be mediated by the GPCR encoded by CG9918 in insulin-producing cells. In addition, the results reveal cellular and molecular features of a cell-cell signaling system in Drosophila with likely homologies to a mammalian entero-insular axis (Alfa, 2015).

Reduction of nutrient-derived secretogogues, like glucose, is a primary mechanism for attenuating insulin output during starvation in humans and flies. Consistent with this, it was found that circulating Ilp2HF levels were reduced to a similar degree in lst mutant or control flies during prolonged fasting. Therefore, lst was dispensable for Ilp2 reduction during fasting. However, lst mutants upon refeeding or during subsequent ad libitum feeding had enhanced circulating Ilp2HF levels compared to controls, findings that demonstrate a requirement for Lst to restrict insulin output in fed flies. Thus, while induced by nutrient restriction, Lst decretin function was revealed by nutrient challenge. This linkage of feeding to decretin regulation of insulin output is reminiscent of incretin regulation and action (Campbell, 2013; Alfa, 2015 and references therein).

Recent studies have demonstrated functional conservation in Drosophila of fundamental hormonal systems for metabolic regulation in mammals, including insulin, glucagon, and leptin (Rajan, 2012). This study used Drosophila to identify a hormonal regulator of insulin output, glucose, and lipid metabolism without an identified antecedent mammalian ortholog -- emphasizing the possibility for work on flies to presage endocrine hormone discovery in mammals. Gain of Lst function in these studies led to reduced insulin signaling, and hyperglycemia, consistent with prior work. By contrast, loss of Lst function led to excessive insulin production and secretion, hypoglycemia, and elevated triglycerides, phenotypes consistent with the recognized anabolic functions of insulin signaling in metazoans, and with the few prior metabolic studies of flies with insulin excess (Alfa, 2015).

Prior studies show that somatostatin and galanin are mammalian gastrointestinal hormones that can suppress insulin secretion. Somatostatin-28 (SST-28) is a peptide derivative of the pro-somatostatin gene that is expressed widely, including in gastrointestinal cells and pancreatic islet cells. Islet somatostatin signaling is thought to be principally paracrine, rather than endocrine, and serum SST-28 concentrations increase post-prandially. Galanin is an orexigenic neuropeptide produced throughout the CNS and in peripheral neurons and has been reported to inhibit insulin secretion. Unlike enteroendocrine-derived hormones that act systemically, galanin is secreted from intrapancreatic autonomic nerve terminals and is thought to exert local effects. In addition, Galanin synthesis and secretion are increased by feeding and dietary fat. Thus, like incretins, output of SST- 28 and galanin are induced by feeding, but in contrast to incretins, these peptides suppress insulin secretion. Further studies are needed to assess the roles of these peptide regulators in the modulation of insulin secretion during fasting (Alfa, 2015).

While sequence-based searches did not identify vertebrate orthologs of Lst, this study found that the postulated Lst receptor in IPCs, encoded by CG9918, is most similar to the GPCRs NMUR1 and NMUR2. In rodents, NMU signaling may be a central regulator of satiety and feeding behavior, and this role may be conserved in other organisms. In addition, NMU mutant mice have increased adiposity and hyperinsulinemia, but a direct role for NMU in regulating insulin secretion by insulin-producing cells was not identified. In rodents, the central effects of NMU on satiety are thought to be mediated by the receptor NMUR2; however, hyperphagia, hyperinsulinemia, and obesity were not reported in NMUR2 mutant mice. Together, these studies suggest that a subset of phenotypes observed in NMU mutant mice may instead reflect the activity of NMU on peripheral tissues like pancreatic islets, but this has not been previously shown. Notably, humans harboring the NMU R165W allele displayed obesity and elevated insulin C-peptide levels, without evident hyperphagia -- further suggesting that the central and peripheral effects of NMU reflect distinct pathways that may be uncoupled. This study has shown that NMU is produced abundantly in human foregut organs and suppresses insulin secretion from pancreatic b cells, supporting the view that NMU has important functions outside the CNS in regulating metabolism. Thus, like the incretin GLP-1, NMU may have dual central and peripheral signaling functions in the regulating metabolism. Demonstration that NMU is a mammalian decretin will require further studies on NMU regulation and robust methods to measure circulating NMU levels in fasting and re-feeding. In summary, these findings should invigorate searches for mammalian decretins with possible roles in both physiological and pathological settings (Alfa, 2015).

A subset of enteroendocrine cells is activated by amino acids in the Drosophila midgut

The intestine is involved in digestion and absorption, as well as the regulation of metabolism upon sensation of the internal intestinal environment. Enteroendocrine cells are thought to mediate these internal intestinal chemosensory functions. Using the CaLexA (calcium-dependent nuclear import of LexA) method, this study examined the enteroendocrine cell populations that are activated when flies are subjected to various dietary conditions such as starvation, sugar, high fat, protein, or pathogen exposure. A specific subpopulation of enteroendocrine cells in the posterior midgut that express Dh31 and tachykinin was found to be activated by the presence of proteins and amino acids (Park, 2016).

To study the chemosensory functions of enteroendocrine cells in the Drosophila midgut, a method was needed to specifically label as many enteroendocrine cells as possible. In an independent study, Drosophila regulatory peptide genes were sought that are expressed in the enteroendocrine cells. The sum of expression of three regulatory peptide-GAL4 drivers (AstC-, Npf-, and Dh31-GAL4; for convenience, hereafter these three drivers together will be called EE-GAL4) were found to be expressed in about 80% of enteroendocrine cells in the midgut (Park, 2016).

To visualize in vivo changes in cytoplasmic calcium ion concentration, the CaLexA (calcium-dependent nuclear import of LexA) system, which uses a calcium ion-sensitive synthetic transcription factor, was used. When calcium levels rise, modified nuclear factor of activated T cells (NFAT) is imported into the nucleus to transcriptionally activate GFP reporter expression. This CaLexA system has been successfully used to visualize neuronal activation and calcium ion changes in fat body tissue (Park, 2016).

First, whether the CaLexA system could be used to monitor enteroendocrine cell activation, was tested. For this purpose, modified NFAT was expressed in enteroendocrine cells using EE-GAL4. GFP expression in the midgut was monitored after feeding adult flies with 200 mm sucrose for 5 days after eclosion. Sucrose was provided as a minimal nutrient source. Strong GFP expression was observed only in enteroendocrine cells in the middle midgut, while most enteroendocrine cells in the anterior and posterior midgut did not express GFP. Next, whether various dietary conditions could activate the enteroendocrine cells was tested, as well as whether stimuli or enteroendocrine cell specificity exists. Enteroendocrine cell activation in the middle midgut was observed for every tested condition including starvation, indicating that the middle midgut enteroendocrine cells are constitutively activated. The middle midgut is an acidic region, and acid secretion from the enteroendocrine cells likely constitutively occurs to maintain such an environment (Park, 2016).

To quantitate enteroendocrine cell activation using the CaLexA system, the numbers of GFP-expressing cells was counted. GFP-expressing cells in the caudal half of the posterior midgut were counted, since enteroendocrine cell activation upon exposure to various dietary conditions was concentrated to this region. Only GFP-expressing cells costaining with Prospero were counted, excluding autofluorescent signals from food particles. These nonspecific signals can be observed as white dots that do not costain with Prospero, as seen for example in starvation or 50 mm NaCl conditions. When flies were provided with a single sugar diet composed of only sucrose, 14 ± 1.5 SEM GFP-expressing cells were observed in the caudal half of the posterior midgut. This can be considered a baseline for all of the conditions tested, with the exception of starvation, normal fly food, and high fat diet, since 200 mm sucrose was provided in all but these three conditions to induce a quantifiable level of feeding even in adverse conditions. A small number of posterior midgut enteroendocrine cells were activated when cornmeal-based fly food, commonly used in the lab, was provided. Significant activation was not observed when flies were exposed to conditions such as a diet of normal food with high fat composition, starvation, or a diet of 200 mm sucrose with the addition of 50 mm NaCl. In contrast, many enteroendocrine cells were activated in the posterior midgut upon oral infection with Erwinia carotovora carotovora 15 (Ecc15), which causes a gut immune response. Enteroendocrine cell activation was observed at a similar level when flies were fed Pseudomonas aeruginosa, indicating that the enterondocrine cell response is not specific to a particular bacterial species. Enteroendocrine cell activation was also observed upon feeding on heat-inactivated Ecc15 or P. aeruginosa, at slightly decreased levels compared to untreated bacteria, but still higher than the 200 mm sucrose control flies. In contrast, enteroendocrine cells were not activated upon flies being fed uracil, which causes a gut immune response through acting as a DUOX-activating ligand in the Drosophila gut epithelia. These results indicated that enteroendocrine cell activation is not due to the detection of pathogenicity. Supporting this conclusion, ingestion of nonpathogenic E. coli or yeast also caused enteroendocrine cell activation. On the basis of these results, it is hypothesized that the enteroendocrine cells were being activated by the presence of protein. Enteroendocrine cell activation was observed in a dose-dependent manner when flies were fed various concentrations of casein peptone, providing evidence that protein cues were activating the enteroendocrine cells. Next, the twenty amino acids were all individually tested for enteroendocrine cell activation. The amino acids caused enteroendocrine cell activation, indicating that enteroendocrine cells are activated upon detection of most, if not all, amino acids. Aspartic acid and glutamic acid, which contain negatively charged side chains, caused a relatively low number of activated cells, but this is likely due to the lower concentration used due to solubility issues. The number of activated cells also increased in a dose-dependent manner, suggesting that these amino acids act as cues to activate enteroendocrine cells. For similar reasons, poorly water-soluble tyrosine also cannot be excluded as a potential activation cue for enteroendocrine cells (Park, 2016).

Enteroendocrine cells in the posterior midgut are activated by protein and amino acids. GFP expression pattern induced by the CaLexA system in the caudal half of the posterior midgut upon exposure to the indicated dietary conditions (Park, 2016).

Enteroendocrine cell activation by microorganisms, casein peptone, and amino acids was concentrated in enteroendocrine cells of the posterior midgut, in particular the caudal half. Enteroendocrine cells in this region can be largely divided into two populations, with one population expressing the regulatory peptides AstA and AstC, and the other expressing Dh31 and tachykinin. To examine which population of enteroendocrine cells in the caudal half of the posterior midgut are activated by microorganisms, casein peptone, and amino acids, the enteroendocrine cells were costained with AstA or Dh31 antisera. Although EE-GAL4 is expressed in both populations of enteroendocrine cells in the caudal half of the posterior midgut, most of the activated enteroendocrine cells belonged to the Dh31-expressing cell population. When Ecc15 was provided, an average of 68 cells label with Dh31 antiserum in the corresponding region, and 54 out of 61 cells that express GFP through the CaLexA system co-stain with Dh31 antiserum. Under the same conditions, an average of 96 cells are labeled by AstA antiserum, and only two of 64 cells expressing GFP through the CaLexA system co-stain with AstA antiserum . In conclusion, amino acids in proteins activate a specific subset of Dh31- and tachykinin-expressing enteroendocrine cells in the posterior midgut through an as yet unknown mechanism. Since the activation of these cells appears to be unrelated to pathogenicity of the protein source, it seems unlikely that these enteroendocrine cells are directly involved in eliciting an immune response to pathogens. The role of this enteroendocrine cell subgroup thus appears to mainly be detection of a potential nutrient source through its protein content. It is unclear whether activation of these cells by amino acids influences the production and/or secretion of Dh31 or other regulatory peptides. Weaker Dh31 antibody staining was not observed in GFP-expressing cells, which would be expected if Dh31 secretion was enhanced in the activated cells. It is formally possible that Dh31 secretion is enhanced in activated cells, and production is increased to compensate for the increased secretion, but the data are insufficient to provide support for either scenario. Tachykinin production is enhanced in the midgut upon nutrient deprivation, resulting in repression of lipogenesis in enterocytes. However, in this study, the enteroendocrine cells activated by proteins and amino acids were not activated upon starvation, and sucrose was coprovided as a minimal nutrient in all dietary conditions providing proteins and amino acids, with the sucrose-only basal level acting as a baseline for activation. This indicates that proteins and amino acids, and not other cues such as carbohydrate deprivation, act as specific cues to activate a particular subset of enteroendocrine cells. The molecular identity of potential protein or amino acid receptors in the enteroendocrine cells is as yet unknown. In mammals, several amino acids including L-glutamine have been shown to stimulate GLP1 secretion in vitro. CaSR, a primarily Gq-coupled calcium-sensing receptor, is expressed in the enteroendocrine cells, and CaSR activation has been associated with amino acid-stimulated gut hormone secretion. The umami taste receptor dimer T1R1/T1R3 and GPRC6A are also additional candidates for mediating GLP1 release in response to amino acids. This work provides an in vivo enteroendocrine system to investigate such possible amino acid receptors (Park, 2016).

This study has shown that the CaLexA system can be used to monitor the activation of Drosophila midgut enteroendocrine cells upon exposure to specific stimuli. Activation of enteroendocrine cells upon exposure to sugar, fat, and bitter compounds was not observed using this method. It was found that proteins and most amino acids are capable of activating enteroendocrine cells in the posterior midgut. These activated cells are limited to a subpopulation of enteroendocrine cells that secrete specific regulatory peptides including Dh31 and tachykinin. This study provides an important step in studying the chemosensory functions of enteroendocrine cells (Park, 2016).

A subset of neurons controls the permeability of the peritrophic matrix and midgut structure in Drosophila adults

The metazoan gut performs multiple physiologic functions, including digestion and absorption of nutrients, and also serves as a physical and chemical barrier against ingested pathogens and abrasive particles. Maintenance of these functions and structures is partly controlled by the nervous system, yet the precise roles and mechanisms of the neural control of gut integrity remain to be clarified in Drosophila. This study screened for GAL4 enhancer-trap strains and labeled specific subsets of neurons. The strong inward rectifier potassium channel Kir2.1 was used to inhibit their neuronal activity. An NP3253 line was identified that is susceptible to oral infection by Gram-negative bacteria. The subset of neurons driven by the NP3253 line includes some of the enteric neurons innervating the anterior midgut, and these flies have a disorganized proventricular structure with high permeability of the peritrophic matrix and epithelial barrier. The findings of the present study indicate that neural control is crucial for maintaining the barrier function of the gut, and provide a route for genetic dissection of the complex brain-gut axis in the model organism Drosophila adults (Kenmoku, 2016).

Metamorphosis of the Drosophila visceral musculature and its role in intestinal morphogenesis and stem cell formation

This study has combined the use of specific markers with electron microscopy to follow the formation of the adult visceral musculature and its involvement in gut development during metamorphosis. Unlike the adult somatic musculature, which is derived from a pool of undifferentiated myoblasts, the visceral musculature of the adult is a direct descendant of the larval fibers, as shown by activating a lineage tracing construct in the larval muscle and obtaining labeled visceral fibers in the adult. However, visceral muscles undergo a phase of remodeling that coincides with the metamorphosis of the intestinal epithelium. During the first day following puparium formation, both circular and longitudinal syncytial fibers dedifferentiate, losing their myofibrils and extracellular matrix, and dissociating into mononuclear cells ("secondary myoblasts"). Towards the end of the second day, this process is reversed, and between 48 and 72h after puparium formation, a structurally fully differentiated adult muscle layer has formed. The musculature, the intestinal epithelium is completely renewed during metamorphosis. The adult midgut epithelium rapidly expands over the larval layer during the first few hours after puparium formation; in case of the hindgut, replacement takes longer, and proceeds by the gradual caudad extension of a proliferating growth zone, the hindgut proliferation zone (HPZ). The subsequent elongation of the hindgut and midgut, as well as the establishment of a population of intestinal stem cells active in the adult midgut and hindgut, requires the presence of the visceral muscle layer, based on the finding that ablation of this layer causes a severe disruption of both processes (Aghajanian, 2016).

DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm

Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. This study used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). This study confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, this paper reports a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterizes the role of Kah in the regulation of embryonic midgut morphogenesis (Mendoza-Garcia, 2021).

Specification of FCs in the VM depends on Alk signaling in response to Jeb secretion from the somatic mesoderm Signaling via Alk activates the Ras/MAPK pathway, translocating the FCM fate-promoting TF Lameduck (Lmd) from the nucleus to the cytoplasm. A similar mechanism has been suggested for a still unknown FC-fate repressor triggering the FC-specific transcriptional program in the VM. This transcriptional program remains relatively unexplored with only a few identified targets reported, such as Hand, org-1, kirre, dpp and Alk itself. Although ChIP has been the predominant approach for mapping protein-chromatin interactions, it requires significant amounts of starting material and specific antibodies. RNA-seq has also been intensely employed for transcriptomic analyses and, although straightforward for cell culture studies, isolation of the VM would be required for its use in identifying Alk transcriptional targets in Drosophila . Therefore, in efforts to identify novel Alk transcriptional targets in the VM, TaDa was used, allowing genome-wide RNA PolII occupancy to be investigated in the specific tissue of choice. TaDa requires less starting material and provides cell-type specificity, although resolution can be less accurate compared with RNA-seq and ChIP-seq due to its dependency on frequency of GATC sites in the genome (Mendoza-Garcia, 2021).

The experimental design used in this study was based on either activating or inhibiting Alk signaling throughout the VM, followed by TaDa analysis. Comparison of the TaDa dataset with previously published RNA-seq data from cells isolated from the mesoderm suggest the data recapitulated endogenous binding of RNA PolII. The dataset also agreed with current understanding of Alk signaling and induction of cell fate specification in the trunk VM, including observed differential expression of previously identified Alk transcriptional targets, such as Hand, org-1, kirre and dpp . However, these FC-enriched targets were not the most significantly expressed genes within the TaDa dataset, perhaps reflecting lower levels or smaller temporal windows of active transcription that may additionally be complicated by differentially stable mRNA or protein products. Taken together, a combination of different analyses supported this approach as replicating transcriptional events in the VM and led to validation of TaDa-identified genes in the VM as targets of Alk-driven signaling events (Mendoza-Garcia, 2021).

A number of differentially expressed genes were validated by in situ hybridization during embryogenesis. mRNA was indeed visualized in the visceral musculature, and VM expression was confirmed in the HandC-GFP scRNA-seq dataset for fax, Sumo and CG11658. Expression of fax was observed in the VM and CNS. Interestingly, Fax has been identified in a screen for diet-regulated proteins in the Drosophila ovary. Insulin signaling in response to diet promotes activation of the Ribosomal protein S6 Kinase (S6K), which drives fax expression. Notably, Alk modulates insulin signaling in the brain during nutrient restriction, making Fax interesting for further study. Another interesting candidate is Sumo, which encodes the Drosophila SUMO-1 homologue. Sumo is not exclusively expressed in the VM, but is rather expressed maternally and ubiquitously throughout the embryo. Functional studies have identified a role for Sumo in the post-translational modification of several TFs, as well as in modulation of signaling in the fly. Overall, the TaDa analysis identified numerous uncharacterized genes, and further investigation will be crucial to decipher their role in the developing visceral muscles (Mendoza-Garcia, 2021).

One interesting uncharacterized target was Kah, which encodes a Snail family TF. Kah overexpression in the thorax has been reported to block development of thoracic bristles, revealing a potential to drive changes in cell identity (Singari, 2014). It was possible to validate Kah as an Alk target locus in the embryonic VM, with differences in Kah expression when Alk signaling was either blocked or activated. However, Alk-independent Kah transcription in the early VM was noted in addition to the Alk-modulated transcription that is reminiscent of the VM expression reported for org-1. Currently, the TFs downstream of Alk that regulate Kah transcription are unknown, although this will be interesting to study in the future (Mendoza-Garcia, 2021).

Alk signaling in the VM drives FC specification via Ras/MAPK pathway activation, leading to the transcription of FC-specific genes such as Hand, org-1, kirre, dpp and also Kah. Loss of Hand, org-1 and dpp does not alter FC specification in the VM, suggesting a complex temporal regulation that assures FC specification and eventually formation of visceral muscles. Characterization of Kah mutant alleles suggest that, similar to Hand and org-1, Kah is dispensable for VM FC specification, although formally Kah could be responsible for FC-specific transcriptional changes of yet unidentified targets (Mendoza-Garcia, 2021).

Kah mutants exhibit defects in midgut constriction formation. A number of players are implicated in this event, such as Wg, Dpp, Ultrabithorax (Ubx), Pnt, Extra macrochaetae (Emc) and Org-1. Interestingly, Alk signaling activity is important for VM Dpp expression and maintenance of Org-1 in FCs. Although it is difficult to define a role for Alk in VM events post FC specification, this study shows that Alk may indeed play a role in later midgut development. It is also noted that inappropriate activation of Alk signaling via Jeb expression results in a range of later gut defects. Thus, perturbation of Alk signaling appears to disrupt later events in midgut constriction. However, whether this is a consequence of earlier, Alk-dependent, visceral FC specification or refers to an independent role of Alk signaling in the VM is unclear. This study was particularly interested in Pnt, as this ETS domain TF has been reported by the FlyBi-project to bind Kah in high-throughput Y2H, and pnt mutant embryos also exhibit a midgut constriction phenotype. Like Kah, Pnt is not required for VM FC specification. The observation of low penetrance midgut constriction defects in transheterozygous KahΔATG/pntΔ88 embryos suggest that Kah and Pnt may function together in this process, which is further supported by live imaging, through which stronger midgut constriction phenotypes were observed in Kah, pntΔ88 double mutants than in pntΔ88 alone. Employing publicly available Kah-ChIP datasets, it was possible to define a Kah-binding motif similar to that of Snail. Analysis of publicly available Pnt-ChIP datasets identified numerous targets of both Kah and Pnt, including Kah itself, nej, put, Mad and Ras85D. Interestingly, although several components of the Dpp signaling appear to be misregulated in Kah mutants, wg and dpp expression appear normal, and robust pMAD activity is observed in both KahΔATG and KahΔZnF mutants. Earlier work reported that wg and dpp expression are also normal in the VM of pnt mutant embryos. It is likely that as yet unidentified players function downstream of Kah in this process, and it was not yet possible to identify a key component downstream of Kah that could explain the underlying molecular mechanisms. It is also important to note that, although this work focused on a role in the VM, Kah is also expressed in the embryonic SM and CNS. This analysis of Kah mutant RNA-seq together with Kah-ChIP and Pnt-ChIP datasets identifies candidates to be focused on in future studies (Mendoza-Garcia, 2021).

The TaDa approach successfully allowed identification of transcriptional targets of Alk signaling in the developing mesoderm, including the transcriptional regulator Kahuli. Many of these targets are currently uncharacterized and future studies should allow their function(s) in the VM to be elucidated. The in-depth study of Kah highlights a role for this TF in later visceral musculature development, where it appears to work in concert with other factors, including Pnt, to regulate midgut constriction. Combined ChIP and RNA-seq analyses highlights a group of interesting and largely uncharacterized genes, which should shed further light on the midgut constriction process (Mendoza-Garcia, 2021).

Regional cell-specific transcriptome mapping reveals regulatory complexity in the adult Drosophila midgut

Deciphering contributions of specific cell types to organ function is experimentally challenging. The Drosophila midgut is a dynamic organ with five morphologically and functionally distinct regions (R1-R5), each composed of multipotent intestinal stem cells (ISCs), progenitor enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs), and visceral muscle (VM). To characterize cellular specialization and regional function in this organ, RNA-sequencing transcriptomes were generated of all five cell types isolated by FACS from each of the five regions, R1-R5. In doing so, transcriptional diversities were identified among cell types, and regional differences within each cell type were documented that define further specialization. Cell-specific and regional Gal4 drivers were validated; roles for transporter Smvt and transcription factors GATAe, Sna, and Ptx1 in global and regional ISC regulation were demonstrated, and the transcriptional response of midgut cells upon infection was studied. The resulting transcriptome database (http://flygutseq.buchonlab.com) will foster studies of regionalization, homeostasis, immunity, and cell-cell interactions (Dutta, 2015).

In this analysis, genes highly expressed in ISCs were uncovered and enriched cis-regulatory motifs upstream of these "ISC-high" genes were identified. This approach identified GATAe, sna, Fos (Kayak), and Ptx1 as central regulators of ISC behavior and has paved the way for future studies on more factors that could influence ISC functionality. sna was confirmed as a regional ISC-high gene that regulates stem cell differentiation in the midgut similar to its paralog, escargot (esg). In contrast, GATAe functions in ISC maintenance, much like its mammalian homolog, GATA6, which is involved in the maintenance and proliferation of stem cells and colorectal cancer. Kay (the fly homolog of Fos) and Da had previously been identified as TFs that control ISC activity and fate. Thus, the results indicate that stem cell activity is controlled not by a single factor but by a combinatorial network of autonomously acting TFs such as GATAe, kayak, and sna and previously studied da, esg, and sc, which in concert regulate stemness in the midgut. This dataset could be a useful resource for identifying mammalian homologs with similar functions in stem and cancer cells and in understanding TF regulatory networks in mammals (Dutta, 2015).

The primary source of biotin for Drosophila is dietary yeast and enteric bacteria. Interestingly, this study shows that the biotin transporter Smvt is essential for ISC maintenance and homeostasis, signifying the importance of nutrients derived from symbiotic organisms in regulating intestinal homeostasis and ISC function (Dutta, 2015).

An evolutionarily conserved feature of the gastrointestinal tract is the division of function between specialized regions. This study reveals that all cell types of the Drosophila midgut have profound regional variation in their gene expression. Nevertheless, differences between intestinal cell types were even more pronounced than regional variations within a specific cell type. For all cell types, it was consistently found the cells of regions R1 and R3 to be vastly different from those of the other regions. This suggests that the specialization of all cells in a region is coordinated (Dutta, 2015).

In the gut epithelium proper, differentiated cells showed the most regionalization, with EEs being the most variant between regions. In agreement with recent studies documenting enteroendocrine cell diversity in the midgut, the results indicate that there are multiple subtypes of the hormone secreting EEs that likely have regionalized functions. EBs and ECs also showed clear regional specificities, including altered expression of genes involved in metabolism and digestion. Although lipases, glycoside hydrolases, and glucose transporters were highly expressed in the anterior midgut, serine endopeptidases and amino acid transporters were primarily expressed in the posterior midgut cells, consistent with the premise that digestion is highly compartmentalized (Dutta, 2015).

Remarkably, multipotent ISCs also displayed transcriptional variation along the length of the gut. Different ISC populations differ in the levels of effectors and targets in key signaling pathways, such as EGFR/Ras/MAPK, Wnt, and JAK/STAT, also showing distinct qualitative differences. Of note, ISCs of the acidic R3 express vacuolar H+ ATPases, indicating an adaptation of ISCs to their local environment. The tests showed that the R3-specific TFs Ptx1 and lab are important for maintaining these regional ISC regional properties. Additional regionalized TFs like exex and Drm were identified, and some of these are likely to control other regional properties. While regionalized gene expression almost certainly determines regional differences in cell morphology and function, enteric environment factors (e.g., microbiota and nutrition distributions) within the gut might also influence regional characteristics (Dutta, 2015).

The transcriptome of visceral muscles varied strongly by region, and studies have suggested that regional identities in the gut are maintained by gradients of morphogens. Accordingly, the morphogens Wg, Wnt 4, Wnt 6, WntD, Dpp, and Vn were expressed in gradients in VM cells, suggesting roles for these components of the stem cell niche in defining a region's transcriptional signature. However, it was noticed that spatial expression of lowly expressed ligands like Dpp and Upd3 varied from previous reports, and thus, this dataset should be used with discretion in such cases. Solely based on gene expression data, it cannot be determined whether the ISCs or niche cells such as VMs are primary in establishing and maintaining regionalization. It will be interesting to test whether differences in the niche are driven by gut-extrinsic factors or whether ISCs engineer their own niche through self-reinforcing feedback, for instance, by epigenetic programming of daughter cells. It is hoped that these data will guide future experiments that test the cross-talk between stem cells and the niche in midgut regionalization (Dutta, 2015).

This study uncovered an unexpected role for EEs as potential players in the immune response to pathogens by inducing AMP expression along with ECs and EBs. Interestingly, the different cell types produce different combinations of AMPs, suggesting a specialization by cell type in the immune response, as also found in the mammalian digestive tract. A model is proposed wherein infection either directly activates EEs to express AMPs or the damaged ECs signal ISCs to proliferate and EEs to produce AMPs (see Cell-Type-Specific Responses upon Infection with P. entomophila). Further studies will be required to clearly define the response of EEs to infection (Dutta, 2015).

In conclusion, this study has systematically characterized transcription by genomic analysis of regions and cell types in the Drosophila midgut. Gut regionalization is a critical factor for human health, as diseases of intestinal origin are often regionalized. It is hoped that the provided dataset (http://flygutseq.buchonlab.com/) will help to pave the way for future studies that elucidate the complex interplay among midgut cells, regions, and microbes that will promote understanding of gut physiology and homeostasis (Dutta, 2015).

Misregulation of an adaptive metabolic response contributes to the age-related disruption of lipid homeostasis in Drosophila

Loss of metabolic homeostasis is a hallmark of aging and is commonly characterized by the deregulation of adaptive signaling interactions that coordinate energy metabolism with dietary changes. The mechanisms driving age-related changes in these adaptive responses remain unclear. This study characterized the deregulation of an adaptive metabolic response and the development of metabolic dysfunction in the aging intestine of Drosophila. Activation of the insulin-responsive transcription factor Foxo in intestinal enterocytes was found to be required to inhibit the expression of evolutionarily conserved lipases as part of a metabolic response to dietary changes. This adaptive mechanism becomes chronically activated in the aging intestine, mediated by changes in Jun-N-terminal kinase (JNK) signaling. Age-related chronic JNK/Foxo activation in enterocytes is deleterious, leading to sustained repression of intestinal lipase expression and the disruption of lipid homeostasis. Changes in the regulation of Foxo-mediated adaptive responses thus contribute to the age-associated breakdown of metabolic homeostasis (Karpac, 2013).

This work identifies Foxo-mediated repression of intestinal lipases as a critical component of an adaptive response to dietary changes in Drosophila. Interestingly, misregulation of this metabolic response also contributes to the age-associated breakdown of lipid homeostasis, as elevated JNK signaling leads to chronic Foxo activation and subsequent disruption of lipid metabolism due to chronic repression of lipases. This age-related deregulation of an adaptive metabolic response is reminiscent of insulin resistance-like phenotypes in vertebrates, which can also be triggered by chronic activation of JNK, and thus highlights the antagonistic pleiotropy inherent in metabolic regulation. The adaptive nature of signaling interactions that drive pathology (such as JNK-mediated insulin resistance) has remained elusive in many instances, and this work provides a model for age-related changes in an adaptive regulatory process that ultimately lead to a pathological outcome. It is believed that this system can serve as a productive model to address a number of interesting questions with relevance to the loss of metabolic homeostasis in aging organisms (Karpac, 2013).

In mammals, JNK has been shown to promote insulin resistance both cell-autonomously and systemically (through inflammation), subsequently affecting lipid homeostasis in various tissues. The current results further introduce a mechanism by which JNK can alter cellular and systemic lipid metabolism through the regulation of lipases, independent of changes in IIS. Thus, JNK-mediated Foxo activation in select tissues may be able to alter intracellular lipid metabolism, changing metabolic fuel substrates and disrupting metabolic homeostasis in other tissues without altering insulin responsiveness (Karpac, 2013).

Whereas the current data show that Foxo activation leads to the transcriptional repression of intestinal lipases, especially LipA/Magro, it remains unclear if this control is direct or indirect. Foxo is classically described as an activator of transcription, but recent reports have shown that Foxo can transcriptionally repress genes through direct association with promoters. The promoter regions of LipA/Magro and CG6295 do not contain conserved Foxo transcription factor binding sites, suggesting that the regulation of these genes may be indirect, potentially through Foxo-regulated expression of secondary effectors. Thus, tissue-specific control of lipid homeostasis by IIS/Foxo might be achieved through the regulation of lipogenic or lipolytic transcription factors that can elicit global and direct changes in cellular lipid metabolism. Previous reports have shown that the nuclear receptor dHR96, a critical regulator of lipid and cholesterol homeostasis, promotes lipA/magro expression. However, dhr96 expression is upregulated in aging intestines, suggesting that the age-related repression of intestinal lipases is not merely due to decreases in dHR96 levels. dhr96 transcript levels are strongly induced in genetic conditions where Foxo is activated and intestinal lipases are repressed, again suggesting that Foxo does not mediate its effects on lipase transcription by antagonizing dhr96 expression. Furthermore, age-related changes that are independent of JNK/Foxo activation may also contribute to the repression of intestinal lipase expression and disruption of lipid metabolism, such as an age-associated decline in feeding/food intake (Karpac, 2013).

The reasons for the increase in JNK and Foxo activity in aging enterocytes remain to be explored. Buchon (2009) has also shown that age-related activation of JNK in the intestinal epithelium is dependent on the presence of commensal bacteria, as maintaining animals axenically reduces activation of JNK in the first 30 days of life. Thus, bacteria-induced inflammation and subsequent JNK activation appears to be a likely cause, in part, for age-related increases in Foxo activity. In a separate study, however, this laboratory found that Foxo activation still occurs in intestines of old (40-day-old), axenically reared flies, suggesting that age-related activation of Foxo may also occur through JNK-independent processes. Supporting this idea, the results show that inhibiting JNK function in enterocytes can attenuate, although not completely inhibit, this Foxo activation. Additional factors, such as sirtuins or histone deacetylases, recently shown to deacetylate and activate Foxo in response to endocrine signals, may also lead to age-related increases in intestinal Foxo activity (Karpac, 2013).

Interactions between JNK and IIS/Foxo are mediated by various mechanisms. In mammals, JNK phosphorylates the insulin receptor substrate (IRS), inhibiting insulin signaling transduction. Whereas JNK has clearly been shown to antagonize IIS (activate Foxo) in C. elegans and Drosophila, that exact mechanism by which Foxo activation is achieved may be divergent in mammals. For example, no IRS homolog has been identified in worms, and the JNK phosphorylation site in mammalian IRS is not conserved in flies. The current data show that JNK-mediated Foxo activation in the aging fly intestine is not achieved through IIS antagonism upstream of Akt, suggesting either a direct interaction between Foxo and JNK or changes in other regulators of Foxo. Recent studies have shown that JNK-mediated phosphorylation of 14-3-3 proteins results in the release of their binding partners, including Foxo. The conservation of 14-3-3 proteins between vertebrates and invertebrates makes 14-3-3 an interesting candidate in promoting Foxo function via JNK in the aging fly intestine. This chronic intestinal Foxo activation and subsequent metabolic changes, provide a physiological system in Drosophila to genetically dissect the crosstalk between IIS/Foxo and JNK signaling. Detailed analysis of these signaling interactions promises to provide important insight into the pleiotropic effects of IIS/Foxo function and the pathogenesis of age-related metabolic diseases (Karpac, 2013).

The data further reveal the pleiotropic consequences of Foxo activation in regard to healthspan and longevity in Drosophila. Overexpressing Foxo in the fat body or muscle of flies leads to lifespan extension. The data presented here show that chronic Foxo activation in intestinal enterocytes disrupts lipid metabolism by deregulating intestinal lipases and thus highlight how cell- and tissue-specific consequences of Foxo function play an important role in determining either the beneficial (i.e., lifespan extension) or pathological (i.e., disruption of lipid metabolism) outcome of Foxo activation (Karpac, 2013).

Recent work in C. elegans has begun to explore the relationship between lipid metabolism and longevity, revealing that increases in intestinal lipase expression can extend lifespan. The beneficial effects of elevated lipase expression appear to be mediated by increases in specific types of fatty acids, which can activate autophagy and lead to lifespan extension. The current study identifies Foxo-mediated repression of intestinal lipases as a critical component of an adaptive response to dietary changes in Drosophila. Interestingly, misregulation of this metabolic response also contributes to the age-associated breakdown of lipid homeostasis, as elevated JNK signaling leads to chronic Foxo activation and subsequent disruption of lipid metabolism due to chronic repression of lipases. This age-related deregulation of an adaptive metabolic response is reminiscent of insulin resistance-like phenotypes in vertebrates, which can also be triggered by chronic activation of JNK, and thus highlights the antagonistic pleiotropy inherent in metabolic regulation. The adaptive nature of signaling interactions that drive pathology (such as JNK-mediated insulin resistance) has remained elusive in many instances, and the current work provides a model for age-related changes in an adaptive regulatory process that ultimately lead to a pathological outcome. This system can serve as a productive model to address a number of interesting questions with relevance to the loss of metabolic homeostasis in aging organisms (Karpac, 2013).

The results further introduce a mechanism by which JNK can alter cellular and systemic lipid metabolism through the regulation of lipases, independent of changes in IIS. Thus, JNK-mediated Foxo activation in select tissues may be able to alter intracellular lipid metabolism, changing metabolic fuel substrates and disrupting metabolic homeostasis in other tissues without altering insulin responsiveness (Karpac, 2013).

Whereas the data show that Foxo activation leads to the transcriptional repression of intestinal lipases, especially LipA/Magro, it remains unclear if this control is direct or indirect. Foxo is classically described as an activator of transcription, but recent reports have shown that Foxo can transcriptionally repress genes through direct association with promoters. The promoter regions of LipA/Magro and CG6295 do not contain conserved Foxo transcription factor binding sites, suggesting that the regulation of these genes may be indirect, potentially through Foxo-regulated expression of secondary effectors. Thus, tissue-specific control of lipid homeostasis by IIS/Foxo might be achieved through the regulation of lipogenic or lipolytic transcription factors that can elicit global and direct changes in cellular lipid metabolism. Previous reports have shown that the nuclear receptor dHR96, a critical regulator of lipid and cholesterol homeostasis, promotes lipA/magro expression. However, dhr96 expression is upregulated in aging intestines, suggesting that the age-related repression of intestinal lipases is not merely due to decreases in dHR96 levels. dhr96 transcript levels are strongly induced in genetic conditions where Foxo is activated and intestinal lipases are repressed, again suggesting that Foxo does not mediate its effects on lipase transcription by antagonizing dhr96 expression. Furthermore, age-related changes that are independent of JNK/Foxo activation may also contribute to the repression of intestinal lipase expression and disruption of lipid metabolism, such as an age-associated decline in feeding/food intake (Karpac, 2013).

The reasons for the increase in JNK and Foxo activity in aging enterocytes remain to be explored. Age-related activation of JNK in the intestinal epithelium is dependent on the presence of commensal bacteria, as maintaining animals axenically reduces activation of JNK in the first 30 days of life. Thus, bacteria-induced inflammation and subsequent JNK activation appears to be a likely cause, in part, for age-related increases in Foxo activity. In a separate study, however, it was found that Foxo activation still occurs in intestines of old (40-day-old), axenically reared flies, suggesting that age-related activation of Foxo may also occur through JNK-independent processes. Supporting this idea, the current results show that inhibiting JNK function in enterocytes can attenuate, although not completely inhibit, this Foxo activation. Additional factors, such as sirtuins or histone deacetylases, recently shown to deacetylate and activate Foxo in response to endocrine signals, may also lead to age-related increases in intestinal Foxo activity (Karpac, 2013).

Interactions between JNK and IIS/Foxo are mediated by various mechanisms. In mammals, JNK phosphorylates the insulin receptor substrate (IRS), inhibiting insulin signaling transduction. JNK has also been shown to directly phosphorylate and activate Foxo in mammalian cell culture, that exact mechanism by which Foxo activation is achieved may be divergent in mammals. For example, no IRS homolog has been identified in worms, and the JNK phosphorylation site in mammalian IRS is not conserved in flies. The data show that JNK-mediated Foxo activation in the aging fly intestine is not achieved through IIS antagonism upstream of Akt, suggesting either a direct interaction between Foxo and JNK or changes in other regulators of Foxo. Recent studies have shown that JNK-mediated phosphorylation of 14-3-3 proteins results in the release of their binding partners, including Foxo. This chronic intestinal Foxo activation and subsequent metabolic changes, provide a physiological system in Drosophila to genetically dissect the crosstalk between IIS/Foxo and JNK signaling. Detailed analysis of these signaling interactions promises to provide important insight into the pleiotropic effects of IIS/Foxo function and the pathogenesis of age-related metabolic diseases (Karpac, 2013).

The data further reveal the pleiotropic consequences of Foxo activation in regard to healthspan and longevity in Drosophila. Overexpressing Foxo in the fat body or muscle of flies leads to lifespan extension. Overexpression of selected cytoprotective Foxo target genes in stem cells, on the other hand, is sufficient to prevent age-associated dysplasia and extend lifespan. The data presented here show that chronic Foxo activation in intestinal enterocytes disrupts lipid metabolism by deregulating intestinal lipases and thus highlight how cell- and tissue-specific consequences of Foxo function play an important role in determining either the beneficial (i.e., lifespan extension) or pathological (i.e., disruption of lipid metabolism) outcome of Foxo activation (Karpac, 2013).

Recent work in C. elegans has begun to explore the relationship between lipid metabolism and longevity, revealing that increases in intestinal lipase expression can extend lifespan. The beneficial effects of elevated lipase expression appear to be mediated by increases in specific types of fatty acids, which can activate autophagy and lead to lifespan extension. Interventions that promote lipid homeostasis with age, such as JNK/Foxo inhibition in intestinal enterocytes, might thus affect healthspan and/or longevity through means other than primarily maintaining energy homeostasis (Karpac, 2013).

High sugar diet disrupts gut homeostasis though JNK and STAT pathways in Drosophila

The incidence of diseases associated with a high sugar diet has increased in the past years, and numerous studies have focused on the effect of high sugar intake on obesity and metabolic syndrome. However, how a high sugar diet influences gut homeostasis is still poorly understood. This study used Drosophila melanogaster as a model organism and supplemented a culture medium with 1 M sucrose to create a high sugar condition. The results indicate that a high sugar diet promoted differentiation of intestinal stem cells through upregulation of the JNK pathway and downregulation of the JAK/STAT pathway. Moreover, the number of commensal bacteria decreased in the high sugar group. These data suggests that the high caloric diet disrupts gut homeostasis and highlights Drosophila as an ideal model system to explore gastrointestinal disease (Zhang, X. 2017).

The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila

Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. This study investigated stem-cell death in the digestive system of adult Drosophila melanogaster. It was found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively kills normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spares differentiated cells. The dying stem cells are engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduce CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers (Singh, 2016).

To investigate the molecular mechanism behind the resistance of CSCs to therapeutics, the death of stem cells with different degrees of quiescence was studied in the adult Drosophila digestive system, including intestinal stem cells (ISCs). Expression of the proapoptotic genes rpr and p53 effectively ablated differentiated cells but had little effect on stem cells (Singh, 2016).

In mammals, treatment-resistant leukaemic stem cells (LSCs) can be eliminated by a two-step protocol involving initial activation by interferon-α (IFNα) or colony-stimulating factor (G-CSF), followed by targeted chemotherapy. In Drosophila, activation of the hopscotch (also known as JAK)-Stat92E signalling pathway induces hyperplastic stem cells, which are overproliferating, but retain their apico-basal polarity and differentiation ability. A slightly different two-step protocol was conducted in Drosophila stem cells by overexpressing the JAK-Stat92E pathway ligand unpaired (upd) and rpr together. The induction of upd + rpr using the temperature-sensitive (ts) mutant esg-Gal4 (esg^ts > upd + rpr effectively ablated all of the ISCs and RNSCs through apoptosis within four days. Consistent with this result, expressing a gain-of-function Raf mutant (Raf^gof) also accelerated apoptotic cell death of hyperplastic ISCs (Singh, 2016).

Expressing a constitutively active form of Ras oncogene at 85D (also known as Ras^V12) in RNSCs and the knockdown of Notch activity in ISCs can transform these cell types into CSC-like neoplastic stem cells, which were not only overproliferating, but also lost their apico-basal polarity and differentiation abilit. It ws found that expressing rpr in Ras^V12-transformed RNSCs or in ISCs expressing a dominant-negative form of Notch (N^DN) caused the ablation of only a proportion of the transformed RNSCs and few transformed ISCs and it did not affect differentiated cells; substantial populations of the neoplastic stem cells remained even seven days after rpr induction (Singh, 2016).

These results suggest that the activation of proliferation can accelerate the apoptotic cell death of hyperplastic stem cells, but that a proportion of actively proliferating neoplastic RNSCs and ISCs are resistant to apoptotic cell death. Neoplastic tumours in Drosophila are more similar to high-grade malignant human tumours than are the hyperplastic Drosophila tumours (Singh, 2016).

Vesicle-mediated COPI and COPII are essential components of the trafficking machinery for vesicle transportation between the endoplasmic reticulum and the Golgi. In addition, the COPI complex regulates the transport of lipolysis enzymes to the surface of lipid droplets for lipid droplet usage. In a previous screen, it was found that knockdown of COPI components (including Arf79F, the Drosophila homologue of ADP-ribosylation factor 1 (Arf1)) rather than COPII components resulted in stem-cell death, suggesting that lipid-droplet usage (lipolysis) rather than the general trafficking machinery between the endoplasmic reticulum and Golgi is important for stem-cell survival (Singh, 2016).

To further investigate the roles of these genes in stem cells, a recombined double Gal4 line of esg-Gal4 and wg-Gal4 was used to express genes in ISCs, RNSCs, and HISCs (esg^ts wg^ts > X). Knockdown of these genes using RNA interference (RNAi) in stem cells ablated most of the stem cells in 1 week. However, expressing Arf79F^RNAi in enterocytes or in differentiated stellate cells in Malpighian tubules did not cause similar marked ablation. These results suggest that Arf79F knockdown selectively kills stem cells and not differentiated cells (Singh, 2016).

It was also found that expressing Arf79F^RNAi in Ras^V12-transformed RNSCs ablated almost all of the transformed stem cells. Similarly, expressing Arf79F^RNAi in N^DN-transformed ISCs ablated all of the cells within one week, but restored differentiated cells to close to their normal levels within one week (Singh, 2016).

δ-COP- and γ-COP-mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique, and it was found that the COPI complex cell-autonomously regulated stem cell survival. In summary, knockdown of the COPI-Arf79F complex effectively ablated normal and transformed stem cells but not differentiated enterocytes or stellate cells (Singh, 2016).

In the RNAi screen acyl-CoA synthetase long-chain (ACSL), an enzyme in the Drosophila lipolysis-β-oxidation pathway, and bubblegum (bgm), a very long-chain fatty acid-CoA ligase, were also identified. RNAi-mediated knockdown of Acsl and bgm effectively killed ISCs and RNSCs, but killed HISCs less effectively. Expressing Acsl^RNAi in Ras^V12-transformed RNSCs also ablated almost all of the transformed RNSCs in one week (Singh, 2016).

Brummer (bmm) is a triglyceride lipase, the Drosophila homologue of mammalian ATGL, the first enzyme in the lipolysis pathway. Scully (scu) is the Drosophila orthologue of hydroxy-acyl-CoA dehydrogenase, an enzyme in the β-oxidation pathway. Hepatocyte nuclear factor 4 (Hnf4) regulates the expression of several genes involved in lipid mobilization and β-oxidation. To determine whether the lipolysis-β-oxidation pathway is required for COPI-Arf79F-mediated stem cell survival, upstream activating sequence (UAS)-regulated constructs (UAS-bmm, UAS-Hnf4, and UAS-scu) were also expressed in stem cells that were depleted of Arf79F, β-COP, or ζ-COP. Overexpressing either scu or Hnf4 significantly attenuated the stem cell death caused by knockdown of the COPI-Arf79F complex. Expressing UAS-Hnf4 MARCM clones also rescued the stem cell death phenotype induced by γ-COP knockdown. However, bmm overexpression did not rescue the stem-cell death induced by Arf79F knockdown. Since there are several other triglyceride lipases in Drosophila in addition to bmm, another lipase may redundantly regulate the lipolysis pathway (Singh, 2016).

To further investigate the function of lipolysis in stem cells, the expression of a lipolysis reporter (GAL4-dHFN4; UAS-nlacZ which consisted of hsp70-GAL4-dHNF4 combined with a UAS-nlacZ reporter gene was investigated. The flies were either cultured continuously at 29°C or heat-shocked for 30 min at 37°C, 12 h before dissection. Without heat shock, the reporter was expressed only in ISCs and RNSCs of mature adult flies, but not in enteroendocrine cells, enterocytes, quiescent HISCs or quiescent ISCs of freshly emerged young adult flies (less than 3 days old. Expressing δ-COP^RNAi almost completely eliminated the reporter expression, suggesting that the reporter was specifically regulated by the COPI complex. After heat shock or when a constitutively active form of JAK (hop^Tum-l) was expressed, the reporter was strongly expressed in ISCs, RNSCs and HISCs, but not in enteroendocrine cells or enterocytes. These data suggest that COPI-complex-regulated lipolysis was active in stem cells, but not in differentiated cells, and that the absence of the reporter expression in quiescent HISCs at 29°C was probably owing to weak hsp70 promoter activity rather than to low lipolysis in these cells (Singh, 2006).

Lipid storage was futher investigated, and it was found that the size and number of lipid droplets were markedly increased in stem cells after knockdown of Arf79F (Singh, 2016).

Arf1 inhibitors (brefeldin A, golgicide A, secin H3, LM11 and LG8) and fatty-acid-oxidation (FAO) inhibitors (triacsin C, mildronate, etomoxir and enoximone) were used, and it was found that these inhibitors markedly reduced stem-cell tumours in Drosophila through the lipolysis pathway but had a negligible effect on normal stem cells (Singh, 2016).

These data together suggest that the COPI-Arf1 complex regulates stem-cell survival through the lipolysis-β-oxidation pathway, and that knockdown of these genes blocks lipolysis but promotes lipid storage. Further, the transformed stem cells are more sensitive to Arf1 inhibitors and may be selectively eliminated by controlling the concentration of Arf1 inhibitors (Singh, 2016).

These data suggest that neither caspase-mediated apoptosis nor autophagy-regulated cell death regulates the stem-cell death induced by the knockdown of components of the COPI-Arf79F complex. Therefore whether necrosis regulates the stem-cell death induced by knockdown of the COPI-Arf79F complex was investigated. Necrosis is characterized by early plasma membrane rupture, reactive oxygen species (ROS) accumulation and intracellular acidification. Propidium iodide detects necrotic cells with compromised membrane integrity, the oxidant-sensitive dye dihydroethidium (DHE) indicates cellular ROS levels and LysoTracker staining detects intracellular acidification. The membrane rupture phenotype was detected only in esg and the propidium iodide signal was observed only in ISCs from flies that had RNAi-induced knockdown of expression of COPI-Arf79F components, and not in cells from wild-type flies. In the esg^ts wg^ts > Acsl^RNAi flies, all of the ISCs and RNSCs were ablated after four days at 29°C, but a fraction of the HISCs remained, and these were also propidium iodide positive, indicating that the HISCs were dying slowly. This slowness may have been due to either a lower GAL4 (wg-Gal4) activity in these cells compared to ISCs and RNSCs (esg-Gal4) or quiescence of the HISCs. Furthermore, strong propidium iodide signals were detected in transformed ISCs from esg^ts > N^DN + Arf79F^RNAi but not esg^ts flies, indicating that the transformed stem cells were dying through necrosis (Singh, 2016).

Similarly, DHE signals were detected only in ISCs from esg^ts > Arf79F^RNAi flies, indicating that the dying ISCs had accumulated ROS and were intracellularly acidified. Overexpressing catalase (a ROS-chelating enzyme) rescued the stem-cell death specifically induced by the γ-COP mutant clone, and the ROS inhibitor NAC blocked the Arf1 inhibitor-induced death of Ras^V12-induced RNSC tumours. These data together suggest that knockdown of the COPI-Arf1 complex induced the death of stem cells or of transformed stem cells (Ras^V12-RNSCs, N^DN-ISCs) through ROS-induced necrosis. Although ISCs, RNSCs, and HISCs exhibit different degrees of quiescence, they all rely on lipolysis for survival, suggesting that this is a general property of stem cells (Singh, 2016).

Cases were noticed where the GFP-positive material of the dying ISCs was present within neighbouring enterocytes, suggesting that these enterocytes had engulfed dying ISCs (Singh, 2016).

The JNK pathway, autophagy and engulfment genes are involved in the engulfment of dying cells. Therefore, whether these genes are required for COPI-Arf79F-regulated ISC death was investigated. The following was found: (1) ISC death activated JNK signalling and autophagy in neighbouring enterocytes; (2) knockdown of these genes in enterocytes but not in ISCs rescued ISC death to different degrees; (3) the drpr-mbc-Rac1-JNK pathway in enterocytes is not only necessary but also sufficient for ISC death; and (4) inhibitors of JNK and Rac1 could block Arf1-inhibitor-induced cell death of the Ras^V12-induced RNSC tumours. These data together suggest that the drpr-mbc-Rac1-JNK pathway in neighbouring differentiated cells controls the engulfment of dying or transformed stem cells (Singh, 2016).

The finding that the COPI-Arf79F-lipolysis-β-oxidation pathway regulated transformed stem-cell survival in the fly led to an investigation of whether the pathway has a similar role in CSCs. WTwo Arf1 inhibitors (brefeldin A and golgicide A) and two FAO inhibitors (triascin C and etomoxir) were tested on human cancer cell lines, and it was found that the growth, tumoursphere formation and expression of tumour-initiating cell markers of the four cancer cell lines were significantly suppressed by these inhibitors, suggesting that these inhibitors suppress CSCs. In mouse xenografts of BSY-1 human breast cancer cells, a novel low-cytotoxicity Arf1-ArfGEF inhibitor called AMF-26 was reported to induce complete regression in vivo in five days. Together, this report and the current results suggest that inhibiting Arf1 activity or blocking the lipolysis pathway can kill CSCs and block tumour growth (Singh, 2016).

Stem cells or CSCs are usually localized to a hypoxic storage niche, surrounded by a dense extracellular matrix, which may make them less accessible to sugar and amino acid nutrition from the body's circulatory system. Most normal cells rely on sugar and amino acids for their energy supply, with lipolysis playing only a minor role in their survival. The current results suggest that stem cells and CSCs are metabolically unique; they rely mainly on lipid reserves for their energy supply, and blocking COPI-Arf1-mediated lipolysis can starve them to death. It was further found that transformed stem cells were more sensitive than normal stem cells to Arf1 inhibitors. Thus, selectively blocking lipolysis may kill CSCs without severe side effects. Therefore, targeting the COPI-Arf1 complex or the lipolysis pathway may prove to be a well-tolerated, novel approach for eliminating CSCs (Singh, 2016).

Disruption of the lipolysis pathway results in stem cell death through a sterile immunity-like pathway in adult Drosophila

Previous work has shown that the Arf1-mediated lipolysis pathway sustains stem cells and cancer stem cells (CSCs); its ablation resulted in necrosis of stem cells and CSCs, which further triggers a systemic antitumor immune response. This study shows that knocking down Arf1 in intestinal stem cells (ISCs) causes metabolic stress, which promotes the expression and translocation of ISC-produced damage-associated molecular patterns (DAMPs; Pretaporter [Prtp] and calreticulin [Calr]). DAMPs regulate macroglobulin complement-related (Mcr) expression and secretion. The secreted Mcr influences the expression and localization of enterocyte (EC)-produced Draper (Drpr) and LRP1 receptors (pattern recognition receptors [PRRs]) to activate autophagy in ECs for ATP production. The secreted ATP possibly feeds back to kill ISCs by activating inflammasome-like pyroptosis. This study identified an evolutionarily conserved pathway that sustains stem cells and CSCs, and its ablation results in an immunogenic cascade that promotes death of stem cells and CSCs as well as antitumor immunity (Aggarwal, 2022).

Previous work showed that the Arf1-mediated lipolysis pathway is specifically activated in stem cells and sustains stem cells in adult Drosophila (Singh, 2016). Arf1 is one of the most evolutionarily conserved genes between Drosophila and mouse, with an amino acid identity of 95.6% between the two species. It was found recently that Arf1-mediated lipid metabolism sustains cancer stem cells (CSCs) and that its ablation triggers immunogenic-like death (immunogenic cell death [ICD]) of CSCs and induces antitumor immunity by exposing damage-associated molecular patterns (DAMPs; calreticulin [Calr], high-mobility group box 1 [HMGB1], and ATP) (Aggarwal, 2022).

However, the molecular mechanism that coordinates stem cells/CSCs with neighboring cells to execute the biological processes (stem cell necrosis or anti-tumor immunity) is still unclear. This study dissected the molecular mechanism using the Drosophila genetic system. Knockdown of the pathway was found to promote stem cell death through an immunogenic-like and aging cascade. Ablation of Arf1-mediated lipid metabolism in Drosophila ISCs resulted in several aging-like hallmarks, including lipid droplet (LD) accumulation, Reactive oxygen species (ROS) accumulation, mitochondrial defects, mitophagy activation, and lysosomal protein aggregates, followed by an immunogenic-like cell death (Aggarwal, 2022).

ICD is a process that releases DAMPs and activates immune responses to destroy damaged or stressed cells in the absence of microbial components. These molecules are often present in a given cell compartment and are not expressed or are only somewhat expressed under physiological conditions but strongly induced and then translocated to the cell surface or extracellular space under conditions of stress, damage, or injury. The most important DAMPs are (1) pre-apoptotic exposure of the ER-sessile molecular chaperone Calr on the cell surface, (2) release of the non-histone nuclear protein HMGB1 into the extracellular space, and (3) active secretion of ATP. With respect to tumors, the surface-exposed Calr facilitates engulfment of tumor-associated antigens by binding to LRP1/CD91 receptors (pattern recognition receptors [PRRs]) on dendritic cells (DCs). During ICD, Calr interacts with another protein, ERp57, and the two are rapidly translocated to the cell surface from the ER lumen before the cells exhibit any sign of apoptosis. ERp57 is a disulfide isomerase that has several thioredoxin-like domains and regulates cell redox homeostasis. Knocking down Arf1-mediated lipolysis in ISCs was found to promote the expression and translocation of ISC-produced DAMPs (Pretaporter [Prtp] and Calr). Like ERp57, Prtp is a disulfide isomerase with several thioredoxin-like domains. The DAMPs may then regulate the expression and secretion of the protein macroglobulin complement-related (Mcr; a complement C5 homolog). The secreted Mcr possibly further controls the expression and localization of EC-produced Draper [Drpr] and LRP1 receptors (PRRs) to activate autophagy in ECs for ATP production. The secreted ATP likely feeds back to kill ISCs by activating inflammasome-like pyroptosis. Therefore, Arf1-mediated lipid metabolism is crucial for stem cell maintenance, and its ablation promotes stem cell decay and anti-tumor immunity through an immunogenic aging cascade (Aggarwal, 2022).

Stem cell functional decay or decline may be one of the important causes of organismal aging and disease. This study demonstrated that Arf1-mediated lipid metabolism sustains stem cells and that its ablation triggers an immunogenic-like stem cell death cascade. The dying stem cells display the following features: LD accumulation, mitochondrial defects, ROS production, ER stress and release of DAMPs to activate PRRs in neighboring ECs, mitophagy activation, lysosomal protein aggregations, and ISC necrosis through inflammasome-like pyroptosis. These features are similar to hallmarks of aging. Arf1 ablation in ISCs might trigger a stem cell aging and death cascade. The gold standard method for evaluating ICD is in vivo tumor vaccination. Previously an experiment of vaccination was performed in Arf1-ablated mice. The current study has demonstrated that many of the factors that contribute to ICD are expressed and function in Arf1-ablated flies, indicating that the pathway is partially conserved between Drosophila and mammals. However, it is important to confirm conserved biological functions of the ICD in Drosophila in future experiments. Similarly, inflammasome pyroptosis is only partially conserved between Drosophila and mammals. It is important to confirm the pathway by using inflammasome markers and demonstrate conserved biological functions of the pathway in Drosophila in future experiments (Aggarwal, 2022).

A previous report demonstrated that Mcr, through Drpr, cell non-autonomously regulates autophagy during wound healing and salivary gland cell death in Drosophila and that Prtp is not involved in this Mcr-Drpr-mediated autophagy induction. Mcr is an analog of mammalian C1q/C5. C1q binds to the Calr-LRP1 coreceptor in mammals, and Mcr binds to LRP1 (Flybase) in Drosophila. This study found that Calr and Prtp function in parallel or downstream of the Arf1-lipolysis pathway and regulate the expression of Mcr and LRP1. Mcr and LRP1 further regulate each other and control the expression of Drpr. Calr and Prtp also regulate the expression of their respective receptors, LRP1 and Drpr. This information suggests that two interconnected complexes, Calr-Mcr-LRP1 and Prtp-Drpr, function downstream of the Arf1-lipolysis pathway and coordinately regulate ISC death (Aggarwal, 2022).

In the mammalian immune system, DCs are activated after DAMPs bind to PRRs on their surface. The activated DCs present antigens to T cells, and the activated T cells kill damaged cells. The current study found that ablation of the COPI/Arf1-mediated lipolysis-β-oxidation pathway in stem cells induced expression of DAMPs, which then activate the phagocytic ECs through PRRs (LRP1 and Drpr) on the ECs to kill the stem cells. These findings suggest that such a coordinated cell death process is not limited to mammalian immune responses. In another naturally occurring example, Drpr pathway phagocytosis genes in follicle cells (FCs) non-autonomously promote nurse cell (NC) death in the developing Drosophila ovary. Although it is not clear how the stretch FCs time the precise developmental death of NCs, in light of the present findings, it is possible that a metabolic or stress signal during this developmental stage increases DAMPs in NCs to activate the Drpr pathway in FCs and non-autonomously promote NC death. DAMPs are also induced in organs during organ transplantation as a result of ischemic damage from the interrupted blood supply while the organ is outside of the body. The DAMPs induced in a graft stimulate immune responses mediated by host innate cells at the site of the graft and the donor's innate immune system and contribute to graft rejection. Drpr-mediated phagocytosis is also an essential process during development and in maintenance of tissue homeostasis in several systems. As mentioned above, the Mcr-Drpr pathway is involved in autophagy induction during wound healing and salivary gland cell death in Drosophila. It is proposed that such a coordinated cell death (CCD) is a novel and general cell death process in which death of abnormal or altered cells occurs by first sending danger signals (such as DAMPs) and then activating neighboring cells to execute the death process. The abnormality or alteration can be metabolic stress (such as disruption of Arf1-mediated lipid metabolism in stem cells), developmental changes (such as NC death during Drosophila ovary development or salivary gland cell death during metamorphosis), or damage during wound healing or circulation blockage during ischemic damage or pathogen infection. The danger signals then activate phagocytes and other cells (such as T cells) to cell non-autonomously promote targeted cell death. CCD may mediate cell aging/death and organ degeneration under physiological conditions or CSC death and anti-tumor immunity under pathological conditions (Aggarwal, 2022).

The finding that the DAMP-Mcr-LRP1/Drpr pathway connects metabolically stressed stem cells after Arf1 ablation to activation of phagocytic ECs to kill the stem cells will enable further dissection of the CCD mechanism in Drosophila. Arf1 is one of the most evolutionarily conserved genes, and the DAMP-Mcr/C1q-LRP1/Drpr pathway is well conserved throughout evolution. CCD involves coordination or communication of two or more different cells. Model organisms such as Drosophila, with their advanced genetic tractability and well-characterized cellular histology, will serve as valuable in vivo models for dissecting the detailed cellular and molecular mechanisms of CCD. These findings may lead to new therapeutic strategies for many human diseases, such as induction of anti-tumor immunity in individuals with cancer and the blocking of neuronal death in individuals with neurodegenerative conditions (Aggarwal, 2022).

This study has identified an evolutionarily conserved pathway that sustains stem cells, and its ablation results in an ICD cascade that promotes death of stem cells through inflammasome-like pyroptosis. It was demonstrated that many of the factors that contribute to ICD and inflammasome-like pyroptosis are expressed and function in Arf1-ablated flies. However, the gold standard method for evaluating ICD is in vivo tumor vaccination. The components of ICD and inflammasome-like pyroptosis are only partially conserved between Drosophila and mammals. It is important to further confirm the pathway by using inflammasome markers and demonstrate conserved biological functions of the pathway in Drosophila in future experiments (Aggarwal, 2022).

Reduced gut acidity induces an obese-like phenotype in Drosophila melanogaster and in mice

In order to identify genes involved in stress and metabolic regulation, this study carried out a Drosophila P-element-mediated mutagenesis screen for starvation resistance. A mutant, m2, was isolated that showed a 23% increase in survival time under starvation conditions. The P-element insertion was mapped to the region upstream of the vha16-1 gene, which encodes the c subunit of the vacuolar-type H+-ATPase. It was found that vha16-1 is highly expressed in the fly midgut, and that m2 mutant flies are hypomorphic for vha16-1 and also exhibit reduced midgut acidity. This deficit is likely to induce altered metabolism and contribute to accelerated aging, since vha16-1 mutant flies are short-lived and display increases in body weight and lipid accumulation. Similar phenotypes were also induced by pharmacological treatment, through feeding normal flies and mice with a carbonic anhydrase inhibitor (acetazolamide) or proton pump inhibitor (PPI, lansoprazole) to suppress gut acid production. This study may thus provide a useful model for investigating chronic acid suppression in patients (Lin, 2015).

Hs3st-A and Hs3st-B regulate intestinal homeostasis in Drosophila adult midgut

Intrinsic and extrinsic signals as well as the extracellular matrix (ECM) tightly regulate stem cells for tissue homeostasis and regenerative capacity. Little is known about the regulation of tissue homeostasis by the ECM. Heparan sulfate proteoglycans (HSPGs), important components of the ECM, are involved in a variety of biological events. Two heparin sulfate 3-O sulfotransferase (Hs3st) genes, Hs3st-A and Hs3st-B, encode the modification enzymes in heparan sulfate (HS) biosynthesis. This study demonstrates that Hs3st-A and Hs3st-B are required for adult midgut homeostasis. Depletion of Hs3st-A in enterocytes (ECs) results in increased intestinal stem cell (ISC) proliferation and tissue homeostasis loss. Moreover, increased ISC proliferation is also observed in Hs3st-B null mutant alone, or in combination with Hs3st-A RNAi. Hs3st-A depletion-induced ISC proliferation is effectively suppressed by simultaneous inhibition of the EGFR signaling pathway, suggesting that tissue homeostasis loss in Hs3st-A-deficient intestines is due to increased EGFR signaling. Furthermore, this study found that Hs3st-A-depleted ECs are unhealthy and prone to death, while ectopic expression of the antiapoptotic p35 is able to greatly suppress tissue homeostasis loss in these intestines. Together, these data suggest that Drosophila Hs3st-A and Hs3st-B are involved in the regulation of ISC proliferation and midgut homeostasis maintenance (Guo, 2014).

More Drosophila enteroendocrine peptides: Orcokinin B and the CCHamides 1 and 2

Antisera to orcokinin B, CCHamide 1, and CCHamide 2 recognize enteroendocrine cells in the midgut of the Drosophila and its larvae. Although the antisera to CCHamide 1 and 2 are mutually cross-reactive, polyclonal mouse antisera raised to the C-terminals of their respective precursors allowed the identification of the two different peptides. In both larva and adult, CCHamide 2 immunoreactive endocrine cells are large and abundant in the anterior midgut and are also present in the anterior part of the posterior midgut. The CCHamide 2 immunoreactive endocrine cells in the posterior midgut are also immunoreactive with antiserum to allatostatin C. CCHamide 1 immunoreactivity is localized in endocrine cells in different regions of the midgut; those in the caudal part of the posterior midgut are identical with the allatostatin A cells. In the larva, CCHamide 1 enteroendocrine cells are also present in the endocrine junction and in the anterior part of the posterior midgut. Like in other insect species, the Drosophila orcokinin gene produces two different transcripts, A and B. Antiserum to the predicted biologically active peptide from the B-transcript recognizes enteroendocrine cells in both larva and adult. These are the same cells as those expressing beta-galactosidase in transgenic flies in which the promoter of the orcokinin gene drives expression of this enzyme. In the larva, a variable number of orcokinin-expressing enteroendocrine cells are found at the end of the middle midgut, while in the adult, those cells are most abundant in the middle midgut, while smaller numbers are present in the anterior midgut. In both larva and adult, these cells also express allatostatin C. A specific polyclonal antiserum was also made to the NPF precursor in order to determine more precisely the expression of this peptide in the midgut. Using this antiserum, expression in the midgut was found to be the same as described previously using transgenic flies, while in the adult, midgut expression appears to be concentrated in the middle midgut, thus suggesting that in the anterior midgut only minor quantities of NPF are produced (Veenstra, 2014).

Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch

This study used tracing methods that allow simultaneously capturing the dynamics of intestinal stem and committed progenitor cells (called enteroblasts) and intestinal cell turnover with spatiotemporal resolution. Intestinal stem cells (ISCs) divide 'ahead' of demand during Drosophila midgut homeostasis. Their newborn enteroblasts, on the other hand, take on a highly polarized shape, acquire invasive properties and motility. They extend long membrane protrusions that make cell-cell contact with mature cells, while exercising a capacity to delay their final differentiation until a local demand materializes. This cellular plasticity is mechanistically linked to the epithelial-mesenchymal transition (EMT) programme mediated by escargot, a snail family gene. Activation of the conserved microRNA miR-8/miR-200 in 'pausing' enteroblasts in response to a local cell loss promotes timely terminal differentiation via a reverse EMT by antagonizing escargot. These findings unveil that robust intestinal renewal relies on hitherto unrecognized plasticity in enteroblasts and reveal their active role in sensing and/or responding to local demand (Antonello, 2015).

The robustness of intestinal cell renewal relies on cellular plasticity in committed progenitor cells and a rather loose regulation of ISCs proliferation. One key finding is that stem cells divide continually and generate a 'stock' of committed progenitor cells that do not terminally differentiate right away but postpone their final differentiation for long time intervals in the absence of a local epithelial cell loss. Accordingly, one noticeable change in newborn progenitor cells after their (enterocyte) fate commitment is their transformation from rounded cells to spindle-shaped cells that appear to actively monitor their surroundings by extending long membrane actin-rich protrusions that make cell-cell contact with mature epithelial cells and their mother ISCs. Timely terminal differentiation with epithelial cell loss is orchestrated by activation of a conserved pro-epithelial microRNA, in turn, directly repressing the repressors of differentiation. A microRNA-induced repression of the repressors of differentiation provides a faster mechanism than one involving a transcriptional regulator since synthesizing a miRNA likely requires less time than synthesizing a protein. Importantly, mutual antagonism between the microRNA (MiR-8/miR-200) and its targets (Escargot/Snail2 and Zfh1/ZEB) may serve to slow down the mesenchymal-to-epithelial process inside individual mesenchymal/progenitor cells until they are successfully integrated in the epithelium. Consistently, abrupt transition as in mir-8 overexpressing midguts results in erroneous tissue repair (Antonello, 2015).

Supply and demand in business production involves frequently two alternative solutions called 'make-to-stock' and 'make-to-order'. In 'make-to-stock' or MTS, production is continuous so that response to customers can be supplied immediately. However, as production is not based on actual demand, the MTS solution is not robust against fluctuations in demand and errors in forecasting can result in shortages (if there is insufficient residual stock) or overproduction. In 'make-to-order', or MTO, production only starts upon receiving a customer's order, thereby precisely matching production to demand. However, the MTO generates a delay in the response and can be less efficient and competitive than the MTS paradigm. The dynamics of stem cells and committed progenitor cells in the midgut suggests a hybrid solution between MTS and MTO -- reminiscent to the business solution known as delayed differentiation. Thus, in basal homeostasis, production of new cells to replace cell loss occurs in two stages: (1) a 'make-to-stock' stage where committed progenitor cells are continually generated and 'stocked' in an 'undifferentiated' state; and (2) a 'make-to-order' stage where terminal differentiation takes place only in response to a local demand. In mice and humans, the rapid turnover that occurs in the small intestinal epithelium is thought to be the result of continual shedding of superficial cells balanced by the continual stem cell production. The mechanism described in this study may be more general than expected and could account for how murine cells after fate commitment like the secretory-committed cells defer for long periods their terminal differentiation (Buczacki et al, 2013; Antonello, 2015).

Escargot/Snail2 sustains the undifferentiated state and self-renewing divisions of midgut intestinal stem cells. However, the committed progenitor cells also express escargot and apparently at higher levels than the stem cells. It is hypothesized that below a certain threshold level, Escargot maintains stemness and a partial EMT that may facilitate regular cell division and a topologically confined position at the base of the intestinal epithelium. Conversely, when Escargot surpasses a certain threshold level, it promotes a full EMT that confers invasive properties and motility for the successful response and integration of the newly differentiated cells in the preexisting epithelium. Intriguingly, the enteroendocrine cells appear to escape from this block in terminal differentiation and differentiate at the normal rate in the absence of escargot. There is as yet no explanation for the behaviour of these progenitor cells (Antonello, 2015).

Mechanistically, the different levels of escargot could be achieved via Notch signalling pathway, which is prominently activated in enterocyte-committed progenitors. Notch signalling activates directly zfh1 gene and Zfh1, a homolog of the mammalian stemness and EMT-determinant Zeb1,2, and binds to the escargot promoter region, and this study shows that Zfh1 acts genetically upstream of escargot. Thus, progenitor cells receiving Notch signalling might enhance escargot transcriptional levels via Notch-induced zfh1 transcription. Such regulatory mechanism would explain, for example, that loss of Notch results in stem-like/round cells (Antonello, 2015).

In mammalian cell culture, the EMT process has been linked to the acquisition of stem-like nature via an interplay between the ZEB1,2 and Snail transcription factors and the microRNAs of the miR-200 family. Moreover, EMT determinants often regulate each other to promote EMT. Thus, the interactions between Escargot/Snail2, zfh1/Zeb and miR-8/miR-200 that were identified in this study exemplify the conservation of the regulatory mechanisms involved in EMT/MET and stemness in an in vivo context and a normal physiology of an adult organism. However, this study shows that escargot-zfh1 promotes stemness and full EMT/invasive properties in distinct cell populations and likely at different concentration levels, highlighting the utility of Drosophila midgut as a model to dissect out mechanisms linking physiological EMT to cellular plasticity and stemness as well as provide novel insights linking polyploidy and EMT towards stemness (Antonello, 2015).

Although midgut mesenchymal/progenitor cells have motility, most of them maintain their own local area as clearly defined by Flybow clonal analysis. This situation is similar to the leading edge mesenchymal cells during collective cell migration. Midgut enteroblasts retain contact via E-cadherin with their mother ISC, a process that might be regulated by escargot as in tracheal cells. Cell-cell contact is crucial to sustain Notch signalling in committed progenitor cells and likely to help to stabilize polarity of enteroblasts and their membrane protrusions that contact mature cells. Through these protrusions, mesenchymal/enteroblasts might actively monitor their surroundings. When a protrusion detects changes in tension and mechanical forces generated during the elimination of a dying cells, a positive input might be created that triggers the activation of expression of the microRNA mir-8 in the particular progenitor cell which, in turn, promotes the epithelial state and integration of the newly differentiated cell in the epithelium. Adhesion via E-cadherin could facilitate communication between an epithelial cells and a mesenchymal/progenitor cell in its vicinity so that a single, newly differentiated cell fills the gap left by the cleared cell (Antonello, 2015).

Dynamic pseudopodia in migrating cells have been proposed as a mechanism for temporal and spatial sensing during cell migration. Direction sensing is also consistent with time-lapse data showing individual progenitor cells re-adjusting position in the homeostatic midguts. Transduction of mechanical cues via YAP and TAZ (called Yorkie in flies) is functionally involved in differentiation of mesenchymal stem cells. Hence, Drosophila Hippo/Yorkie-YAP in mature enterocytes is a primary candidate pathway for a potential transduction of mechanical cues activating mir-8 in response to cell death (Antonello, 2015).

In summary, the miR-8-escargot-zfh1 axis and the EMT/MET programme provides a conceptual shift of the current stem cell-centred view of tissue renewal and offers a starting point for investigating how mature cells speak with neighbouring committed progenitor cells to ensure that epithelial cell loss and cell addition are kept in balance (Antonello, 2015).

miR-263a regulates ENaC to maintain osmotic and intestinal stem cell homeostasis in Drosophila

Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinal stem cell (ISC) homeostasis. The Drosophila genome encodes an exceptionally large number of DEG/ENaC subunits termed Pickpocket (Ppk) 1-31. This study found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na+ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Evidence is provided that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC (Kim, 2016).

The Drosophila intestinal system is an attractive model for studying signaling events that control stem cell homeostasis given its anatomical and functional similarities to human epithelial systems, including the intestine. The adult midgut is continuously damaged during feeding as well as by chemicals and pathogens they encounter in the food, and thus needs to be constantly renewed. The renewal process requires tight regulation of the activities of multiple conserved signaling pathways in response to various types of intestinal epithelial injuries. These responses promote both intestinal stem cell (ISC) proliferation and enteroblast (EB) differentiation, expediting the rapid generation of new midgut epithelial cells to replace damaged (Kim, 2016).

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. In the past few years, miRNAs have been shown to play an important role in stem cell homeostasis by regulating differentiation and self-renewal. This study found that a well-conserved miRNA, miR-263a, is necessary for maintaining ISC homeostasis. Deletion of miR-263a in the adult midgut enterocytes (ECs) activates a stress response that, in turn, activates signaling pathways required for ISC proliferation, resulting in midgut hyperplasia. Well-conserved subunits of the epithelial sodium channel (ENaC) were found to be biologically important targets of miR-263a,and regulation of these subunits by miR-263a was found to be critical for maintaining proper osmotic homeostasis in the midgut epithelium. Remarkably, many of the phenotypes of miR-263a mutants are reminiscent of the pathophysiology of cystic fibrosis (CF), an autosomal recessive disorder caused by mutations in the gene encoding the chloride channel CF transmembrane conductance regulator (CFTR). In CF patients, loss-of-function mutations in the CFTR can elevate the activity of ENaC through a mechanism that is not fully understood. ENaC is present at the apical plasma membrane in many epithelial tissues throughout the body to regulate sodium reabsorption, and control total body salt and water homeostasis. The most common symptoms of CF are potential lethal blockages of distal small intestines, airway mucus obstruction, and chronic airway inflammation, which are consistent with the model that upregulation in ENaC activity increases sodium and water reabsorption, ultimately leading to dehydration of the intraluminal surface and reduction in mucus transport. Interestingly, this study provides evidence that overexpression of miR-183, the human ortholog of miR-263a, can also directly target all three subunits of human ENaC to regulate its activity. Altogether, these findings describe the role of a miRNA in regulating ENaC levels and suggest that the Drosophila intestine could be used as a model for CF (Kim, 2016).

In CF, two different models have been proposed regarding the role of hydration and salt concentration in normal airway defense. The hydration model proposes that increased absorption of fluid by the epithelium leads to dehydrated mucus and impaired mucociliary clearance that contributes to the establishment of an environment promoting colonization of the lungs by bacteria. In contrast, the salt model proposes that the salt content of airway fluid in CF is too high and thus prevents salt-sensitive defensin molecules in the airway surface liquid from killing bacteria, leading to increased susceptibility to lung infections. In Drosophila, the phenotypes associated with perturbation of ENaC are consistent with the hydration model, as misregulation of ENaC subunits in miR-263a mutants result in increased sodium reabsorption across the midgut epithelium. Furthermore, a dehydration-like phenotype of the PM, which is analogous to mucous secretions in the vertebrate digestive tract, was observed. Consistent with the PM providing protection against abrasive food particles and pathogens, miR-263a mutants appear more susceptible to bacterial infections as they succumb to P. aeruginosa infection more rapidly than the controls. In addition, increased bacterial load and antimicrobial peptide levels, and disruption of the intestinal pH were observed in miR-263a mutants. Interestingly, ECs in miR-263a mutants appear swollen, which is likely due to increased water reabsorption through osmosis. Finally, an activation was observed of stressed pathways characteristic of damaged ECs, which correlates with increased proliferation of ISCs (Kim, 2016).

Consistent with previous reports that cell swelling can activate the JNK pathway, JNK signaling is activated in miR-263a mutants that have large ECs. In addition, the JAK/STAT and EGFR pathways that regulate ISC proliferation are hyperactivated. Similarly, in CF airway and small intestine epithelia, cells in the airway epithelium and submucosal glands are more proliferative than cells in non-CF airways. In addition, in all CF mouse models in which CFTR has been deleted, goblet cell hyperplasia was observed in the small intestine (Kim, 2016).

Although the existence of Drosophila CFTR is yet to be determined, given its phenotypic similarities to the pathophysiology of CF, miR-263a mutants may provide a cost-effective and high-throughput animal model for identifying potential therapeutics that can specifically target ENaC in vivo, as the Drosophila gut is amenable to large-scale small-molecule screens. In addition, miR-183 might itself be a potential therapeutic agent for regulating ENaC activity in CF, based on the data that overexpression of miR-183 can directly target the expression of all three ENaC subunits in CFBE41o cells. Thus, possibly a combinational therapy for CF using the CFTR potentiator, Ivacaftor (also known as Kalydeco, which improves the transport of chloride through the mutated CFTR, together with overexpression of miR-183, could be imagined (Kim, 2016).

Control of lipid metabolism by Tachykinin in Drosophila

The intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. This study demonstrates that Tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila and that TKs repress lipogenesis in enterocytes (ECs) associated with TKR99D receptor and protein kinase A (PKA) signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions (Song, 2014).

Previous studies in mammals have indicated that a few gut secretory hormones, like GLP1 and GLP2, are involved in intestinal lipid metabolism. However, due to gene and functional redundancy, mammalian genetic models for gut hormones and/or their receptors with severe metabolic defects are not available. This study has establish that Drosophila TKs produced from EEs coordinate midgut lipid metabolic processes. The studies clarify the roles of TK hormones in intestinal lipogenesis and establish Drosophila as a genetic model to study the regulation of lipid metabolism by gut hormones (Song, 2014).

Six mature TKs, TK1-TK6, are processed and secreted from TK EEs in both the brain and midgut (Reiher, 2011). Using a specific Gal4 driver line, gene expression in TK EEs was specifically manipulated, leading to the demonstration that loss of gut TKs results in an increase in midgut lipid production. Further, this study showed that TKs regulate intestinal lipid metabolism associated with TKR99D, but not TKR86C, which is consistent with the expression of these receptors. Consistent with previous reports that TK/TKR99D signaling regulates cAMP level and PKA activation, loss of gut TKs is associated with a reduction in PKA activity in ECs, and overexpression of a PKA catalytic subunit was able to reverse the increased intestinal lipid production associated with loss of TKR99D. In addition, the transcription factor SREBP that triggers lipogenesis was controlled by TK/TKR99D/PKA signaling. Taken together, these results suggest that TKs produced from EEs regulate midgut lipid metabolism via TKR99D/PKA signaling and regulation of, at least, SREBP-induced lipogenesis in ECs (Song, 2014).

Interestingly, this study reveals that TKs derived from either the brain or gut exhibit distinct functions: TKs derived from gut control intestinal lipid metabolism, whereas TKs derived from brain control behavior. This is reminiscent of the distinct functions of mammalian secreted regulatory peptides, where different spatial expressions or deliveries of peptides like Ghrelin can result in distinct physiological functions. In addition, some prohormones encode multiple mature peptides that can have multiple functions. For example, processing of proglucagon in the pancreas α cells preferentially gives rise to glucagon, which antagonizes the effect of insulin. In intestine L cells, however, proglucagon is mostly processed into GLP1 to promote insulin release. These studies of TKs exemplify how secreted regulatory peptides derived from different tissues can be associated with fundamentally diverse physiological functions. Clearly, additional studies examining the function of secreted peptides in a cell-type- and tissue-specific manner are needed to fully appreciate and unravel their complex roles both in flies and mammals (Song, 2014).

There is a growing body of studies emphasizing that intestinal lipid metabolism is key to the control of systemic lipid homeostasis. For example, chemicals such as orlistat, designed to inhibit dietary lipid digestion/absorption in the intestine, efficiently reduce obesity. In addition, mammalian inositol-requiring enzyme 1β deficiency-induced abnormal chylomicron assembly in the small intestine results in hyperlipidemia. Similarly, in Drosophila, dysfunction of intestinal lipid digestion/absorption caused by Magro/LipA deficiency eventually decreases whole-body lipid storage and starvation resistance in Drosophila. Further, intestinal lipid transport, controlled by lipoproteins, is essential for systemic lipid distribution and energy supply in other tissues. Consistent with these observations, this study demonstrates that increased midgut lipid synthesis associated with gut TK deficiency is sufficient to elevate systemic lipid storage. Although TK ligands and TK receptors show high homologies between mammals and fruit flies, whether mammalian TK signaling plays a similar role in intestinal lipid metabolism is largely unknown. Future studies will reveal whether mammalian TK signaling affects intestinal lipid metabolism as in Drosophila. If this is the case, it may provide a therapeutic opportunity for the treatment of intestinal lipid metabolic disorder and obesity (Song, 2014).

Production and secretion of gut hormones are precisely regulated under various physiological conditions. Similar to previous observations that starvation induces gut TK secretion in other insects, this study found that nutrient deprivation promotes TK production in EEs. Interestingly, feeding of amino-acid-enriched yeast, but not coconut oil or sucrose, potently suppressed gut TK levels, indicating that amino acids may act directly on TK production in EEs. It has been reported that dietary nutrients regulate gut hormone production through certain receptors located on the cell membrane of EEs in mammals. Future studies will be necessary to elucidate the detailed mechanism by which nutrients regulate TK production from EEs (Song, 2014).

Transforming growth factor beta/Activin signaling functions as a sugar-sensing feedback loop to regulate digestive enzyme expression

Organisms need to assess their nutritional state and adapt their digestive capacity to the demands for various nutrients. Modulation of digestive enzyme production represents a rational step to regulate nutriment uptake. However, the role of digestion in nutrient homeostasis has been largely neglected. This study analyzed the mechanism underlying glucose repression of digestive enzymes in the adult Drosophila midgut. Glucose represses the expression of many carbohydrases and lipases. The data reveal that the consumption of nutritious sugars stimulates the secretion of the transforming growth factor β (TGF-β) ligand, Dawdle, from the fat body. Dawdle then acts via circulation to activate TGF-β/Activin signaling in the midgut, culminating in the repression of digestive enzymes that are highly expressed during starvation. Thus, this study not only identifies a mechanism that couples sugar sensing with digestive enzyme expression but points to an important role of TGF-β/Activin signaling in sugar metabolism (Chng, 2004).

Digestive enzymes expression is subjected to complex regulation. However, apart from the regulation of magro (lipase) by the nutrient-sensitive DHR96 and dFOXO (Karpac, 2013). It is noteworthy that an arbitrary threshold for RNA-seq analysis has rejected several genes whose repression was more subtle. For this, it has been have independently verified Amy-p, Amy-d, CG9466, CG9468, and CG6283 to be repressed by glucose through qRT-PCR. Thus, the actual repertoire of carbohydrases and lipases affected by glucose could be potentially larger (Karpac, 2013).

To date, little is known about the contribution of digestion on sugar homeostasis. It seems likely that glucose repression of carbohydrases and lipases is aimed at reducing the amount of sugars and lipids that are available for absorption. Consistent with this view, glucose transmembrane transporters were also found among genes that were downregulated by dietary glucose. A high-sugar diet in Drosophila is associated with dire consequences such as hyperglycemia, insulin resistance, and increased fat accumulation. Thus, reducing both carbohydrases and lipases expression may restrict the nutritional load available for absorption into the circulation when carbohydrate stores in the organism are sufficient and fats are accumulating. In accordance with this, early postprandial glucose level was elevated in the hemolymph when TGF-β/Activin pathway function was compromised in the midgut, a condition associated with elevated digestive enzymes expression. However, when the levels of TAG, glycogen, glucose, and trehalose were monitored after 2 weeks on a high-sugar diet, no significant differences were observed between flies whereby Smad2 or Babo were knocked down in the midgut and control. Sugar homeostasis is a tightly regulated process involving multiple tissues. One possibility would be that the postprandial increase in glucose was counteracted by early acting satiety response when hemolymph glucose level passed a certain threshold, thus limiting the net amount of glucose entering the circulation. Clearly, the role of glucose repression in sugar homeostasis and metabolism warrants additional research. An understanding of how the repertoire of digestive enzymes respond to other nutriments in the diet will provide insights into how an organism may rebalance its diet after ingestion and improve understanding of nutrients homeostasis (Karpac, 2013).

In this study, it was also shown that digestive enzyme repression is induced only by nutritious carbohydrates in the diet. Arabinose, a sweet-tasting sugar with no nutritional value, and L-glucose, another nonutilizable sugar did not suppress amylase and maltase expression. Hence, postprandial activation of gustatory receptors in the gut are considered to be an unlikely mechanism for glucose repression of digestive enzymes. Instead, all these are suggestive of an underlying sugar-sensing mechanism to ensure that carbohydrate digestive capacity toward utilizable carbohydrate sources are not comprised until nutritional sufficiency is attained (Karpac, 2013).

In Drosophila, sugar homeostasis is often associated with the AKH and insulin signaling, whereas insulin signaling is also modulated by proteins and amino acids in the diet. Recently, it has been shown that Daw expression is modulated by insulin signaling, and Daw was identified as a target of dFOXO (Bai, 2013), raising the possibility that glucose repression may be similarly affected by insulin signaling. Surprisingly, disrupting both AKH and insulin signaling did not compromise glucose repression. Instead, this study identified a key role for TGF-β/Activin signaling in this process. Whereas Daw expression may be modulated by insulin signaling, the results clearly showed that glucose repression is mediated through an insulin-independent mechanism. More recently, Ghosh (2014) has demonstrated that Daw is required for insulin secretion, suggesting that the TGF-β/Activin pathway may function upstream of the insulin signaling. It is also noteworthy that, whereas compromising insulin signaling is known to raise circulating sugar levels, this did not affect the ability of flies to repress digestive enzymes in response to dietary glucose. One possible explanation is that Daw expression in response to glucose is dependent on the nutritional state perceived cell autonomously by the fat body cells. Thus, if nutrient sensing in these cells is not compromised, Daw induction and glucose repression can be achieved. Future research should clarify the mechanism underlying Daw induction by nutritious sugar and define the possible interactions between TGF-β/Activin and other sugar-sensing mechanisms (Karpac, 2013).

The TGF-β/Activin pathway in Drosophila has been previously studied in the context of larval brain development, neuronal remodeling, wing disc development, and, more recently, aging and pH homeostasis. This study addresses the physiological function of the TGF-β/Activin pathway in the adult midgut. When the TGF-β/Activin signaling was disrupted in the adult midgut, glucose repression was abolished. Conversely, increasing TGF-β/Activin signaling in the midgut, through the overexpression of the constitutive active form of Babo or Smad2, was sufficient to repress both amylase and maltase expression. Furthermore, glucose repression is mediated by the TGF-β ligand Daw, produced and secreted from the fat body, a metabolic tissue functionally analogous to the mammalian liver and adipose tissue. Thus, this study uncovers a physiological role for the TGF-β/Activin pathway in adapting carbohydrate and lipase digestion in response to the nutritional state of the organism. Because many features of digestion and absorption are conserved between flies and mammals, it will be of interest to investigate the role of TGF-β/Activin pathway in mammalian digestion (Karpac, 2013).

Recent studies have attributed a role for Daw in aging and pH homeostasis, two processes tightly linked to metabolism. Thus, it is likely that Daw induced from the fat body in response to carbohydrate in the diet will induce a more global response instead of a local response, affecting only digestive enzyme expression. As such, Daw may act as a central mediator for glucose homeostasis by regulating sugar level in the circulation. When there are sufficient carbohydrates in the diet, Daw expression restricts the expression of carbohydrase and glucose transporters. Concurrently, at the postabsorption level, Daw in the circulation may act directly or indirectly (via insulin signaling) to maintain circulating sugar level. A broader role for Daw in sugar homeostasis is reinforced by the findings that Daw mutant larvae were more sensitive to a high-sugar diet. Similarly, this study found overexpression of Daw, but not Myo, Mav, or Actβ, renders flies sensitive to sugar starvation. Along this line, in C. elegans, the TGF-β signaling is reported to be elevated and required in neurons for satiety. There were also several observations that hyperglycemia is linked to increased TGF-β activity in mammals. Hence, the role of TGF-β/Activin signaling in sugar homeostasis requires further investigation in Drosophila and other organisms (Karpac, 2013).

In conclusion, this study revealed a remarkable resilience in the regulation of carbohydrate and lipid-acting enzymes expression to ensure that digestive capacity in the midgut is not compromised before certain metabolic criteria in the fat body is attained. The study also unraveled a role of the TGF-β/Activin-signaling pathway in the adult Drosophila midgut, which has not been appreciated. It reinforced the notion that the gut is not a passive tube for nutriment flow. Rather, it dynamically modulates digestive enzyme expression in response to the organism’s nutritional state through endocrine signals derived from other metabolic tissues (Karpac, 2013).

Drosophila Pez acts in Hippo signaling to restrict intestinal stem cell proliferation

The conserved Hippo signaling pathway acts in growth control and is fundamental to animal development and oncogenesis. Hippo signaling has also been implicated in adult midgut homeostasis in Drosophila. Regulated divisions of intestinal stem cells (ISCs), giving rise to an ISC and an enteroblast (EB) that differentiates into an enterocyte (EC) or an enteroendocrine (EE) cell, enable rapid tissue turnover in response to intestinal stress. The damage-related increase in ISC proliferation requires deactivation of the Hippo pathway and consequential activation of the transcriptional coactivator Yorkie (Yki) in both ECs and ISCs. This study identified Pez, an evolutionarily conserved FERM domain protein containing a protein tyrosine phosphatase (PTP) domain, as a novel binding partner of the upstream Hippo signaling component Kibra. Pez function (but not its PTP domain) is essential for Hippo pathway activity specifically in the fly midgut epithelium. Thus, Pez displays a tissue-specific requirement and functions as a negative upstream regulator of Yki in the regulation of ISC proliferation (Poernbacher, 2012).

The WW domain protein Kibra has recently been shown to function as a tumor suppressor in the Hippo pathway. Because Kibra is an adaptor molecule, attempts were made to identify physical binding partners of Kibra to further explore upstream Hippo signaling. Affinity purification-mass spectrometry (AP-MS) analysis with Kibra as bait identified Pez as a novel interaction partner of Kibra in Drosophila cultured cells. The same result was recently obtained in a large-scale proteomic study of Drosophila cultured cells. The binding between Pez and Kibra was confirmed by reciprocal coimmunoprecipitation (co-IP) experiments with epitope-tagged proteins. Furthermore, a yeast two-hybrid (Y2H) experiment revealed that the Kibra-Pez interaction is robust and direct (Poernbacher, 2012).

To address a possible function of Pez in the Hippo pathway, two loss-of-function alleles of Pez that were generated by different methods. Pez¹ is an EMS-induced allele resulting in an early premature translational stop codon. Pez² was generated by imprecise excision of the P element P{GawB}NP4748, removing most of the Pez coding sequence. Homozygotes for either Pez allele as well as heteroallelic Pez¹/Pez² flies are viable but smaller than controls. Combinations of the Pez alleles with the deficiency Df(2L)ED384 uncovering the Pez locus are also viable and cause a similar reduction in body size as the homozygous or heteroallelic combinations. One copy of a GFP-tagged Pez genomic rescue construct (gPez) restores normal body size. Therefore, both Pez¹ and Pez² are likely to represent strong or null alleles. For further experiments, heteroallelic Pez¹/Pez² flies were used as Pez mutant flies (Poernbacher, 2012).

In addition to their reduced body size, Pez mutant flies exhibit a developmental delay of 2 days and decreased fertility, all hallmarks of starvation. Pez mutant larvae are small and have decreased triglyceride (TAG) stores and increased expression of the starvation marker genes lipase-3 and 4E-BP. Clones of Pez mutant cells in larval fat bodies did not affect lipid droplets, thus excluding a fat body-autonomous requirement for Pez in lipid metabolism. Surprisingly, overexpression of Drosophila Pez in the developing eye or wing decreased the size of the adult organs, indicating that Pez restricts growth rather than promoting it. It is proposed that the starvation-like phenotype of Pez mutants is due to indirect effects on metabolism arising from a failure in nutrient utilization. Clones of Pez mutant cells in wing imaginal discs did not show growth defects in comparison to their corresponding wild-type sister clones. However, Pez mutant flies exhibit hyperplasia and extensive multilayering of the adult midgut epithelium. One copy of gPez restores normal tissue architecture. The structure of the larval midgut epithelium, as well as that of the other larval and adult epithelia, is not disturbed in Pez mutants. Thus, Pez specifically functions to restrict growth of the adult midgut epithelium (Poernbacher, 2012).

The Pez protein contains two conserved structural elements: an amino-terminal FERM domain (band 4.1-ezrin-radixin- moesin family of adhesion molecules) and a carboxyterminal protein tyrosine phosphatase (PTP) domain. A truncated version of the protein lacking the FERM domain (DFERM-Pez) or a phosphatase-dead protein (PezPD) still rescued the Pez mutant gut phenotype when overexpressed in ECs. However, overexpression of DFERM-Pez in the developing wing failed to decrease wing size, whereas overexpression of PezPD or of a truncated protein lacking the PTP domain (DPTP-Pez) caused a similar phenotype as overexpression of wild-type Pez, suggesting that the FERM domain is required for the growth-regulatory function of endogenous Pez but becomes dispensable when DFERM-Pez is overexpressed in ECs. In contrast, the potential phosphatase activity of Pez is clearly not needed for its function in growth control (Poernbacher, 2012).

Two other FERM domain proteins, Merlin (Mer) and Expanded (Ex), act in upstream Hippo signaling to control organ size in Drosophila. Together with the WW domain protein Kibra, Ex and Mer constitute the KEM complex that assembles at the apical junction of epithelial cells and regulates the core Hippo pathway kinase cassette. Overexpression of Kibra, Ex, or Mer in ECs of Pez mutant flies significantly suppressed the Pez gut phenotypes. Thus, Pez is not an essential mediator of Hippo signaling downstream of the KEM complex. Mer and Ex did not detectably coimmunoprecipitate with Pez in Drosophila S2 cells. However, Kibra and Pez coimmunoprecipitated and colocalized in S2 cells. This was dependent on the first WW domain of Kibra, whereas the FERM and PTP domains of Pez as well as two potential ligands of WW domains, a PPPY motif and a PPSGY motif, in the central linker region of Pez were dispensable. A fragment encompassing a proline-rich stretch of Pez (amino acids 368-627; PezPro) was sufficient for the binding to Kibra, whereas the remaining linker region (amino acids 622-967; PezLink) did not bind Kibra. Importantly, knockdown of Kibra via Myo1A-Gal4 caused mild overgrowth of the adult midgut epithelium, and overexpressed Kibra recruited gPez-GFP from the cell cortex of ECs into cytoplasmic punctae. The subcellular localizations of overexpressed Kibra, Ex, or Mer were not affected when Pez was absent (Poernbacher, 2012).

It is concluded that Pez and Kibra function together in a protein complex to regulate Hippo signaling in adult midgut ECs. The results establish that the Drosophila Pez protein acts as a component of upstream Hippo signaling, restricts transcriptional activity of Yki in epithelial cells of the adult midgut, and plays a crucial role in the control of ISC proliferation. Importantly, the involvement of Hippo signaling in intestinal regeneration is conserved in the mammalian system ] (Poernbacher, 2012).

The two mammalian homologs of Drosophila Pez are the widely expressed, cytosolic nonreceptor tyrosine phosphatases PTPD1/PTPN21 and PTPD2/PTP36/PTPN14/Pez. All three proteins share a similar domain structure including the well-conserved terminal FERM and PTP domains. The central region shows extensive sequence divergence but it contains several shorter regions of conservation that may function as adaptors in signal transduction. PTPD1 is a component of a cortical scaffold complex nucleated by focal adhesion kinase (FAK) and thus regulates a proliferative signaling pathway through a scaffolding function. PTPD2 has been implicated in the regulation of cell adhesion, as an inducer of TGF-β signaling, and in lymphatic development of mammals and choanal development of humans. Interestingly, PTPD2 is a potential tumor suppressor, based on sporadic mutations in breast cancer cells and colorectal cancer cells. It is tempting to speculate that mammalian PTPD2 shares the function of its fly homolog as a component of Hippo signaling that restrains the oncogenic potential of gut regeneration (Poernbacher, 2012).

Suppressor of Deltex mediates Pez degradation and modulates Drosophila midgut homeostasis

Pez functions as an upstream negative regulator of Yorkie (Yki) to regulate intestinal stem cell (ISC) proliferation and is essential for the activity of the Hippo pathway specifically in the Drosophila midgut epithelium. This study reports that Suppressor of Deltex (Su(dx)) acts as a negative regulator of Pez. Su(dx) was shown to target Pez for degradation both in vitro and in vivo. Overexpression of Su(dx) induced proliferation in the fly midgut epithelium, which could be rescued by overexpressed Pez. The study also demonstrated that the interaction between Su(dx) and Pez, bridged by WW domains and PY/PPxY motifs, is required for Su(dx)-mediated Pez degradation. Furthermore, Kibra, a binding partner of Pez, was shown to stabilize Pez via WW-PY/PPxY interaction. Moreover, PTPN14, a Pez mammalian homolog, is degraded by overexpressed Su(dx) or Su(dx) homologue WWP1 in mammalian cells. These results reveal a previously unrecognized mechanism of Pez degradation in maintaining the homeostasis of Drosophila midgut (Wang, 2015).

The protein tyrosine phosphatase Pez is the Drosophila homologue of non-receptor type protein tyrosine phosphatase 14 (PTPN14), a regulator of the TGF-β pathway (Smith, 1995; Wyatt, 2007; Wyatt, 2008). PTPN14 overexpression activates TGF-β signalling and causes epithelial-mesenchymal transition (EMT) (Wyatt, 2007). Its overexpression is also correlated with lymphatic function, choanal development, angiogenesis and hereditary haemorrhagic telangiectasia (Au, 2010; Benzinou, 2012). Recent studies have revealed that PTPN14 negatively regulates the oncogenic function of Yes-associated protein (YAP) through retaining YAP in the cytoplasm and sustaining the phosphorylation state of YAP (Huang, 2013, Liu, 2013; Wang, 2012; Michaloglou, 2013). YAP is the transcription co-activator downstream of Hippo signalling to mediate the expression of various genes to promote growth, and its upregulation was found in a variety of human tumours and cancers (Wang, 2015).

Drosophila midgut, where the intestinal stem cells (ISCs) are under tight control to maintain homeostasis, has been developed as an excellent model to study adult stem cells in recent years. The Hippo signalling pathway has been shown to play an essential role in the regulation of ISC proliferation. Pez has been identified as a negative upstream regulator of Yorkie (Yki), the Drosophila homologue of YAP, and is required for the activity of the Hippo pathway in the regulation of ISC proliferation (Poernbacher, 2012). However, how the stability and function of Pez are regulated remains unclear (Wang, 2015).

Suppressor of Deltex (Su(dx)) is a member of the NEDD4 (neural precursor cell-expressed developmentally downregulated gene 4) family E3 ubiquitin ligase (Cornell, 1999). There are three typical NEDD4 family members in Drosophila, dSmurf, Su(dx) and NEDD4. Each of them contains an N-terminal phospholipid binding C2 domain, four WW domains and a C-terminal HECT-type ligase domain. Su(dx) was first reported as a negative regulator of the Notch signaling pathway (Fostier, 1998). It downregulates the expression of Notch target genes through promoting Notch endosomal sorting (Hori, 2004; Wilkin, 2004; Wang, 2015 and references therein).

This study shows that Su(dx) targets Pez for degradation both in vitro and in vivo. Su(dx) overexpression induces cell proliferation in Drosophila midgut by downregulating Pez protein levels. It was also demonstrated that Su(dx) directly interacts with Pez via its WW domains and Pez's PY/PPPY motifs. This interaction subsequently promotes Pez ubiquitylation. Furthermore, Kibra, a WW domain containing Pez binding partner, was found to stabilize Pez on interaction. Moreover, evidence is provided that overexpression of Su(dx) or its homologue WWP1 is able to degrade PTPN14 in mammalian culture cells, indicating a possibility that a conserved mechanism of Pez degradation may play an essential role in maintaining tissue homeostasis (Wang, 2015).

This study reports the identification of Su(dx) as an E3 ligase of Pez. Su(dx) was first identified as an E3 ligase regulating the Notch signalling pathway. But the direct substrate of Su(dx) was unclear. The present study identified Pez as a Su(dx) substrate. Furthermore, whether Pez regulates the Notch signalling pathway was examined. Pez knockdown induced notched wings and a decrease of Cut in wing discs that is very similar to what have caused by Su(dx) overexpression, indicating a dysfunction of the Notch pathway. However, another typical marker of the Notch pathway, wingless, was not affected by the absence of Pez. It is possible that Pez is not a canonical regulator of the Notch pathway and Su(dx) might have other substrates under this circumstance (Wang, 2015).

According to the observations, Su(dx) overexpression in ECs only induced midgut epithelial proliferation to some extent, and it did not fully mimic the loss of pez induced phenotypes. It is speculated that the difference was largely due to the incomplete degradation efficiency of Pez by Su(dx) overexpression in ECs (Wang, 2015).

PTPN14 has been reported as an inhibitor of YAP1 in mammalian cells. It can suppress the activity of YAP1 through retaining YAP1 in the cytoplasm and sustaining the phosphorylation state of YAP1 (Wang, 2012). However, in the current experiments, Pez overexpression slightly upregulates Yki phosphorylation level without obvious Yki localization change in S2 cells. It is speculated that the mechanism of YAP regulation by PTPN14 may not be conserved in Drosophila (Wang, 2015).

Furthermore, this work presents evidence that Kibra, a WW domain containing Pez partner, stabilizes Pez, providing an interesting model that WW–PY/PPxY interaction play a role in the regulation of protein stabilization. In addition, it was found that other WW-containing proteins, such as Sav, were unable to stabilize Pez. On the basis of these observations, the regulation of Pez stabilization by Su(dx) and Kibra is speculated to be a specific event (Wang, 2015).

It was also found that PTPN14, the human homologue of Pez, can be degraded by overexpressed Su(dx) and its human homologue WWP1. However, in the following experiments, it was found that WWC1, the human homologue of Kibra, did not stabilize PTPN14. These data suggest that, although the similar regulation of Pez/PTPN14 by degradation exists in Drosophila and mammalian cells, the detailed mechanism may vary (Wang, 2015).

It has been reported that PTPN14 sporadic mutations were found in breast cancer cells and colorectal cancer cells, indicating a potential tumour suppressor function of PTPN14. Moreover, amplification and overexpression of WWP1 has been found in breast and prostate cancers. Therefore, the current study may provide new insights into cancer development. Further characterization of the relationship of Su(dx)-Pez in mice and examination of their correlation in clinical cancers may provide potential targeting therapy for cancer treatments (Wang, 2015).

Windpipe controls Drosophila intestinal homeostasis by regulating JAK/STAT pathway via promoting receptor endocytosis and lysosomal degradation

The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. This study has identified windpipe (wdp) as a novel component of the JAK/STAT pathway. Wdp was positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity resulted in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibited JAK/STAT signaling activity. Importantly, Wdp interacted with the receptor Domeless (Dome), and promoted its internalization for subsequent lysosomal degradation. Together, these data led the study to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis (Ren, 2015).

This study has provided evidence that the LRR protein Wdp is a novel component of the JAK/STAT pathway that acts in a negative feedback manner to modulate JAK/STAT signaling activity and control intestinal homeostasis. In vivo and in vitro data indicate that wdp expression levels are positively regulated by JAK/STAT signaling. Loss of wdp disrupts midgut homeostasis under both physiological and damage conditions. Conversely, ectopic expression of Wdp leads to the reduction of JAK/STAT signaling activity. Mechanistically, it was shown that Wdp can interact with Dome, and promote Dome internalization and lysosomal degradation, thereby reducing JAK/STAT signaling activity (Ren, 2015).

Midgut homeostasis is tightly controlled by different signaling pathways. During tissue damage, JAK/STAT, EGFR, JNK and Hippo signaling pathways are required for ISC proliferation and midgut regeneration. On the other hand, other signaling pathways, such as BMP signaling, may negatively regulate intestinal homeostasis after injury, although there exists some controversy about the function of BMP signaling during Drosophila intestinal development. However, the mechanism of how ISC activity returns to quiescence after injury remains largely unknown. This study demonstrates that Wdp controls intestinal homeostasis through interfering with JAK/STAT signaling activity to avoid tissue hyperplasia (Ren, 2015).

The data indicate that loss of Wdp disrupts midgut homeostasis under normal conditions and potentiates tissue regeneration under damage conditions. The proliferation rate of ISCs mutant for wdp is increased, while the differentiation of EC and ee cells is not inhibited. In addition, ectopic Wdp expression suppressed the damage induced tissue regeneration. The data further demonstrate that Wdp controls intestinal homeostasis through interfering with JAK/STAT signaling activity. First, Wdp acts as a JAK/STAT downstream target and its expression levels are positively regulated by JAK/STAT signaling. Second, Wdp functions in a negative feedback loop to modulate JAK/STAT signaling activity. It is interesting to note that JAK/STAT signaling is mainly activated in ISCs and EBs. However, it was found that Wdp expression levels seem higher in ECs compared with progenitor cells. One explanation is that low levels of Wdp in progenitors may guarantee high levels of JAK/STAT signaling, while high levels of Wdp in ECs may serve to reduce Dome levels thereby making ECs insensitive to Upd ligands. Consistent with this view, previous work showed that Dome is mainly expressed in the progenitors but not in their progeny. Moreover, it was found Wdp knock down using EC specific Myo1A^ts also leads to the disruption of midgut homeostasis and the presence of 10xSTAT GFP in putative EC cells, suggesting that JAK/STAT signaling is activated upon wdp knockdown in ECs. On the other hand, it was found Wdp expression was reduced but not totally eliminated in JAK/STAT signaling deficient cells, suggesting that the basal level of Wdp in intestines (especially in ECs) may also be regulated by other regulatory mechanisms or signaling pathways. Further experiments are needed to clarify this issue (Ren, 2015).

It’s important to mention that Wdp expression could be induced under injury conditions, such as DSS or bleomycin treatment. Consistent with the results, two recent studies also identified wdp as an upregulated gene upon Ecc15 and Pseudomonas entomophila (P.e) infection through their microarray data respectively. These stress conditions are also associated with the activation of JAK/STAT signaling. Therefore, their findings are consistent with the view that Wdp can be induced by the JAK/STAT pathway and then restrict its signaling activity in restoring intestinal homeostasis after tissue damage (Ren, 2015).

It was further demonstrated the regulation of Wdp to JAK/STAT signaling in eye discs and S2 cells. 10xSTAT GFP activity was decreased in eye discs overexpressing Wdp while increased in wdp mutant eye discs. Similarly, a reduction of 10×STAT luciferase activity was also observed in S2 cells transfected with Wdp. Thus, it is proposed that Wdp is also likely to modulate JAK/STAT signaling activity for proper development of other tissues (Ren, 2015).

Taken together, it is concluded that Wdp is involved in controlling intestinal homeostasis through interfering with JAK/STAT signaling in a negative feedback manner (Ren, 2015).

Previously, several studies have addressed the roles of endocytosis in regulating JAK/STAT signal pathway. The Noselli lab found blocking internalization led to an inhibition of JAK/STAT signaling activity, while the Zeidler group reported the opposite results. Moreover, several recent studies demonstrate that loss of ept/tsg101 or Rabex-5, two endocytic tumor suppressor genes, also induced JAK/STAT signaling activation and tissue overgrowth. Yet, the regulatory mechanism of how Dome receptors are internalized remains largely unknown. This study demonstrates that Wdp promotes Dome endocytosis and subsequent lysosomal degradation. First, in S2 cells Wdp ectopic expression induces the formation of Dome endocytotic vesicles which were colocalized with the early endosome marker and lysosome marker. Second, it was found Wdp expression can also promote Dome endocytosis in wing and eye imaginal discs. Furthermore, the decreased Dome levels caused by Wdp expression can be suppressed by CQ treatment. All of these data argue that Wdp acts to promote Dome endocytosis from the cell membrane, first into the early endosomes, and finally into the lysosomes for degradation. Previous work are mainly about Dome receptors undergo ligands induced endocytosis, while this work showd that Wdp is able to promote Dome internalization in a Upd independent manner. Coimmnoprecipitation data indicate Wdp can interact with Dome. Moreover, Dome-GFP is aggregated on the cell membrane before they are internalized in the presence of Wdp. Therefore, one possible mechanism is that Wdp interacts with Dome, induces the aggregation of Dome on the cell membrane and then promotes Dome endocytosis. Further experiments are needed to define the detailed mechanism (Ren, 2015).

On the basis of these findings, the following model is proposed (see Model for the function of Wdp): Wdp regulates intestinal homeostasis through its modulation of JAK/STAT signaling. Under physical conditions, low levels of Wdp in progenitors are needed to maintain proper levels of JAK/STAT signaling activity, while high levels of Wdp in ECs reduce Dome levels to ensure these cells are insensitive to JAK/STAT signaling. When midgut epithelium is damaged by environmental challenges, high levels of JAK/STAT signaling activity are induced to replenish the damaged midgut. Then Wdp expression is highly induced in the intestines to reduce Dome levels, thereby switching off the overactivated JAK/STAT signaling. Through this way, ISC proliferative rate returns to normal levels to avoid tissue hyperplasia. While other mechanisms or regulators are likely to be involved in regulating intestinal homeostasis, the data suggest that Wdp is one of the key regulators in this process through interfering with JAK/STAT signaling activity (Ren, 2015).

A tetraspanin regulates septate junction formation in Drosophila midgut

Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes via the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of SJs are observed: pleated SJs (pSJs) and smooth SJs (sSJs), which are present in ectodermally- and endodermally-derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and Snakeskin (Ssk), in Drosophila indicates that the molecular compositions of sSJs and pSJs differ. A deficiency screen based on immunolocalization of Mesh, identified a tetraspanin family protein, Tetraspanin 2A (Tsp2A), as a novel protein involved in sSJ formation in Drosophila. Tsp2A specifically localizes at sSJs in the midgut and Malpighian tubules. Compromised (Tsp2A) expression caused by RNAi or the CRISPR/Cas9 system is associated with defects in the ultrastructure of sSJs, changes localization of other sSJ proteins, and impairs barrier function of the midgut. In most Tsp2A-mutant cells, Mesh fails to localize to sSJs and is distributed through the cytoplasm. Tsp2A forms a complex with Mesh and Ssk and these proteins are mutually interdependent for their localization. These observations suggest that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).

Epithelia separate distinct fluid compartments within the bodies of metazoans. For this epithelial function, specialized intercellular junctions, designated as occluding junctions, regulate the free diffusion of solutes through the paracellular pathway. In vertebrates, tight junctions act as occluding junctions, whereas, in invertebrates, septate junctions (SJs) are the functional counterparts of tight junctions. SJs form circumferential belts around the apicolateral regions of epithelial cells. In transmission electron microscopy, SJs are observed between the parallel plasma membranes of adjacent cells, with ladder-like septa spanning the intermembrane space. SJs are subdivided into several morphological types that differ among different animal phyla, and several phyla possess multiple types of SJs that vary among different types of epithelia (Izumi, 2016).

In arthropods, two types of SJs exist: pleated SJs (pSJs) and smooth SJs (sSJs). pSJs are found in ectodermally-derived epithelia and surface glia surrounding the nerve cord, while sSJs are found mainly in endodermally-derived epithelia, such as the midgut and the gastric caeca. The outer epithelial layer of the proventriculus (OELP) and the Malpighian tubules also possess sSJs, although these epithelia are ectodermal derivatives. The criteria distinguishing these two types of SJs are the arrangement of the septa. In oblique sections of lanthanum-treated preparations, the septa of pSJs are visualized as regular undulating rows but those in sSJs are observed as regularly spaced parallel lines. In freeze-fracture images, the rows of intramembrane particles in pSJs are separated from one another, whereas those in sSJs are fused into ridges. To date, more than 20 pSJ-related proteins, including pSJ components and regulatory proteins involved in pSJ assembly, have been identified and characterized in Drosophila. In contrast, few genetic and molecular analyses have been carried out on sSJs. Recently, two sSJ-specific membrane proteins, Ssk and Mesh, have been identified and characterized (Izumi, 2014; Izumi, 2012; Yanagihashi, 2012). Ssk consists of 162 amino acids and has four membrane-spanning domains, two short extracellular loops, cytoplasmic N- and C-terminal domains, and a cytoplasmic loop (Yanagihashi, 2012). Mesh has a single-pass transmembrane domain and a large extracellular region containing a NIDO domain, an Ig-like E set domain, an AMOP domain, a vWD domain, and a sushi domain (Izumi, 2012). Mesh transcripts are predicted to be translated into three isoforms of which the longest isoform consists of 1,454 amino acids. In Western blot studies, Mesh is detected as a main ~90 kDa band and a minor ~200 kDa band (Izumi, 2012). Compromised expression of ssk or mesh causes defects in the ultrastructure of sSJs and in the barrier function of the midgut against a 10-kDa fluorescent tracer (Izumi, 2012; Yanagihashi, 2012). Ssk and Mesh physically interact with each other and are mutually dependent for their sSJ localization (Izumi, 2012). Thus, Mesh and Ssk play crucial roles in the formation and barrier function of sSJs (Izumi, 2016).

Tetraspanins are a family of integral membrane proteins in metazoans with four transmembrane domains, N- and C-terminal short intracellular domains, two extracellular loops and one short intracellular turn. Among several protein families with four transmembrane domains, tetraspanins are characterized especially by the structure of the second extracellular loop. It contains a highly conserved cysteine-cysteine-glycine (CCG) motif and 2 to 4 other cysteine residues. These cysteines form 2 or 3 disulfide bonds within the loop. Tetraspanins are believed to play a role in membrane compartmentalization and are involved in many biological processes, including cell migration, cell fusion and lymphocyte activation, as well as viral and parasitic infections. Several tetraspanins regulate cell-cell adhesion but none are known to be involved in the formation of epithelial occluding junctions. In the Drosophila genome, there are 37 tetraspanin family members, and some have been characterized by genetic analyses. Lbm, CG10106 and CG12143 participate in synapse formation. Sun associates with light-dependent retinal degeneration. TspanC8 subfamily members, including Tsp3A, Tsp86D and Tsp26D, are involved in the Notch-dependent developmental processes via the regulation of a transmembrane metalloprotease, ADAM10 (Dornier, 2012). However, the functions of most other Drosophila tetraspanins remain obscure (Izumi, 2016).

This study identified a tetraspanin family protein, Tsp2A, as a novel molecular component of sSJs in Drosophila. Tsp2A is required for sSJ formation and for the barrier function of Drosophila midgut. Tsp2A and two other sSJ-specific membrane proteins Mesh and Ssk show mutually dependent localizations at sSJs and form a complex with each other. Therefore, it is concluded that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).

Of the sSJ-specific components, Mesh is a membrane-spanning protein and has an ability to induce cell-cell adhesion, implying that it is a cell adhesion molecule and may be one of the components of the electron-dense ladder-like structures in sSJs (Izumi, 2012). In contrast, both Ssk and Tsp2A are unlikely to act as cell adhesion molecules in sSJs because each of the two extracellular loops of Ssk (25 and 22 amino acids, respectively) appear to be too short to bridge the 15-20-nm intercellular space of sSJs. Furthermore, overexpression of EGFP-Tsp2A in Drosophila S2 cells did not induce cell aggregation, which is a criterion for cell adhesion activity (Izumi, 2016).

Several observations in Tsp2A-mutants may provide clues for understanding the role of Tsp2A in sSJ formation. In most Tsp2A-mutant midgut epithelial cells, Mesh fails to localize to the apicolateral membranes but was distributed in the cytoplasm, possibly to specific intracellular membrane compartments. To further examine where Mesh was localized in Tsp2A-mutant cells, the midgut was doublestained with the anti-Mesh antibody and the antibodies against typical markers of various intracellular membrane compartments, including the Golgi apparatus (anti-GM130), early endosomes (anti-Rab5), recycling endosomes (anti-Rab11) and lysosomes (anti-LAMP1). However, it was not possible to detect any overlap between staining by these markers and that of Mesh. The staining pattern in Tsp2A-mutant midgut epithelial cells produced with the anti-KDEL antibody, which labels endoplasmic reticulum, was similar, although not identical with that produced by the anti-Mesh antibody (Izumi, 2016).

Interestingly, some tetraspanins are known to control the intracellular trafficking of their partners. For instance, a mammalian tetraspanin, CD81 is necessary for normal trafficking or for surface membrane stability of a phosphoglycoprotein, CD19, in lymphoid B cells. The TspanC8 subgroup proteins, which all possess eight cysteine residues in their large extracellular domain, regulate the exit of a metalloproteinase, ADAM10, from the ER and differentially control its targeting to either late endosomes or to the plasma membrane. Consequently, TspanC8 proteins regulate Notch signaling via the activation of ADAM10 in mammals, Drosophila and Caenorhabditis elegans. If Mesh is retained in the trafficking pathway from endoplasmic reticulum to plasma membrane in Tsp2A-mutant cells, Tsp2A may have an ability to promote the intracellular trafficking of Mesh in the secretory pathway. To clarify the role of Tsp2A in sSJ formation, it will be necessary to determine the intracellular membrane compartment where Mesh was localized in Tsp2A-mutant cells (Izumi, 2016).

Tsp2A, Mesh and Ssk are mutually dependent for their localization at sSJs. Consistent with this intimate relationship, the co-immunoprecipitation experiment revealed that Tsp2A physically interacts with Mesh and Ssk in vivo. However, the amount of Ssk observed in the co-immunoprecipitation with EGFP-Tsp2A was barely enriched relative to that in the extracts of embryos expressing EGFP-Tsp2A. This was particularly striking in comparison to the degree of enrichment of Mesh in the co-immunoprecipitation with EGFP-Tsp2A. To interpret these results, the detailed manner of the interaction between Tsp2A, Mesh and Ssk proteins needs to be further clarified. Many tetraspanin family proteins are known to interact with one another and with other integral membrane proteins to form a dynamic network of proteins in cellular membranes. Tetraspanins are also believed to have a role in membrane compartmentalization. Given such functional properties of tetraspanins, Tsp2A may determine the localization of sSJs at the apicolateral membrane region by membrane domain formation (Izumi, 2016).

In the Tsp2A-mutant midgut epithelial cells, Lgl was distributed throughout the basolateral membrane region, whereas it was localized in the apicolateral membrane region in the wild-type. In view of the role of Lgl in the formation of the apical-basal polarity of ectodermally-derived epithelial cells, it is of interest to consider whether this abnormal localization of Lgl in the Tsp2A-mutant affects epithelial polarity. However, in the Tsp2A-mutant midgut epithelial cells, Dlg still showed polarized concentration into the apicolateral membrane region and the Lgl never leaked into the apical membrane domain. These observations suggest that the lack of Tsp2A does not affect the gross apical-basal polarity of the midgut epithelial cells (Izumi, 2016).

Some tetraspanins have been reported to be involved in the regulation of cell-cell adhesion. A mammalian tetraspanin, CD151, regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin reorganization, or through complex formation with α3γ1 integrin. A mammalian tetraspanin, CD9, is concentrated in the axoglial paranodal region in the brain and in the peripheral nervous system, and CD9 knockout mice display defects in the formation of paranodal septate junctions and in the localization of paranodal proteins. Paranodal septate junctions have electron-dense ladder-like structures and their molecular organization is similar to that of pSJs but tetraspanins involved in pSJ formation have not been reported in Drosophila (Izumi, 2016).

Interactions between several tetraspanins and claudins, the key integral membrane proteins involved in the organization and function of tight junctions, are also known. Claudin-11 forms a complex with OAP-1/Tspan-3 and chemical crosslinking reveals a direct association between claudin-1 and CD9. Furthermore, the interaction between claudin-1 and CD81 is shown to be required for hepatitis C virus infectivity. To date, no tight junction defect has been reported in CD9 knockout mice, CD81 knockout mice, or CD9/CD81 double knockout mice. Further investigation is necessary to clarify whether the interactions between tetraspanins and tight junction proteins are involved in the formation and function of tight junctions (Izumi, 2016).

A novel membrane protein Hoka regulates septate junction organization and stem cell homeostasis in the Drosophila gut

Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal tract in arthropods. In Drosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh, and Tsp2A. This study reports a novel sSJ membrane protein Hoka, which has a single membrane-spanning segment with a short extracellular region, and a cytoplasmic region with the Tyr-Thr-Pro-Ala motifs. The larval midgut in hoka-mutants shows a defect in sSJ structure. Hoka forms a complex with Ssk, Mesh, and Tsp2A and is required for the correct localization of these proteins to sSJs. Knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, and stem cell overproliferation. In hoka-knockdown midguts, aPKC is up-regulated in the cytoplasm and the apical membrane of epithelial cells. The depletion of aPKC and yki in hoka-knockdown midguts results in reduced stem cell overproliferation. These findings indicate that Hoka cooperates with the sSJ-proteins Ssk, Mesh, and Tsp2A to organize sSJs, and is required for maintaining intestinal stem cell homeostasis through the regulation of aPKC and Yki activities in the Drosophila midgut (Izumi, 2021).

Epithelia separate distinct fluid compartments within the bodies of metazoans. For this epithelial function, occluding junctions act as barriers that control the free diffusion of solutes through the paracellular pathway. Septate junctions (SJs) are occluding junctions in invertebrates and form circumferential belts along the apicolateral region of epithelial cells. In transmission electron microscopy, SJs are observed between the parallel plasma membranes of adjacent cells, with ladder-like septa spanning the intermembrane space. Arthropods have two types of SJs: pleated SJs (pSJs) and smooth SJs (sSJs). pSJs are found in ectoderm-derived epithelia and surface glia surrounding the nerve cord, whereas sSJs are found mainly in the endoderm-derived epithelia, such as the midgut and gastric caeca. Despite being derived from the ectoderm, the outer epithelial layer of the proventriculus (OELP) and the Malpighian tubules also possess sSJs. Furthermore, pSJs and sSJs are distinguished by the arrangement of septa. For example, the septa of pSJs form regular undulating rows, whereas those in sSJs form regularly spaced parallel lines in the oblique sections in lanthanum-treated preparations. To date, more than 20 pSJ-related proteins have been identified and characterized in Drosophila. In contrast, only three membrane-spanning proteins, Ssk, Mesh and Tsp2A, have been reported as specific molecular constituents of sSJs (sSJ proteins) in Drosophila. Therefore, the mechanisms underlying sSJ organization and the functional properties of sSJs remain poorly understood compared with pSJs. Ssk has four membrane-spanning domains; two short extracellular loops, cytoplasmic N- and C-terminal domains, and a cytoplasmic loop. Mesh is a cell-cell adhesion molecule, which has a single-pass transmembrane domain and a large extracellular region containing a NIDO domain, an Ig-like E set domain, an AMOP domain, a vWD domain and a sushi domain. Tsp2A is a member of the tetraspanin family of integral membrane proteins in metazoans with four transmembrane domains, N- and C-terminal short intracellular domains, two extracellular loops and one short intracellular turn. The loss of ssk, mesh and Tsp2A causes defects in the ultrastructure of sSJs and the barrier function against a 10 kDa fluorescent tracer in the Drosophila larval midgut. Ssk, Mesh and Tsp2A interact physically and are mutually dependent for their sSJ localization. Thus, Ssk, Mesh and Tsp2A act together to regulate the formation and barrier function of sSJs. Furthermore, Ssk, Mesh and Tsp2A are localized in the epithelial cell-cell contact regions in the Drosophila Malpighian tubules in which sSJs are present. Recent studies have shown that the knockdown of mesh and Tsp2A in the epithelium of Malpighian tubules leads to defects in epithelial morphogenesis, tubule transepithelial fluid and ion transport, and paracellular macromolecule permeability in the tubules. Thus, sSJ proteins are involved in the development and maintenance of functional Malpighian tubules in Drosophila (Izumi, 2021).

The Drosophila adult midgut consists of a pseudostratified epithelium, which is composed of absorptive enterocytes (ECs), secretory enteroendocrine cells (EEs), intestinal stem cells (ISCs), EC progenitors (enteroblasts: EBs) and EE progenitors (enteroendocrine mother cells: EMCs). The sSJs are formed between adjacent ECs and between ECs and EEs. To maintain midgut homeostasis, ECs and EEs are continuously renewed by proliferation and differentiation of the ISC lineage through the production of intermediate differentiating cells, EBs and EMCs. Recently, it has been reported that the knockdown of sSJ proteins Ssk, Mesh and Tsp2A in the midgut causes intestinal hypertrophy accompanied by the overproliferation of ECs and ISC. These results indicate that sSJs play a crucial role in maintaining tissue homeostasis through the regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut. Furthermore, it has been reported that the loss of mesh and Tsp2A in adult midgut epithelial cells causes defects in cellular polarization, although no remarkable defects in epithelial polarity were found in the first-instar larval midgut cells of ssk, mesh and Tsp2A mutants. Thus, sSJs may contribute to the establishment of epithelial polarity in the adult midgut (Izumi, 2021).

During the regeneration of the Drosophila adult midgut epithelium, various signaling pathways are involved in the proliferation and differentiation of the ISC lineage. Atypical protein kinase C (aPKC) is an evolutionarily conserved key determinant of apical-basal epithelial polarity . Importantly, it has been reported that aPKC is dispensable for the establishment of epithelial cell polarity in the Drosophila adult midgut. It has been reported that aPKC is required for differentiation of the ISC linage in the midgut. The Hippo signaling pathway is involved in maintaining tissue homeostasis in various organs. In the Drosophila midgut, inhibition of the Hippo signaling pathway activates the transcriptional co-activator Yorkie (Yki), which results in accelerated ISC proliferation via the Unpaired (Upd)-Jak-Stat signaling pathway. Recent studies have shown that Yki is involved in ISC overproliferation caused by the depletion of sSJ proteins in the midgut. Furthermore, it has been shown that aPKC is activated in the Tsp2A-RNAi-treated midgut, leading to activation of its downstream target Yki and causing ISC overproliferation through the activation of the Upd-Jak-Stat signaling pathway. Thus, crosstalk between aPKC and the Hippo signaling pathways appears to be involved in ISC overproliferation caused by Tsp2A depletion (Izumi, 2021).

To further understand the molecular mechanisms underlying sSJ organization, a deficiency screen was performed for Mesh localization, and the integral membrane protein Hoka was identified as a novel component of Drosophila sSJs. Hoka consists of a short extracellular region and the characteristic repeating 4-amino acid motifs in the cytoplasmic region, and is required for the organization of sSJ structure in the midgut. Hoka and Ssk, Mesh, and Tsp2A show interdependent localization at sSJs and form a complex with each other. The knockdown of hoka in the adult midgut results in intestinal barrier dysfunction, aPKC- and Yki-dependent ISC overproliferation, and epithelial tumors. Thus, Hoka plays an important role in sSJ organization and in maintaining ISC homeostasis in the Drosophila midgut (Izumi, 2021).

The identification of Ssk, Mesh and Tsp2A has provided an experimental system to analyze the role of sSJs in the Drosophila midgut. Recent studies have shown that sSJs regulate the epithelial barrier function and also ISC proliferation and EC behavior in the midgut. Furthermore, sSJs are involved in epithelial morphogenesis, fluid transport and macromolecule permeability in the Malpighian tubules. This study reports the identification of a novel sSJ-associated membrane protein Hoka. Hoka is required for the efficient accumulation of other sSJ proteins at sSJs and the correct organization of sSJ structure. The knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, increased ISC proliferation mediated by aPKC and Yki activities, and epithelial tumors. Thus, Hoka contributes to sSJ organization and the maintenance of ISC homeostasis in the Drosophila midgut (Izumi, 2021).

Arthropod sSJs have been classified together based on their morphological similarity. The identification of sSJ proteins in Drosophila has provided an opportunity to investigate whether sSJs in various arthropod species share similarities at the molecular level. However, Hoka homolog proteins appear to be conserved only in insects upon a database search, suggesting compositional variations in arthropod sSJs (Izumi, 2021).

Interestingly, the cytoplasmic region of Hoka includes three YTPA motifs. The same or similar amino acid motifs are also present in the Hoka homologs of other holometabolous insects, such as other Drosophila species, the mosquito, beetle (YTPA motif), butterfly, ant, bee, sawfly, moth (YQPA motif) and flea (YTAA motif), although the number of these motif(s) vary (1 to 3 in Drosophila species, 1 in other holometabolous insects). In contrast, the motif is not present in hemimetabolous insects. The extensive conservation of the YTPA/YQPA/YTAA motif in holometabolous insects suggests that the motif was evolutionarily acquired and plays a critical role in the molecular function of Hoka. It would be interesting to investigate the role of the YTPA/YQPA/YTAA motif in sSJ organization of holometabolous insects (Izumi, 2021).

The extracellular region of Hoka appears to be composed of 13 amino acids alone after the cleavage of the signal peptide, which is too short to bridge the 15-20 nm intercellular space of sSJs. Thus, Hoka is unlikely to act as a cell adhesion molecule in sSJs. Indeed, the overexpression of Hoka-GFP in Drosophila S2 cells did not induce cell aggregation, which is a criterion for cell adhesion activity (Izumi, 2021).

The loss of an sSJ protein results in the mislocalization of other sSJ proteins, indicating that sSJ proteins are mutually dependent for their sSJ localization. In thessk -deficient midgut, Mesh and Tsp2A were distributed diffusely in the cytoplasm. In the mesh mutant midgut, Ssk was localized at the apical and lateral membranes, whereas Tsp2A was distributed diffusely in the cytoplasm. In the Tsp2A-mutant midgut, Ssk was localized at the apical and lateral membranes, whereas Mesh was distributed diffusely in the cytoplasm. Among these three mutants, the mislocalization of Ssk, Mesh or Tsp2A is consistent; Mesh and Tsp2A were distributed in the cytoplasm, whereas Ssk was localized at the apical and lateral membranes. However, in the hoka-mutant larval midgut, Mesh and Tsp2A were distributed along the lateral membrane, whereas Ssk was mislocalized to the apical and lateral membranes. Interestingly, in some hoka mutant midguts, Ssk, Mesh and Tsp2A were localized to the apicolateral region, as observed in the wild-type midgut. Differences in subcellular misdistribution of sSJ proteins between the hoka mutant and the ssk, mesh and Tsp2A-mutants indicate that the role of Hoka in the process of sSJ formation is different from that of Ssk, Mesh or Tsp2A. Ssk, Mesh and Tsp2A may form the core complex of sSJs, and these proteins are indispensable for the generation of sSJs, whereas Hoka facilitates the arrangement of the primordial sSJs at the correct position, i.e. the apicolateral region. This Hoka function may also be important for rapid paracellular barrier repair during the epithelial cell turnover in the adult midgut. Notably, during the sSJ formation process of the outer epithelial layer of the proventriculus (OELP, the sSJ targeting property of Hoka was similar to that of Mesh, implying that Hoka may have a close relationship with Mesh, rather than Ssk and Tsp2A during sSJ development (Izumi, 2021).

The knockdown of hoka in the adult midgut leads to a shortened lifespan in adult flies, intestinal barrier dysfunction, increased ISC proliferation and the accumulation of ECs. These results are consistent with the recent observation for ssk, mesh and Tsp2A-RNAi in the adult midgut. The intestinal barrier dysfunction caused by RNAi for sSJ proteins may permit the leakage of particular substances from the midgut lumen, which may induce particular cells to secrete cytokines and growth factors for ISC proliferation. Alternatively, sSJs or sSJ-associated proteins may be directly involved in the secretion of cytokines and growth factors through the regulation of intracellular signaling in the ECs. In the latter case, it has been shown that Tsp2A knockdown in ISCs/EBs or ECs hampers the endocytic degradation of aPKC, thereby activating the aPKC and Yki signaling pathways, leading to ISC overproliferation in the midgut. Therefore, it has been proposed that sSJs are directly involved in the regulation of aPKC and the Hippo pathway-mediated intracellular signaling for ISC proliferation. This study has shown that the expression of hoka-RNAi together with aPKC-RNAi or yki-RNAi in ECs significantly reduced ISC overproliferation caused by hoka-RNAi. Thus, aPKC- and Yki-mediated ISC overproliferation appears to commonly occur in sSJ protein-deficient midguts. However, the possibility that the leakage of particular substances through the paracellular route may be involved in ISC overproliferation in the sSJ proteins-deficient midgut cannot be excluded (Izumi, 2021).

It has been reported that apical aPKC staining is observed in ISCs but is barely detectable in ECs. This study found that the expression of hoka-RNAi in ECs increased aPKC staining in the midgut. Additionally, in the hoka-RNAi midgut, apical aPKC staining was observed in ISCs and in differentiated cells, including EC-like cells. Thus, apical and increased cytoplasmic aPKC may contribute to ISC overproliferation. Interestingly, EC-like cells in the hoka-RNAi midgut do not always localize aPKC to the apical regions. Apical aPKC staining was detected in EC-like cells mounted by other cells but was barely detectable in the lumen-facing EC-like cells. These mounted cells are thought to be newly generated cells after the induction of hoka-RNAi, which may not be able to exclude aPKC from the apical region in the crowded cellular environment. A recent study showed that aberrant sSJ formation caused by Tsp2A-depletion impairs aPKC endocytosis and increases aPKC localization in the membrane of cell borders. The sSJ proteins, including Hoka, may also regulate endocytosis to exclude aPKC from the apical membrane of ECs. The identification of molecules involved in aPKC-mediated ISC proliferation may provide a better understanding of the aPKC-mediated signaling pathway, as well as the mechanisms underlying the increased expression and apical targeting of aPKC in the ECs deficient for sSJ proteins (Izumi, 2021).

Sex differences in intestinal carbohydrate metabolism promote food intake and sperm maturation

Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. This study used the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. Carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signaling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this male-biased intestinal metabolic state in controlling food intake and sperm production through gut-derived citrate. This work uncovers a male gonad-gut axis coupling diet and sperm production, revealing that metabolic communication across organs is physiologically important. The instructive role of citrate in inter-organ communication might be significant in more biological contexts than previously recognized (Hudry, 2019).

Males and females differ in their physiology and disease susceptibility yet the sex of cells and animals has often been neglected in research, or a single sex (male) is preferentially used. This might have prevented identification of sex differences that could inform clinical studies and therapies. Pressure to consider both sexes in basic and clinical research is revealing that sex differences are extensive, yet relatively underexplored (Hudry, 2019).

Sex chromosome sensing in Drosophila melanogaster activates a splicing cascade that results in expression of the RNA-binding protein Tra^F only in females, leading to sex-specific splicing of the transcription factors Doublesex (Dsx) and Fruitless (Fru) in a subset of cells, which sculpt sexually dimorphic anatomical features, reproductive systems, and behavior. Although superficially distinct from mammalian mechanisms involving gonadal release of sex hormones, Drosophila and mammalian sex differentiation shares common effectors such as the Dmrt/Dsx family of transcription factors. Furthermore, mouse models have revealed a cell-intrinsic contribution of sex chromosome complements to sex differences in body size and adiposity in mammals, and studies in flies have hinted at cell-extrinsic contributions to sex-biased phenotypes. Thus, sex differentiation in both insects and mammals appears to be a complex process integrating intrinsic and extrinsic inputs (Hudry, 2019).

Like its mammalian counterpart, the adult Drosophila digestive tract is a plastic and functionally regionalized organ, harboring microbiota and cell types akin to those found in humans, including self-renewing epithelial progenitors, digestive and absorptive enterocytes (ECs), and hormone-secreting enteroendocrine cells.Recently work has revealed sex differences in intestinal stem cell proliferation, which are adult-reversible and intrinsic to the stem cells (Hudry, 2016). During the course of these experiments, intestinal sex differences were observed in metabolic gene expression (Hudry, 2016), suggesting that sex-biased intestinal metabolism might contribute to sex differences in whole-body physiology (Hudry, 2019).

The intestine communicates with other organs, and peptide hormones are well established mediators. However, intermediate products of intracellular, housekeeping metabolic pathways are detected in the circulation, and recent work is revealing that both healthy tissues and tumors can use (and sometimes require) such exogenous, circulating metabolites. Consequently, there is considerable interest in exploring the instructive potential of metabolites in the context of inter-organ signaling (Hudry, 2019).

This study has uncover bi-directional communication between the male gonad and an adjacent intestinal region. This communication affects both gut and testes function and is mediated by cytokine signaling and the metabolite citrate (Hudry, 2019).

Regional differences in gene expression are observed along animal gastrointestinal tracts, suggestive of functional specializations. This study now provides evidence for region- and cell-type-specific carbohydrate metabolism. Intestinal carbohydrate metabolism also differs between the sexes, illustrating how sex differences can be confined to specific organ portions; even when digestive enzymes are more broadly expressed along the midgut, their male upregulation is posterior midgut (R4)-specific. It is suggested that specific gut portions might be physiologically 'sexualized' to subserve reproductive needs -- in this case spermatogenesis. The posterior midgut might be more broadly sexually dimorphic than other intestinal regions; oxidative stress response proteins are male biased and Yp1 is female biased in this same region (Hudry, 2016). In female flies, posterior midgut ECs adjust their lipid metabolism after mating to maximize reproductive output. It will be of interest to explore whether this requires their female identity; if it does, is female identity the 'ground state' in the absence of a male gonad, or does it result from an ovary signal? Comparative studies could also explore contributions of intestinal sex differences to reproductive success in animals other than Drosophila and whether the evolution of a placenta (an organ purpose-built for reproduction) replaced or reinforced such intestinal contributions in female mammals (Hudry, 2019).

The male gonad controls sex differences in intestinal carbohydrate metabolism through male-biased cytokine signaling activity. Drosophila Upd proteins belong to the type I family of cytokines, like mammalian interleukins and leptin. In both humans and rodents, leptin expression is sexually dimorphic. Males and females also differ in their interleukin repertoire, which contributes to sex differences in immunity and autoimmune disease. A possible contribution of cytokines such as leptin to sex differences in organ physiology deserves further investigation, particularly in light of leptin's known reproductive and gastrointestinal roles (Hudry, 2019).

The gonadal regulation of intestinal sugar metabolism contrasts with the intrinsic, sex-chromosome-dependent control of sex differences in gut stem cell proliferation (Hudry, 2016). This illustrates the complexity of an organ's 'sexual identity;' two lineage-related cells within an epithelium (stem cells and their EC progeny) acquire sex-specific functions (proliferation and carbohydrate metabolism) through two distinct mechanisms. Sexual identity is reversible in both cases and needs to be actively maintained in adults, raising the question of whether adult plasticity in sexual identity might be adaptive. Environmental factors could modulate the expression or penetrance of sex determinants -- possibly tissue specifically. There is some evidence in support of this idea: male flies that lack Fru^M are defective in courtship but learn to court when housed in groups with wild-type flies in a Dsx^M-dependent manner. Early life exposure to nutrient scarcity also affects neuronal wiring selectively of male C. elegans. In light of these findings, it will be of interest to explore how plastic sex differences in physiology are and why (Hudry, 2019).

Gut-gonad communication is bi-directional; the male gonad communicates with a specific gut portion, which responds by secreting citrate. Gut-derived citrate in turn promotes food intake and maturation of male gametes. How might it do so? Import of exogenous citrate might help sustain the high TCA cycle requirements of developing sperm. Sertoli cells are highly glycolytic and have been proposed to act as a paracrine source of lactate for developing gametes. It is therefore conceivable that citrate acts as another exogenous carbon source. Consistent with this idea, the mitochondrial citrate carrier is present and active in human sperm, and boar sperm can metabolize exogenous citrate through the Krebs cycle in vitro. Alternatively, import of gut-derived citrate might sustain membrane formation through its conversion to acetyl-CoA by ATCPL, then used for fatty acid synthesis; both spermatid elongation and individualization require extensive membrane biosynthesis and remodeling. Citrate could also support epigenetic changes relevant to male gamete maturation through its conversion to acetyl-CoA, used as a donor for histone acetyl transferase-mediated histone acetylatione (Hudry, 2019).

The effects of gut-derived citrate on sperm production can be uncoupled from its orexigenic actions. Preventing citrate import into neurons reduces food intake, suggesting that its promotion of feeding might result from its actions in the nervous system. Given that preventing gut-derived citrate efflux does not affect circulating citrate, it is tempting to speculate that local gut and/or testis-innervating neurons might harbor the citrate sensors. This effect of citrate on food intake is male specific: reducing gut-derived citrate efflux does not reduce feeding in females. This ongoing work is revealing that, in females, gonad to gut communication also promotes feeding, but via a different mechanism and possibly as a result of different dynamics and/or metabolic requirements of male and female gamete production (Hudry, 2019).

More generally, this study provides evidence that citrate functions in communication between organs. In mammals, plasma levels of citrate are among the highest among TCA cycle intermediates. Organ-specific differences in citrate production and consumption have been reported, but little is known about its roles and regulation by diet, age, or sex. Bone, an organ that controls male fertility through an endocrine hormone, produces unusually high amounts of citrate. In the context of male gametes, the prostate should also be considered as a potentially relevant citrate source; it secretes large amounts of citrate into the seminal fluid that developing sperm will come into contact with. The roles of prostate citrate have been investigated in the context of the metabolic rewiring of prostate tumors. Less is known about its roles in the context of sperm production, partly because surgical interventions such as prostatectomy impair other aspects of testis physiology. Contributions of exogenous citrate to sperm-mediated transgenerational effects also deserve further investigation in light of citrate's epigenetic effects. It will also be of interest to characterize the transporters for citrate import into the germline to control spermatogenesis and/or into neurons to control food intake; CG7309 and Indy-2 genes code for putative citrate transporters and have testis-specific expression. In mammals, the Indy homolog NaCT is specifically expressed in testis, liver, and brain, and NaCT knockout mice are protected from diet- and age-induced adiposity and insulin resistance (Hudry, 2019).

The physical proximity between the male gonad and the gut portion to which it signals raises the possibility that the relative positioning of internal organs is physiologically significant. Although this particular association is not conserved in adult humans, testis development is a complex process from a three-dimensional perspective, which in all placental mammals involves descent of testes from a position near the kidneys, perhaps providing opportunities for inter-organ communication. More generally, a spectrum of conditions (so-called heterotaxy syndromes) resulting from the abnormal arrangement of internal organs including the gastrointestinal tract can lead to serious disease manifestations. Subtler, likely undiagnosed defects in intestinal positioning could result in milder gastrointestinal symptoms and/or contribute to differences in whole-body physiology across individuals (Hudry, 2019).

Notch and EGFR regulate apoptosis in progenitor cells to ensure gut homeostasis in Drosophila

The regenerative activity of adult stem cells carries a risk of cancer, particularly in highly renewable tissues. Members of the family of inhibitor of apoptosis proteins (IAPs) inhibit caspases and cell death, and are often deregulated in adult cancers; however, their roles in normal adult tissue homeostasis are unclear. This study show that regulation of the number of enterocyte-committed progenitor (enteroblast) cells in the adult Drosophila involves a caspase-mediated physiological apoptosis, which adaptively eliminates excess enteroblast cells produced by intestinal stem cells (ISCs) and, when blocked, can also lead to tumorigenesis. Importantly, it was found that Diap1 is expressed by enteroblast cells and that loss and gain of Diap1 led to changes in enteroblast numbers. Antagonistic interplay between Notch and EGFR signalling was found to govern enteroblast life/death decisions via the Klumpfuss/WT1 and Lozenge/RUNX transcription regulators, which also regulate enteroblast differentiation and cell fate plasticity. These data provide new insights into how caspases drive adult tissue renewal and protect against the formation of tumours (Reiff, 2019).

IRBIT directs differentiation of intestinal stem cell progeny to maintain tissue homeostasis

The maintenance of the intestinal epithelium is ensured by the controlled proliferation of intestinal stem cells (ISCs) and differentiation of their progeny into various cell types, including enterocytes (ECs) that both mediate nutrient absorption and provide a barrier against pathogens. The signals that regulate transition of proliferative ISCs into differentiated ECs are not fully understood. IRBIT (CG9977) is an evolutionarily conserved protein that regulates ribonucleotide reductase (RNR), an enzyme critical for the generation of DNA precursors. This study shows that IRBIT expression in ISC progeny within the Drosophila midgut epithelium cells regulates their differentiation via suppression of RNR activity. Disruption of this IRBIT-RNR regulatory circuit causes a premature loss of intestinal tissue integrity. Furthermore, age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny. Collectively, these findings demonstrate a role of the IRBIT-RNR pathway in gut homeostasis (Arnaoutov, 2020).

An intestinal zinc sensor regulates food intake and developmental growth

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes. This study used a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. These findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis (Redhai, 2020).

To investigate nutrient sensing in enterocytes, 111 putative nutrient sensors in D. melanogaster were selected on the basis of their intestinal expression and their predicted structure or function. Using two enterocyte-specific driver lines, their expression was downregulated in midgut enterocytes throughout development under two dietary conditions, nutrient-rich and nutrient-poor; it was reasoned that dysregulation of nutrient-sensing mechanisms may increase or reduce the normal period of larval growth, and might do so in a diet-dependent manner. Enterocyte-specific knockdown of the gene CG11340, also referred to as pHCl-22, resulted in developmental delay. This delay was exacerbated, and was accompanied by significantly reduced larval viability, under nutrient-poor conditions; these phenotypes were confirmed using a second RNAi transgene and a new CG11340 mutant. In the tradition of naming Drosophila genes according to their loss-of-function phenotype, CG11340 was named 'hodor', an acronym for 'hold on, don't rush', in reference to the developmental delay (Redhai, 2020).

A transcriptional reporter revealed that Hodor was expressed in the intestine. A new antibody revealed that Hodor expression was confined to enterocytes in two midgut portions that are known to store metals: the copper cell region and the iron cell region. Within the copper cell region, Hodor was expressed only in so-called interstitial cells. hodor-Gal4 was also present in the interstitial cells of the copper cell region; however, in the experimental conditions used in this study and in contrast to published results, it was not detected in the iron cell region. Apart from the intestine, Hodor was found only in principal cells of the excretory Malpighian tubules. To identify the cells from which Hodor controls systemic growth, region- or cell-type-specific downregulation and rescue experiments were conducted. Only fly lines in which hodor was downregulated in interstitial cells showed slowed larval development. This developmental delay persisted when hodor knockdown was induced post-embryonically during larval growth, and was rescued only in fly lines in which hodor expression was re-instated in cell types that included interstitial cells. The fat body (analogous to liver and adipose tissue) has long been known to couple nutrient availability with developmental rate; however, recent studies have revealed contributions from the intestine, particularly in nutrient-poor conditions. The current findings confirm a role for the intestine in coupling nutrient availability with larval growth, and further implicate a subpopulation of enterocytes-interstitial cells-as important mediators. Interstitial cells were described decades ago in blowfly, but had remained relatively uncharacterized since; their name refers only to their position, interspersed among the acid-secreting copper cells that control microbiota loads (Redhai, 2020).

This study established that the lethality of hodor mutation or knockdown was apparent only during the larval period. The development of hodor mutants was slower throughout larval life, and surviving mutants attained normal pupal and adult sizes. Consistent with previous findings, hodor mutation or knockdown was found to reduce luminal acidity in the copper cell region, suggesting a role specifically for interstitial cells in this process. hodor mutants also had increased gut bacterial titres, which is consistent with the observed functional defects in the copper cell region. Enlarged volumes of both the lumen of the copper cell region and the interstitial cells were also apparent after 1-3 days of (delayed) larval development; ultrastructurally, this was apparent in interstitial cells as a reduction in the complexity of their characteristic basal infoldings. This study was, however, able to rule out all of these defects as reasons for the developmental delay (Redhai, 2020).

During the course of these experiments, it was observed that hodor mutant larvae were more translucent than control larvae. This was suggestive of peripheral lipid depletion, which was confirmed by quantifying and staining for triacylglycerides. Reduced lipid stores did not result from disrupted enterocyte integrity: the intestinal barrier of mutants was intact, both anatomically and functionally. It was observed that hodor mutants had less food in their intestines and accumulated insulin-like peptide Ilp2 in their brains (nutrient-dependent Ilp2 secretion promotes larval development; its accumulation in the brain is commonly interpreted as peptide retention in the absence of transcriptional changes). Consistent with reduced systemic insulin signalling, hodor mutant larval extracts had reduced levels of phospho-Akt and phospho-S6 kinase. As these are all indicators of starvation, food intake was quantified, and it was observed to be reduced in both hodor mutant larvae and in hodor knockdowns targeting interstitial cells. Reduced food intake was apparent soon after hatching and persisted throughout larval development. Ectopic expression of Ilp2-which rescues developmental delay in larvae that lack insulin-like peptides-in hodor mutants partially rescued their developmental delay, but did not increase their food intake. An 'instructive' link between intestinal Hodor and food intake was further suggested by the overexpression of hodor in otherwise wild-type enterocytes; this resulted in larvae that ate more and developed at a normal rate, but had increased lipid stores. Therefore, Hodor controls larval growth from a subset of enterocytes by promoting food intake and systemic insulin signalling. In its absence, larvae fail to eat sufficiently to proceed through development at the normal rate and are leaner. When present in excess, Hodor causes larvae to eat more and accumulate the energy surplus as fat (Redhai, 2020).

In fly adipose tissue, amino acid availability activates Tor signalling to promote systemic growth. This study therefore combined hodor knockout or knockdown with genetic manipulations to alter Tor signalling. In flies with reduced or absent Hodor function, decreasing or increasing Tor signalling in hodor-expressing cells exacerbated or rescued the developmental delay, respectively. The reduced food intake of hodor mutants was also significantly rescued by activation of Tor signalling in hodor-expressing cells. Genetic targeting of Rag GTPases or the Gator1 complex in these cells failed to affect the developmental delay of hodor mutants, which could suggest non-canonical regulation of Tor signalling in Hodor-expressing cells. The systemic effects of Hodor on food intake and larval growth are therefore modulated by Tor signalling within Hodor-expressing interstitial cells (Redhai, 2020).

Hodor belongs to the (typically neuronal) Cys-loop subfamily of ligand-gated ion channels, and is predicted to be a neurotransmitter-gated anion channel. It is known to show activity in response to alkaline conditions in Xenopus oocytes, but the acidic pH of the copper cell region prompted a search for additional ligands. Although alkaline pH-induced Hodor activity was confirmed in oocyte expression systems, Hodor did not respond to typical Cys-loop receptor ligands such as neurotransmitters or amino acids. Instead, the screen identified zinc as an unanticipated ligand, which elicited a strong dose-dependent response only in Hodor-expressing oocytes; this response to zinc showed peak current amplitude values much greater than those observed in response to pH or to other metals such as iron or copper. Force-field-based structural stability and binding affinity calculations identified the amino acid pair E255 and E296 as a potential binding site for the divalent zinc ion. Mutating these residues did not abrogate the zinc-elicited currents, but did result in currents with faster rise time and deactivation kinetics, which supports the idea that zinc is a relevant Hodor ligand. On the basis of its sequence and conductance properties, Hodor has been proposed to transport chloride (Feingold, 2016; Remnant, 2016), and the zinc-elicited currents that were observed in oocytes had a reversal potential that is consistent with chloride selectivity. In vivo experiments in flies showed that supplementation of a low-yeast diet with zinc led to a reduction of chloride levels in interstitial cells, whereas hodor mutation increased chloride levels. Thus, Hodor is a pH-modulated, zinc-gated chloride channel (Redhai, 2020).

Attempts were made to establish the relevance of zinc binding in vivo. Zinc enrichment is observed in both the copper and iron cell regions of the larval gut, revealing an unrecognized role for these Hodor-expressing regions in zinc handling. Mutation of hodor failed to affect this zinc accumulation, although dietary yeast levels did, which is consistent with a role for Hodor in sensing rather than transporting zinc. (Notably, the white mutation-which is frequently used in the genetic background of Drosophila experiments-results in a small but significant reduction in both intestinal zinc accumulation and larval growth rate, although the status of the w gene neither exacerbated nor masked the more substantial, hodor-induced developmental delay. Furthermore, larvae that were fed a low-yeast diet ate significantly more when the diet was supplemented with zinc; this effect was abrogated in hodor mutants. In a food choice experiment, control larvae developed a preference for zinc-supplemented food over time, which suggests that the preference develops after ingestion. Consistent with this idea, zinc preference was specifically abrogated in hodor mutants (their general ability to discriminate between other diets was confirmed. Thus, zinc sensing by Hodor is physiologically relevant in vivo. Metals such as zinc are primarily provided by yeasts in nature; Hodor may be one of several sensors used to direct larvae to nutrient-rich food sources (Redhai, 2020).

The subcellular localization of Hodor suggests that it may normally maintain low cytoplasmic chloride concentrations by transporting it out of the interstitial cells and/or into their lysosomes. In accordance with this, and consistent with its putative lysosomal localization signals, Hodor was specifically enriched in apical compartments containing late endosome or lysosomal markers, as well as decorating the brush border of interstitial cells. The presence of Hodor in a subpopulation of lysosomes was of interest, because chloride transport across lysosomal membranes often sustains the activity of the proton-pumping vacuolar-type ATPase (V-ATPase) that maintains lysosomal acidity and Tor activation on the lysosome. To explore a role for Hodor in enabling Tor signalling, whether the absence of hodor induced autophagy-a hallmark of reduced Tor signalling, was tested. First, the induction of common autophagy markers in interstitial cells after genetic interference with the V-ATPase complex, which is known to promote autophagy by reducing lysosomal acidity and Tor signalling, was confirmed. Similar to reduced V-ATPase function, loss of hodor increased autophagy in interstitial cells. Expression of the dual autophagosome and autolysosome reporter UAS-GFP-mCherry-Atg8a in the intestinal cells of hodor mutants confirmed the induction of autophagy, and revealed two additional features. First, the acidification of autophagic compartments was defective in hodor mutants. Second, the increased autophagy and defective acidification observed in hodor mutants were particularly prominent in the two Hodor-expressing intestinal regions (the copper cell region and the iron cell region), consistent with cell-intrinsic roles for Hodor in these processes. Additional support for the roles of lysosomal function and Tor signalling in controlling whole-body growth from interstitial cells was provided by the finding that most V-ATPase subunits were transcriptionally enriched in the copper cell region. Functionally, the downregulation of V-ATPase subunits specifically in Hodor-expressing cells-and not in other subsets of enterocytes, such as those targeted by R2R4-Gal4-led to developmental delay and reduced food intake, phenotypes comparable to those observed as a result of hodor downregulation. Hence, although the directionality of zinc sensing and chloride transport in interstitial cells remains to be established, the data are consistent with roles for brush-border Hodor in transporting chloride out of interstitial cells-thus maintaining osmolarity and water balance. Lysosomal Hodor may transport chloride into the lysosome to sustain V-ATPase function, lysosomal acidification and TOR signalling, pointing to new links between lysosomal homeostasis in specialized intestinal cells, food intake and systemic growth. Nutrients such as amino acids are important regulators of Tor signalling. The genetic data are consistent with novel input from metals and/or micronutrients into Tor signalling. The nutrient-dependent zinc accumulation in lysosomal organelles-recently described in mammalian cells and nematode worms-suggests that links between zinc, lysosomes and Tor may be of broader importance. Two attractive cell types in which to explore such links are the Paneth cells of the mammalian intestine, which accumulate zinc and regulate intestinal immunity and stem cell homeostasis, and the 'lysosome-rich enterocytes' that have recently been described in fish and mice, which have roles in protein absorption (Redhai, 2020).

An extensive reconstruction of the hodor family tree supported the presence of a single member of the family in the ancestor of insects. Because Hodor-like proteins are present only in insects, they may prove to be highly specific targets for the chemical control of disease vectors, particularly given that mosquito genomes contain a single gene rather than the three paralogues that are found in most flies. To test this idea, CRISPR-Cas9 genome editing was used to generate a mutant that lacks the single hodor-like gene (AGAP009616) in the malaria vector Anopheles gambiae. This gene is also expressed in the digestive tract-specifically in the midgut-and in Malphighian tubules. Three independent deletion alleles revealed that AGAP009616 function is essential for the viability of A. gambiae. A target that is expressed in the intestine, such as Hodor, is particularly attractive for vector control as it may circumvent accessibility issues and could be directly targeted using ingestible drugs such as those applied to larval breeding sites (Redhai, 2020).

Metals have received little attention in the contexts of development or whole-body physiology, and are often regarded as passive 'building blocks'. By revealing the roles of a metal sensor in food intake and growth control, these findings highlight the importance of investigating the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis. These mechanisms could prove to be useful in insect vector control (Redhai, 2020).

Identification of Split-GAL4 Drivers and Enhancers That Allow Regional Cell Type Manipulations of the Drosophila melanogaster Intestine

The Drosophila adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, the intersectional split-GAL4 system was applied to the adult midgut; 653 driver combinations are reported that label cells by region and cell type. 424 split-GAL4 drivers with midgut expression were identified from over 7,300 drivers screened, and then the expression patterns of each of these 424 were evaluated when paired with three reference drivers that report activity specifically in progenitor cells, enteroendocrine cells, or enterocytes. A subset of the drivers expressed in progenitor cells was evaluated for expression in enteroblasts using another reference driver. It was possible to show that driver combinations can define novel cell populations by identifying a driver that marks a distinct subset of enteroendocrine cells expressing genes usually associated with progenitor cells. The regional cell type patterns associated with the entire set of driver combinations are documented in a freely available website, providing information for the design of thousands of additional driver combinations to experimentally manipulate small subsets of intestinal cells. In addition, it was shown that intestinal enhancers identified with the split-GAL4 system can confer equivalent expression patterns on other transgenic reporters. Altogether, the resource reported here will enable more precisely targeted gene expression for studying intestinal processes, epithelial cell functions, and diseases affecting self-renewing tissues (Ariyapala, 2020).

Signaling dynamics control cell fate in the early Drosophila embryo

The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? This study combined optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. Erk activity was found to be sufficient to 'posteriorize' 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. The endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes (Johnson, 2019).

Through a combination of genetic perturbations and time-varying optogenetic stimuli, this study begins to define a model for how Erk is capable of programming three distinct cell fates the early embryo (see A conceptual model of Erk-dependent cell fate control in the early embryo). At the posterior, sustained Erk signaling induces gut endoderm, tissue that is characterized by expression of the Fog/Mist receptor-ligand pair which leads to apical constriction and tissue invagination. The boundary of this tissue is determined by the total Erk dose, and sustained Erk activity at almost any embryonic position is sufficient to trigger gut endoderm gene expression and contractility. A notable exception is the anterior pole, where the combination of Bcd and Erk switches cells to anterior fates. In the middle of the embryo, the combination of transient Erk and Dorsal normally induces the formation of ectoderm-derived neuroblasts, a fate that can be overridden by additional, ectopic Erk activity. By isolating the Erk pathway and titrating the inputs delivered, it was further shown that some cells in the embryo can adopt three distinct responses at different signaling thresholds. Lateral cells shift from no response to form intermediate neuroblasts (characterized by ind expression) or gut endoderm (marked by mist expression and contractility) as a single input parameter - the duration of Erk signaling - is increased. These three transitions are reminiscent of the requirements that define a morphogen, a substance whose concentration determines multiple distinct fates. Yet in the case of Erk it is signaling dynamics, not instantaneous concentration, which is interpreted into a cellular response (Johnson, 2019).

In contrast to the now-classic models for how Erk dynamics are decoded in cultured cells, it study findw that the early Drosophila embryo does not read the duration of a single persistent stimulus, but rather senses the cumulative load of Erk signaling. Two lines of evidence support this conclusion. First, the same overall Erk dose can be delivered in a single bolus or divided into discrete pulses spread out over a 2 h window, leading to the same effect. Second, long low-amplitude light stimuli (as well as gain-of-function mutants that activate Erk to low levels) achieve the same phenotypes as short high-amplitude light pulses. But does this distinction between duration and cumulative load sensing matter? It could be argued that it is quite important, as the putative network architectures that perform these two signal processing functions can be quite different. Persistence detection is thought to rely on network motifs like the coherent feedforward loop, whereas cumulative load detection can be implemented by combining long-term integration with an ultrasensitive downstream step. Obtaining the dynamic input-output response of a biological network can thus be a crucial first step towards a complete understanding of its network architecture and subsequent identification of molecular components. Such insights are sorely needed, as a mechanistic understanding of how signaling pathway activity is decoded into precise, reproducible patterns of gene expression is still lacking (Johnson, 2019).

There are still many unresolved questions regarding Erk-dependent cell fate choices in the early embryo. This study has shown Bicoid is sufficient to prevent posterior fates at the anterior pole, but future work must be done to dissect how Erk dynamics interact with the Bicoid gradient to pattern the formation of different anterior structures. A complete picture of anterior fate choices may benefit from precise control over both Bicoid and Erk using multi-color optogenetics. Moreover, the Erk-dependent terminal gap genes tll and hkb do much more than specify terminal fates, and participate in complex interactions with other gap and segmentation genes. Indeed, brief 15-30 min light stimuli do not affect gastrulation but lead to abdominal segment fusion, suggesting that segmentation gene network is quite sensitive to perturbations in the spatiotemporal profile of Erk signaling. The use of light to deliver quantitative spatial and temporal perturbations to gap gene expression could prove instrumental for a deeper understanding of the segmentation circuit (Johnson, 2019).

The Ras/Erk pathway is only one of many signaling outputs from receptor tyrosine kinases (RTKs), and in general it remains an open question whether light-induced Erk fully reproduces all the nuances of receptor-level stimulation. To further probe this question in the early embryo, whether OptoSOS stimulation recapitulates a classic genetic epistasis result was tested: in embryos expressing a gain-of-function Torso RTK, loss of tll can suppress the TorGOF phenotype. Indeed, this study found that the expected proportion of OptoSOS-tll treated with 45 min of light exhibit the tailless phenotype, not the TorGOF-like phenotype normally observed in OptoSOS embryos. Interestingly, suppression is lost at higher light doses, suggesting that sufficiently strong Erk activity can posteriorize embryos even in the absence of tll. Looking forward, the recent development of light-controlled RTKsopens the door to a full, systematic comparison between stimulation at the levels of the receptor versus Ras, and a quantitative comparison between these approaches is eagerly awaited (Johnson, 2019).

The relationship between Erk dynamics and cellular responses has long been studied in cultured mammalian cells. This study found that principles of dynamic control also operate to control Erk-dependent cell fates in a developing organism. It would be argued that the early Drosophila embryo is an ideal model system for dissecting dynamic control: cell fates specification occurs within 3 hours and is highly reproducible between embryos, gastrulation movements can be observed by brightfield microscopy and provide a spatially-localized readout of cell fate, the list of 'downstream' candidates for decoding dynamics is limited to a few dozen active zygotic genes in the early embryo, and the combination of optogenetic and classical genetic tools enable complex perturbations of network components. Moreover, Erk signaling in the early embryo is likely to be only one of many examples of dynamic cell fate control in vivo. The approaches outlined in this study could prove useful in many additional contexts for dissecting how developmental cell fates are specified (Johnson, 2019).

Fitness trade-offs incurred by ovary-to-gut steroid signalling in Drosophila

Sexual dimorphism arises from genetic differences between male and female cells, and from systemic hormonal differences. How sex hormones affect non-reproductive organs is poorly understood, yet highly relevant to health given the sex-biased incidence of many diseases. This study reports that steroid signalling in Drosophila from the ovaries to the gut promotes growth of the intestine specifically in mated females, and enhances their reproductive output. The active ovaries of the fly produce the steroid hormone ecdysone, which stimulates the division and expansion of intestinal stem cells in two distinct proliferative phases via the steroid receptors EcR and Usp and their downstream targets Broad, Eip75B and Hr3. Although ecdysone-dependent growth of the female gut augments fecundity, the more active and more numerous intestinal stem cells also increase female susceptibility to age-dependent gut dysplasia and tumorigenesis, thus potentially reducing lifespan. This work highlights the trade-offs in fitness traits that occur when inter-organ signalling alters stem-cell behaviour to optimize organ size (Ahmed, 2020).

Steroidal sex hormones including oestrogen, progesterone and testosterone regulate the growth and physiology of reproductive organs during puberty, the oestrus cycle and pregnancy. Consequently, these hormones also promote tumorigenesis in the breast, uterus and prostate. Although sex-specific differences in physiology and disease predisposition extend to nearly all organs, the functions of sex-specific steroids in non-sex organs remain relatively poorly explored and controversial. Drosophila uses one major steroid hormone, 20-hydroxy-ecdysone (ecdysone, also known as 20HE) and its derivatives. Similar to vertebrate steroids, 20HE is synthesized by cytochrome P450 enzymes from cholesterol. The ecdysone receptor comprises a ligand-binding EcR subunit and a DNA-binding Usp subunit-orthologues of human farnesoid X and liver X receptors (FXR and LXR) and retinoid X receptor (RXR), respectively. In juvenile insects, 20HE regulates developmental transitions including moulting, metamorphosis and sexual maturation. In adult Drosophila, 20HE is made by the ovaries after mating, resulting in higher levels in females than in males. It acts in the adult nervous and reproductive systems and affects metabolism and lifespan, but a role in the gut has not been described (Ahmed, 2020).

Drosophila intestinal stem cells (ISCs) are more proliferative in females than in males, and females are more prone to age-dependent gut dysplasia and intestinal tumours. These sex-specific traits could be due to ISC-autonomous and/or systemic factors. Consistent with the former, stress-dependent ISC divisions, which are more frequent in females than in males, are reduced if the ISCs are masculinized by repressing the sex-determination genes sxl or tra2. Mated females support more ISC division than virgin flies, which suggests hormonal influences. Because mated females have higher titres of ecdysteroid than virgins or males, tests were performed to see whether 20HE might affect ISC proliferation. Indeed, feeding virgin females 5 mM 20HE strongly induced ISC divisions. This effect was independent of ISC sex identity, and also occurred in mated females and males. Using reporters of receptor activity, it was confirmed that exogenous 20HE promotes EcR-Usp signalling in midgut ISCs, transient progenitors known as enteroblasts (EBs) and differentiated absorptive enterocytes (ECs) (Ahmed, 2020).

Unlike stress caused by detergents, 20HE treatment induced two successive waves of ISC division. Using RNA interference (RNAi) under the control of conditional cell-type-specific Gal4 drivers, it was found that the first wave (at 6 h after 20HE feeding) required EcR only in ISCs, but that later divisions (at 16 h) also depended partially on EcR in EBs. Neither wave of division required EcR in ECs, enteroendocrine or neural cells. Isoform-specific tests revealed that EcR-A was much more important than EcR-B for the 20HE-induced division of ISCs. 20HE-induced divisions were reversible , which suggests a lack of toxicity. EcR activity was not induced by enteric infection, and EcR was dispensable for infection-induced gut regeneration, which indicates a distinct role for EcR in the gut. Loss of Usp, however, did block infection-induced ISC divisions, which suggests that Usp has EcR-independent functions (Ahmed, 2020).

It was next asked whether ISC activation by 20HE involves the Upd-Jak-Stat or Egfr-ERK signalling pathways, which are known to activate ISCs after stress. Six hours of 20HE feeding induced the Egfr ligands spi and krn and their activating protease rho, but not the upd2 or upd3 cytokines or Stat signalling. Exposure to 20HE for 16 h, however, moderately induced upd2, upd3 and Stat activity. The induction of upd2, upd3 and rho required EcR in ISCs and EBs (that is, 'progenitors'), although not in ECs. The Egfr effector ERK was also mildly activated by 16 h of 20HE exposure, mostly in progenitors but occasionally in ECs. ERK activation required upd2, which suggests a signalling relay. Notably, the induction of all of these targets (upd2, upd3, Socs36E, rho, spi and krn) by 20HE was suppressed by blocking ISC mitoses with RNAi molecules that target string (also known as stg or cdc25) or Egfr. This suggests that the observed increases in Jak-Stat and Egfr-ERK signalling are responses to epithelial stress from the early ISC divisions. In further tests, it was found that Upd2 from EBs and ECs contributed strongly to ISC divisions 16 h after 20HE feeding, but only weakly to the early divisions at 6 h. Egfr and Rho, however, were always required. It is concluded that ISC divisions are initially activated ISC-autonomously via EcR, and require Egfr and Rho, whereas later divisions depend in part on cytokines produced by EBs and ECs, perhaps in response to stress from the first mitoses. The relationship of EcR to Egfr signalling warrants further investigation (Ahmed, 2020).

Because mated females produce more ecdysone than virgins or males, whether 20HE might account for sex-specific differences in the gut was tested. Consistent with this, long-term exposure of males to 20HE phenocopied the female condition, increasing ISC mitoses, stress responsiveness, epithelial turnover and midgut size. Genetically feminizing the male ISCs did not give these effect, which suggests that 20HE acts independently of genetic sex determination. Forced expression of the ISC mitogen13 sSpi also failed to enlarge male midguts, which indicates that 20HE affects more than just the ISC mitotic rate. Long-term 20HE feeding also endowed ISCs in virgin females with proliferative characteristics similar to those seen after mating. By contrast, RNAi lines that antagonized 20HE signalling in ISCs and EBs decreased gut size in mated females and suppressed mitoses in response to detergent stress. Thus, sexually dimorphic proliferative traits of ISCs are determined in part by 20HE signalling (Ahmed, 2020).

Similar to human oestrogen and progesterone, ecdysone promotes behavioural and metabolic changes that enhance female reproduction. Dose-response assays showed that 1 mM 20HE fed to virgin females activated EcR targets and ISC mitoses to similar levels to mating. Hence, this study tested whether endogenous, mating-induced 20HE activates ISCs. Indeed, mating induced a large, transient increase in ISC division and enduring gut enlargement. This was independent of genetic sexual identity. As with exogenously fed 20HE, these effects initially required EcR only in ISCs, although EcR in EBs contributed later on. Similar to exogenous 20HE, mating also induced expression of upd2 and rho, which suggests that these are normal physiological responses (Ahmed, 2020).

To confirm the source of endogenous ecdysone, ovary-specific Gal4 drivers were used to express RNAi transgenes that target the ecdysone synthesis enzymes Dib or Spo. This suppressed mating-induced ISC divisions and midgut growth, both of which could be restored by exogenous 20HE. spo mutants also failed to resize the midgut after mating, confirming these results. To learn how the gut grows in mated females, the effects on cell size and number. Depleting EcR in ECs did not reduce EC size, but mating caused a large 20HE- and EcR-dependent increase in female ISC numbers was investigated. This expansion of the stem-cell pool could cause an increase in the total number of midgut cells. These results indicate that mating-dependent ISC division, ISC expansion and gut growth are driven by 20HE signalling from the ovaries to progenitor cells in the gut (Ahmed, 2020).

Gut growth after mating is expected to increase the absorption of nutrients by the intestine and the supply of nutrients to other organs. Because egg production is limited by nutrient availability to the ovaries, whether 20HE-dependent gut growth affected female fecundity was tested. When gut resizing was blocked by expressing EcR RNAi (EcRRNAi) in midgut ISCs, or in both ISCs and EBs, egg production was reduced by approximately 40%. This suggests that 20HE-dependent gut remodelling maximizes female reproductive fitness. However, it was also noticed that the Gal4 drivers were active in a small number of escort cells in the germarium of the ovary, which raises the possibility that these fecundity defects were due in part to a requirement for EcR in those cells (Ahmed, 2020).

A study of Drosophila juvenile hormone (JH), a sesquiterpenoid, came to conclusions similar to the current study-namely that JH promotes mating-dependent gut growth and fecundity in females. The relative roles of 20HE and JH were therefore invstigated. The JH receptors Gce and Met are essential for ISC divisions in response to not only the JH receptor agonist, methoprene, but also 20HE and infection. The mitogenic effects of methoprene were confirmed, but these were weaker than those of 20HE or mating. Furthermore, it was discovered that methoprene-stimulated divisions require 20HE, and that JH or methoprene could suppress ISC divisions driven by 20HE or other stimuli. Although these results indicate interaction between 20HE and JH, further work is required to determine their precise physiological relationship (Ahmed, 2020).

To better understand how ecdysone activates ISCs, two known EcR targets were tested: the transcription factor Broad, and the nuclear receptor Eip75B, a homologue of human PPARγ and REV-ERB17. Eip75B and broad (br) mRNA were induced in midguts by 20HE or mating, and progenitor-cell-specific depletion of either factor suppressed 20HE-induced mitoses. ISC clonal growth, however, required Eip75B but not br, highlighting that Eip75B is a more essential effector. Overexpression of Eip75B was sufficient to promote ISC division and gut epithelial turnover, whereas Eip75B loss impaired both ISC mitoses and maintenance. Progenitor-specific loss of Eip75B also blocked gut growth after mating, and compromised egg production, phenocopying the effects of EcR loss. Eip75B binds DNA to repress target genes, and also binds the nuclear receptor Hr3 to inhibit Hr3-mediated transcriptional activation. Consistent with this mechanism, overexpression of Eip75B or 20HE feeding suppressed an Hr3 activity reporter, and Hr3 overexpression suppressed ISC proliferation. Moreover, depletion of Hr3 counteracted losses in ISC proliferation caused by Eip75B depletion. Although these results indicate that Hr3 is a crucial Eip75B effector, Hr3 loss was not sufficient to activate ISC division, which indicates that Eip75B has other targets. Further tests revealed that Eip75B and Hr3 mediate 20HE-independent ISC responses to stress. Enteric infection strongly induced levels of Eip75B mRNA and suppressed Hr3 activity. Removing Eip75B or Broad from ISCs by mutation or by RNAi blocked infection-induced ISC mitoses, as did overexpression of Hr3. Eip75B was also required for ISC mitoses in response to the oxidative stress agent paraquat. Furthermore, evidence was ontained consistent with previous work that the action of Eip75B is modulated by haem (a Eip75B ligand) and nitric oxide. Functions for haem and nitric oxide in the fly gut are unknown, but potentially interesting. It is concluded that Eip75B, Broad and Hr3 integrate several inputs in addition to 20HE to control ISC proliferation (Ahmed, 2020).

As females age, they experience progressive gut dysplasia in which ISCs overproliferate and mis-differentiate, leading to high microbiota loads (dysbiosis), barrier breakdown and decreased lifespan. Age-dependent intestinal dysplasia is more pronounced in females than in males, and can be identified by increases in mitoses and mis-differentiated cells doubly positive for ISC and EC markers. Suppressing EcR, Usp or Eip75B in midgut progenitors significantly reduced both parameters of dysplasia in aged flies. Similarly, suppressing ecdysone synthesis enzymes (Dib, Spo) in the ovaries, or ubiquitously, also curtailed age-dependent gut dysplasia. This effect could be reversed by 20HE supplementation. These results indicate that age-dependent gut dysplasia is potentiated by ovary-derived ecdysone, explaining the sex bias of this condition (Ahmed, 2020).

Female Drosophila are known to be more susceptible than males to genetically induced ISC-derived tumours. This study found that ISC/EB-specific RNAi targeting Notch, a receptor required for EC differentiation, drove tumour induction in 100% of mated females but was far less tumorigenic in virgin females or males. Three results indicate that this tumour predisposition is modulated by 20HE. First, in contrast to mated females, virgins were extremely resistant to NotchRNAi-mediated tumorigenesis. Second, targeting 20HE signalling in ISCs with a dominant-negative EcR-A (EcRADN) inhibited tumour growth in mated females. Third, supplementing males or virgin females with 20HE increased tumour initiation and growth. It is surmised that the use of mating-dependent, ovary-derived 20HE to stimulate gut resizing comes at a cost: it predisposes females to gut dysplasia and tumorigenesis (Ahmed, 2020).

Gut dysplasia, tumorigenesis and egg production can all shorten lifespan, which suggests that the effects of ecdysone on the gut might adversely affect longevity. In fact, earlier reports showed that EcR mutants live longer, and proposed that reproduction can shorten lifespan by damaging the soma. The lifespan assays of this study, although subject to the same caveats as previous work, support this view: suppression of EcR in midgut progenitors extended lifespan in females but not males. In evolutionary terms, the disadvantage of a slightly shorter lifespan due to sex-specific hormonal signalling is probably insignificant relative to the reproductive fitness advantage conferred by increased egg production. This may be especially true in the wild, where gut dysplasia-dependent mortality is probably counteracted by nutrient deprivation (Ahmed, 2020).

Similarities in the reproductive biology of Drosophila and mammals suggest that these inter-organ relationships have relevance to human biology. The mitogenic effects of insect ecdysone parallel those of oestrogen and testosterone as drivers of breast, uterine and prostate growth and tumorigenesis. Yet how these steroids affect the human intestine remains poorly explored. Adaptive growth of the intestine is well documented in pregnant and lactating mammals, and might depend on oestrogen and/or progesterone. Laboratory tests with rodents and human cells, as well as some studies with human participants, have linked oestrogen, testosterone and their receptors to gastrointestinal cancers, but epidemiological studies provide conflicting evidence regarding this association. The contributions of sex steroids to intestinal physiology deserve more detailed study (Ahmed, 2020).

Drosophila melanogaster sex peptide regulates mated female midgut morphology and physiology

Drosophila melanogaster females experience a large shift in energy homeostasis after mating to compensate for nutrient investment in egg production. To cope with this change in metabolism, mated females undergo widespread physiological and behavioral changes, including increased food intake and altered digestive processes. The mechanisms by which the female digestive system responds to mating remain poorly characterized. This study demonstrates that the seminal fluid protein Sex Peptide (SP) is a key modulator of female post-mating midgut growth and gene expression. SP is both necessary and sufficient to trigger post-mating midgut growth in females under normal nutrient conditions, and likely acting via its receptor, Sex Peptide Receptor (SPR). Moreover, SP is responsible for almost the totality of midgut transcriptomic changes following mating, including up-regulation of protein and lipid metabolism genes and down-regulation of carbohydrate metabolism genes. These changes in metabolism may help supply the female with the nutrients required to sustain egg production. Thus, this study reports a role for SP in altering female physiology to enhance reproductive output: Namely, SP triggers the switch from virgin to mated midgut state (White, 2021).

Production of progeny requires a significant female energy investment. Consequently, mated females alter aspects of nutrient intake and digestion to maintain energy homeostasis. In Drosophila, such changes help sustain egg production. Given the important role of these responses in supporting reproduction, understanding their mechanisms is crucial to understanding the determinants of reproductive success. This study identified female receipt of the seminal SP as a central signal that triggers post-mating shifts in mated female midgut size and gene expression (White, 2021).

Previous studies have shown that males can modulate female physiology and behavior to enhance reproductive output via SP. These SP-induced post-mating responses that influence female nutrition include increasing female food intake, shifting nutrient preference, and concentrating female excreta. In mated females, SP also regulates levels of JHB3 and 20E, both of which are necessary for midgut resizing. This study shows that SP received during mating is both necessary and sufficient for enlargement of the mated female's midgut and thus that SP is the sole male-derived signal needed to mediate the switch between a female's virgin and mated midgut size. Other SP-related ligands likely play a minimal role in the initiation of post-mating midgut growth. The SP paralog Dup99B is transferred at normal levels within the seminal fluid of SP0 males. Since the midgut lengths and transcriptomes of SP0 mated females were no different from those of virgin females, it is unlikely that Dup99B contributes significantly to stimulating midgut growth. For similar reasons, while any post-mating midgut growth role of myoinhibitory peptides (MIPs), the ancestral ligands of SPR, is unknown, any effect that MIPs might have is likely downstream of SP (White, 2021).

This study shows several additional ways that SP modifies the midgut, beyond the roles that were described above. Midgut size peaks at 6 d post-mating and persists for at least 15 d after mating. This is consistent with the time frame of other SP-mediated long-term post-mating responses, such as reduced receptivity to remating, which persists for ~10 d. Additionally, these experiments utilizing males that transfer SP defective for either sperm binding or release from sperm demonstrate that post-mating midgut resizing requires both long-term storage of SP and the gradual release of its C-terminal domain from sperm (White, 2021).

Consistent with the finding that release of SP's C terminus is necessary for post-mating midgut growth, its receptor SPR was found to play a role in post-mating midgut growth as well. The midguts of females homozygous for a deletion that removes SPR and four other genes did not exhibit post-mating midgut growth. To determine whether their lack of post-mating midgut growth was due to loss of SPR, rather than to loss of any of the other four genes, post-mating midgut grown was also examined in SPR-knockdown females. Although the knockdown females still had small amounts of residual SPR expression, they exhibited suppressed post-mating midgut growth. Taken together, these data indicate that SPR is needed for post-mating midgut growth. SP acting via SPR neuronal signaling has previously been linked to midgut post-mating responses, such as stimulating neuropeptide F release from EEs and increasing intestinal transit time. Additionally, the RNA-seq analysis identified SPR expression in the midgut, suggesting the possibility that SP could act directly on the gut to stimulate growth. Investigating where SPR acts to stimulate post-mating gut growth is an intriguing avenue for future research (White, 2021).

The mechanisms by which SP stimulates post-mating midgut growth are important areas for future investigation. For example, SP could act indirectly, through its regulation of the hormones JHB3 and 20E. SP acts via neuronal SPR to increase 20E synthesis in the ovaries, and ovarian 20E is necessary for post-mating gut growth, together suggesting that SP could stimulate midgut growth by raising ovarian 20E levels. SP's stimulation of JHB3 could also contribute to post-mating midgut growth. During the early hours after mating (short-term response), SP's N-terminal domain (unbound to sperm) can stimulate JHB3 release from corpora allata, potentially inducing midgut growth. However, while JHB3 release may initiate growth in the midgut, this is not sufficient for SP's persistent, long-term effect on midgut growth because no midgut growth was observedafter 3 d in females that had mated to SP-TGQQ or spermless males, both of which deposit SP with an intact N terminus and thus should initiate SP's short-term responses. Moreover, the requirement for SPR implicates SP's C terminus (as opposed to its JHB3-inducing N terminus) in the extended post-mating midgut growth. Thus, any early effects of SP on JHB3 that potentially impacted midgut growth cannot be sustained without activity of SP's C-terminal region, released long-term from sperm (White, 2021).

Additionally, SP-induced gut morphological changes are dependent on the female's nutritional state. Previous studies have shown that sterile OvoD females, who do not increase food intake post-mating, still undergo post-mating gut growth. This study shows that post-mating gut growth is not entirely independent of female nutrition. Midguts of mated females fed a nutrient-poor diet do not grow after mating, despite receiving WT SP from males. This could be the result of nutritional deficiencies negatively regulating production of JHB3 in the corpora allata. Under stress conditions, such as starvation, increased levels of 20E have been proposed to negatively regulate juvenile hormones. Additionally, arrested vitellogenesis of nutritionally deprived females can be rescued by treating them with the juvenile hormone analog methoprene, and insulin receptor mutants exhibit reduced JHB3 synthesis. These findings suggest that signals of nutritional state can affect JHB3 synthesis, and poor nutritional state may suppress SP's effect on JHB3 (White, 2021).

In addition to the effects of SP on post-mating midgut morphology, SP alters midgut physiology by reshaping the intestinal transcriptome. Gioti (2012) and Domanitskaya (2007) profiled transcriptomic responses to SP using microarrays to assay the effects of SP on the head and abdomen at 3 to 6 h after mating. Both studies demonstrated SP-induced changes in the transcriptome, such as induction of genes involved in immune responses. This study shows that SP modulates the messenger RNA (mRNA) complement of a single tissue and demonstrates that SP's effect on midgut transcription can persist for at least 2 d after mating, consistent with the timescale of SP's long-term response. SP was found to underly the vast majority of transcriptional changes in the midgut: Only 11 genes that were significantly differentially expressed between the guts of virgin females and females mated to SP0 males, and none of those genes exhibited a greater than twofold difference in expression between virgin and SP0-mated females. Additionally, the genes differentially regulated between females mated to SPWT and those mated to SP0 males largely overlap with those differentially expressed between virgin and mated females. In other words, the switch from a virgin to a mated state at the RNA level does not occur without SP, analogous to the finding that post-mating midgut size is regulated by SP (White, 2021).

Male proteins transferred during mating may also trigger enteric changes in other insects. In mosquitoes, extracts from male accessory glands can stimulate post-mating responses in females. In the dengue vector Aedes aegypti, male-derived substances can increase blood meal size and promote blood meal digestion in the female. Additionally, post-mating transcriptional changes have been observed in the guts of Anopheles gambiae females. In Anopheles coluzzii females, post-mating transcriptomic changes in the gut are evoked by male transfer of the ecdysteroid hormone 20E, suggesting that male-derived molecules triggering gut remodeling may be common in insects. Given the link between nutrition and egg production, understanding how male mosquitoes influence the female midgut may lead to new strategies for vector control (White, 2021).

The observed post-mating changes in midgut transcriptome parallel post-mating dietary shifts. Mated females showed down-regulation of genes involved in carbohydrate metabolism, such as the maltase-encoding genes and those linked to galactose and glucose metabolism, in the midgut. In conjunction, there was up-regulation of genes required for protein digestion and lipid metabolism. Despite differences in experimental design and execution, other studies examining mating-induced transcriptional changes in whole females or female abdomens have detected similar gene expression changes to those that reported in this study. Down-regulation has been observed of maltase genes in the whole-organism transcriptome of 3- to 5-d-old mated females, and another study also found down-regulation of carbohydrate metabolic genes in female abdomens at 3 h after mating. Two other studies found up-regulation of several proteases, and another study found up-regulation of proteolysis-related genes in female abdomens 3 h after mating. qPCR has been used to detect up-regulation of several fatty acid metabolic genes in the midgut after mating. The results confirm that bgm expression is induced upon mating and add bmm to the list although the current study did not find induction of some other genes (SREBP, Acsl, FAS, and ACC) detected by previously as up-regulated after mating. Discrepancies in exact complements of differentially expressed genes likely reflect methodological differences. Down-regulation was found of genes with GST activity involved in detoxification. Their down-regulation after mating could have consequences for the female's ability to deal with toxic dietary compounds and oxidative stress. Indeed, post-mating gut growth increases a female's propensity to develop life span-shortening gut dysplasia. This may reflect potential trade-offs between the demands of reproduction and somatic maintenance (White, 2021).

These results may reflect a post-mating increase in protein and lipid digestion to help sustain egg production. Previous studies have shown that food intake increases after mating, and sufficient dietary protein and lipids are essential for yolk protein production and female fecundity. However, the up-regulation of protein and lipid metabolic genes is likely not simply a consequence of increased food intake since this study saw a coincident down-regulation of carbohydrate metabolic genes. Rather, it is probable that this study observed the mated female midgut altering digestive parameters to adapt to new nutritional demands (White, 2021).

Although the molecular pathways underlying post-mating midgut transcriptomic changes remain unknown, this study detected enrichment of several transcription factor binding motifs among genes regulated by mating, including those for caudal and GATAe. GATAe has been previously linked to regulating ISC maintenance and differentiation and also plays a key role in maintaining gut homeostasis, suggesting that GATAe could play a role in the increase in ISC proliferation observed after mating, as well as the altered expression of digestive enzymes (White, 2021).

In addition to observing mating-induced changes in the midgut transcriptome, this study found that regions of the midgut display varying degrees of morphological plasticity. Although all regions of the midgut increase in length after mating,this study found that the length of the posterior midgut, a region involved in nutrient absorption, grows more in proportion to total midgut length than any other region. This may be the result of egg production increasing nutrient demand. Alternatively, it could be an indirect result of the proximity of the posterior midgut to the ovaries, or mechanical stress exerted from the ovaries toward the midgut (White, 2021).

This study found that the seminal peptide SP is a key regulator of both female post-mating midgut size and transcription of metabolic pathways. The data show that, in well-nourished females, SP is both necessary and sufficient for post-mating midgut enlargement. Additionally, post-mating midgut growth is a component of SP's long-term response, requiring both long-term SP storage and release from sperm, as well as SPR. Mating also causes a shift in the transcriptome of the midgut, a change due almost completely to SP. The post-mating midgut increased transcription of genes involved in lipid and protein metabolism, while decreasing mRNA levels of sugar metabolic genes, and genes involved in detoxification. These results provide insight into SP's role as a regulator of mated female nutritional homeostasis, by helping the female meet the energetic demands of egg production. Overall, these findings illustrate the dynamic nature of the Drosophila midgut, demonstrating how the male can alter female internal morphology and physiology to enhance reproduction (White, 2021).

A glucose-supplemented diet enhances gut barrier integrity in Drosophila

Dietary intervention has received considerable attention as an approach to extend lifespan and improve aging. However, questions remain regarding optimal dietary regimes and underlying mechanisms of lifespan extension. This study asked how an increase of glucose in a chemically defined diet extends the lifespan of adult Drosophila melanogaster. Glucose-dependent lifespan extension was shown to not be result of diminished caloric intake, or changes to systemic insulin activity, two commonly studied mechanisms of lifespan extension. Instead, this study found that flies raised on glucose-supplemented food increased the expression of cell-adhesion genes, delaying age-dependent loss of intestinal barrier integrity. Furthermore, it was shown that chemical disruption of the gut barrier negated the lifespan extension associated with glucose treatment, suggesting that glucose-supplemented food prolongs adult viability by enhancing the intestinal barrier. It is believed these data contribute to understanding intestinal homeostasis, and may assist efforts to develop preventative measures that limit effects of aging on health (Galenza, 2021).

Basal stem cell progeny establish their apical surface in a junctional niche during turnover of an adult barrier epithelium

Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. New replacement cells-the progeny of basal stem cells-are born without barrier-forming structures such as a specialized apical membrane and occluding junctions. This study investigated how new progeny acquire barrier structures as they integrate into the intestinal epithelium of adult Drosophila. They were found gestate their future apical membrane in a sublumenal niche created by a transitional occluding junction that envelops the differentiating cell and enables it to form a deep, microvilli-lined apical pit. The transitional junction seals the pit from the intestinal lumen until differentiation-driven, basal-to-apical remodelling of the niche opens the pit and integrates the now-mature cell into the barrier. By coordinating junctional remodelling with terminal differentiation, stem cell progeny integrate into a functional, adult epithelium without jeopardizing barrier integrity (Galenza, 2023).

Unraveling the features of somatic transposition in the Drosophila intestine

Transposable elements (TEs) play a significant role in evolution, contributing to genetic variation. However, TE mobilization in somatic cells is not well understood. This study addressed the prevalence of transposition in a somatic tissue, exploiting the Drosophila midgut as a model. Using whole-genome sequencing of in vivo clonally expanded gut tissue, hundreds of high-confidence somatic TE integration sites were mapped genome-wide. Somatic retrotransposon insertions were shown to be associated with inactivation of the tumor suppressor Notch, likely contributing to neoplasia formation. Moreover, applying Oxford Nanopore long-read sequencing technology, evidence is provided for tissue-specific differences in retrotransposition. Comparing somatic TE insertional activity with transcriptomic and small RNA sequencing data, this study demonstrates that transposon mobility cannot be simply predicted by whole tissue TE expression levels or by small RNA pathway activity. Finally, it was revealed that somatic TE insertions in the adult fly intestine are enriched in genic regions and in transcriptionally active chromatin. Together, these findings provide clear evidence of ongoing somatic transposition in Drosophila and delineate previously unknown features underlying somatic TE mobility in vivo (Siudeja, 2021).

Metabolic gene regulation by Drosophila GATA transcription factor Grain

Nutrient-dependent gene regulation critically contributes to homeostatic control of animal physiology in changing nutrient landscape. In Drosophila, dietary sugars activate transcription factors (TFs), such as Mondo-Mlx, Sugarbabe and Cabut, which control metabolic gene expression to mediate physiological adaptation to high sugar diet. TFs that correspondingly control sugar responsive metabolic genes under conditions of low dietary sugar remain, however, poorly understood. This study identified a role for Drosophila GATA TF Grain in metabolic gene regulation under both low and high sugar conditions. De novo motif prediction uncovered a significant over-representation of GATA-like motifs on the promoters of sugar-activated genes in Drosophila larvae, which are regulated by Grain, the fly ortholog of GATA1/2/3 subfamily. grain expression is activated by sugar in Mondo-Mlx-dependent manner and it contributes to sugar-responsive gene expression in the fat body. On the other hand, grain displays strong constitutive expression in the anterior midgut, where it drives lipogenic gene expression also under low sugar conditions. Consistently with these differential tissue-specific roles, Grain deficient larvae display delayed development on high sugar diet, while showing deregulated central carbon and lipid metabolism primarily on low sugar diet. Collectively, this study provides evidence for the role of a metazoan GATA transcription factor in nutrient-responsive metabolic gene regulation in vivo (Kokki, 2021).

Drosophila intestinal homeostasis requires CTP synthase

CTP synthase (CTPS) senses all four nucleotides and forms filamentous structures termed cytoophidia in all three domains of life. How CTPS and cytoophidia function in a developmental context, however, remains underexplored. This study reports that CTPS forms cytoophidia in a subset of cells in the Drosophila midgut. It was found that cytoophidia exist in intestinal stem cells (ISC) and enteroblasts in similar proportions. Both refeeding after starvation and feeding with dextran sulfate sodium (DSS) induce ISC proliferation and elongate cytoophidia. Knockdown of CTPS inhibits ISC proliferation. Remarkably, disruption of CTPS cytoophidia inhibits DSS-induced ISC proliferation. Taken together, these data suggest that both the expression level and the filament-form property of CTPS are crucial for intestinal homeostasis in Drosophila (Zhou, 2021).

Dopamine Modulates Drosophila Gut Physiology, Providing New Insights for Future Gastrointestinal Pharmacotherapy

Dopamine has a variety of physiological roles in the gastrointestinal tract (GI) through binding to Drosophila dopamine D1-like receptors (DARs) and/or adrenergic receptors and has been confirmed as one of the enteric neurotransmitters. To gain new insights into what could be a potential future promise for GI pharmacology, Drosophila was used as a model organism to investigate the effects of dopamine on intestinal physiology and gut motility. GAL4/UAS system was utilized to knock down specific dopamine receptors using specialized GAL4 driver lines targeting neurons or enterocytes cells to identify which dopamine receptor controls stomach contractions. DARs (Dop1R1 and Dop1R2) were shown by immunohistochemistry to be strongly expressed in all smooth muscles in both larval and adult flies, which could explain the inhibitory effect of dopamine on GI motility. Adult males' gut peristalsis was significantly inhibited by knocking down dopamine receptors Dop1R1, Dop1R2, and Dop2R, but female flies' gut peristalsis was significantly repressed by knocking down only Dop1R1 and Dop1R2. These findings also showed that dopamine drives PLC-β translocation from the cytoplasm to the plasma membrane in enterocytes for the first time. Overall, these data revealed the role of dopamine in modulating Drosophila gut physiology, offering us new insights for the future gastrointestinal pharmacotherapy of neurodegenerative diseases associated with dopamine deficiency (El Kholy, 2021).

Identification of a new allele of O-fucosyltransferase 1 involved in Drosophila intestinal stem cell regulation

Adult stem cells are critical for the maintenance of tissue homeostasis. However, how the proliferation and differentiation of intestinal stem cells (ISCs) are regulated remains not fully understood. This study found a mutant, stum 9-3, affecting the proliferation and differentiation of Drosophila adult ISCs in a forward genetic screen for factors regulating the proliferation and differentiation ISCs. stum 9-3 acts through the conserved Notch signaling pathway, upstream of the S2 cleavage of the Notch receptor. Interestingly, the phenotype of stum 9-3 mutant is not caused by disruption of stumble (stum), where the p-element is inserted. Detailed mapping, rescue experiments and mutant characterization show that stum 9-3 is a new allele of O-fucosyltransferase 1 (O-fut1). These results indicate that unexpected mutants with interesting phenotype could be recovered in forward genetic screens using known p-element insertion stocks (Shi, 2021).

Gut cytokines modulate olfaction through metabolic reprogramming of glia

Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. This study shows that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. These findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies (Cai, 2021).

Olfactory perception influences nutrition and promotes physiological and mental well-being. In flies, a dedicated olfactory circuit elicits avoidance behaviours towards geosmin-a volatile compound that is released by mould and some bacteria. Olfactory receptors also mediate an initial attraction to food that contains certain enteropathogens. After infection with these pathogens, however, an avoidance behaviour is triggered by immune receptors in the brain, gustatory bitter neurons, and the neuropeptide leukokinin. Whether changes in olfactory perception contribute to this behavioural switch from attraction to avoidance remains unclear (Cai, 2021).

In Drosophila, odorants are sensed by olfactory receptor neurons in the head, the antenna and the maxillary palp. Olfactory receptor neurons synapse into projection neurons at the antennal lobe (AL), where the signal is converted into a spatiotemporal code in 50 glomerular compartments. Projection neurons axons project to higher olfactory centres to instruct innate and learned behaviour. In this system, glia and neurons operate as a tightly coupled unit to maintain olfactory sensitivity (Cai, 2021).

In ageing flies, olfactory perception of both aversive and attractive odours declines, but the mechanism(s) of this decline remain unclear. Olfactory perception and other neurological processes also decline in ageing mammals, often influenced by gastrointestinal signals (Cai, 2021).

This study investigated the communication between the gut and the brain, and how it influences infection-induced avoidance behaviour, infection tolerance, and olfactory decline during ageing (Cai, 2021).

A modified capillary feeder (CAFE) assay was used to measure choice between food that did or did not contain Erwinia carotovora carotovora 15 (Ecc15), a non-lethal enteropathogen that causes intestinal inflammation. Consistent with recent reports, naive flies consumed more Ecc15-containing food than normal food. However, when orally infected with Ecc15 for 24 h before the feeding assay, flies developed a distinct aversion to food that contained Ecc15. To assess whether this involved changes in olfactory perception, the 'preference index' for attractive (such as putrescine) or aversive (such as 3-octanol) odours was determined in T-maze assays. Preference or aversion for attractive or aversive odours, respectively, declined after infection, which indicates that infection causes a non-selective decline in olfactory discrimination. This was transient, as olfactory discrimination recovered 5 days after infection, coincident with the clearance of bacteria and epithelial regeneration in the intestine. Olfactory discrimination was not influenced by starvation or exposure to heat-killed Ecc15. Consistent with their reported role in sensing pathogenic bacteria, the CO2 receptor Gr63a or the odorant receptor co-receptor Orco was required for the attraction to Ecc15 food: Orco1 and Gr63a1 mutants ingested less Ecc15 food under naive conditions, and when infected, failed to further reduce ingestion of Ecc15-containing food. Together, these observations suggest that after an initial odorant-mediated attraction, flies develop aversion to enteropathogens, through a concerted activation of gustatory and immune receptors and suppression of olfaction (Cai, 2021).

After oral infection with Ecc15, damaged intestinal enterocytes produce the inflammatory IL-6-like cytokines Unpaired 2 and 3 (Upd2 and Upd3) to stimulate intestinal stem-cell proliferation and epithelial regeneration. Proteins of the Upd family activate the JAK-STAT signalling pathway through the receptor Domeless (Dome) and the JAK homologue Hopscotch (Hop). Using the 2xSTAT::GFP reporter for JAK-STAT pathway activity, this study found upregulated GFP expression in the brain 4 h after oral Ecc15 infection, as well as after oral infection with the more lethal enteropathogen Pseudomonas entomophila (PE) that damages the gut epithelium. JAK-STAT activity was observed in a sparse population of cells of the brain that stained positive for the glial marker Repo. Subtype-specific Gal4 drivers revealed that among the five subtypes of Drosophila glia (astrocytes, ensheathing, perineural, subperineural and cortex glia), ensheathing glia (EG) were the main population that upregulate STAT activity in response to Ecc15 infection. This was confirmed using four different Gal4 drivers to label EG and by flow cytometry. Infection did not influence numbers and membranous processes (labelled using UAS::mCD4GFP) of EG at the AL, and glomerular compartmentalization in the AL and lobe size remained unaffected. JAK-STAT activation in EG was sufficient and required for infection-induced changes in olfactory discrimination, as overexpression of constitutively active Hop (hop^tuml) in EG reduced olfactory discrimination, whereas loss of Dome or STAT in all glia (repo::Gal4), or specifically in EG (GMR56F03::Gal4), rescued the decline of olfactory discrimination caused by Ecc15 infection. Overexpression of hop^tuml in EG also reduced ingestion of Ecc15-containing food and promoted survival of flies fed PE-containing food, whereas knockdown of Dome or STAT in EG increased ingestion of Ecc15-containing food in infected flies and increased mortality on PE-containing food. It is proposed that the corresponding changes in ingestion of PE-laced food contribute to reduced mortality, but it is possible that additional genetic background conditions influence mortality, as seen, for example, in Orco-mutant flies, which ingest less bacteria but show increased susceptibility to PE (Cai, 2021).

To test whether gut-derived Upd proteins directly contribute to the infection-induced activation of JAK-STAT signalling in EGs, intestinal enterocyte-specific perturbations were performed using Mex1::Gal4, an enterocyte driver with no expression in the brain. Indeed, JAK-STAT activation in glia at the AL could be triggered in naive flies or prevented in infected flies by overexpression or knockdown, respectively, of Upd2 and Upd3 in enterocytes. Consistently, enterocyte-derived Upd2 and Upd3 were sufficient and required for the modulation of olfactory discrimination caused by infection. Knockdown of Upd2 or Upd3 did not affect olfaction in naive flies, and perturbing these ligands in fatbody (cg::Gal4) or haemocytes (hml::Gal4), tissues that are sources for Upd proteins in other contexts, did not significantly affect STAT activity in glia at the AL (Cai, 2021).

Loss of olfactory sensitivity is an early sign of normal ageing and neurodegeneration. In ageing Drosophila, olfactory perception has been reported to deteriorate before vision, a decline that was possible to recapitulate in T-maze assays. Glomerular compartments in the AL became less organized and less distinct in geriatric (60-70-day-old) flies and AL size increased with age. This correlates with a reduction in the number of EG and of glial membranous processes, changes that are expected to affect AL structure, and thus probably contribute to the age-related decline in olfaction (Cai, 2021).

Ageing in Drosophila is accompanied by the development of intestinal inflammation, and is associated with the constitutive expression and release of Upd cytokines. Consistently, JAK-STAT activity in the AL of EG was increased in old flies, and knockdown of Dome or STAT by RNA interference (RNAi) in EG specifically or in all glia rescued the decline of olfactory discrimination in old flies. The loss of Dome in EG also rescued the age-related decline of EGs and restored the size of the AL. JAK-STAT activation in the EG of old flies is a consequence of intestinal Upd release, as knocking down Upd2 and Upd3 in enterocytes alleviated STAT activation in the AL, and prevented the age-related decline of olfactory discrimination (Cai, 2021).

This age-related decline of olfactory discrimination was independent of the microbiota, as germ-free old flies still exhibited reduced olfaction sensitivity, increased JAK-STAT signalling in the AL, decreased numbers of EG, loss of glial cellular processes, and an enlarged AL. These results are consistent with the observation that the age-related increase in Upd released from the gut is also independent of the microbiota (Cai, 2021).

To understand why EG but not other glia selectively respond to Upd ligands and activate JAK-STAT signalling during ageing or infection, single-cell RNA sequencing (scRNA-seq) was performed on purified glia from young and old flies. Either all glia (labelled using repo::Gal4) or EG selectively (labelled using GMR56F03::Gal4) were profiled using Smart-seq2. The expression of dome was significantly higher in EG than in other glia, consistent with the specific upregulation of socs36E, a known target of JAK-STAT signalling, in EG but not in other glia during ageing. These results are supported by a similar upregulation of Socs36E in EG of old flies observed in a previous scRNA-seq dataset (Cai, 2021).

Bulk RNA sequencing analysis on glia (repo::Gal4, UAS::tdTomato) purified from central brains of flies expressing a 10xSTAT::GFP reporter revealed that the transcriptomes of STAT::GFP⁺ glia from Ecc15 infected and uninfected flies were more similar to each other than to STAT::GFP^- glia of either condition, which indicates that JAK-STAT induction has a stronger influence on glial transcriptomes than other infection-related changes. Differentially expressed genes (866 genes using a cut-off of twofold change, P < 0.001, false discovery rate (FDR) < 0.01 and reads > 0.5) were significantly enriched in genes that encode proteins involved in lipid metabolism and carbohydrate transmembrane transport. These included the lipid binding protein Glial lazarillo (GLaz, a homologue of apolipoprotein D in mammals), which facilitates lipid transport from neurons to glia in flies; the lipid droplet surface binding proteins Lsd-1 and Lsd-2; the diacylglycerol O-acyltransferase Midway, which is a central regulator of triacylglycerol biosynthesis, and Coatomer, which is responsible for protein delivery to lipid droplets (LDs). This induction of lipid storage genes was coupled with induction of the monocarboxylate transporter (MCT) Outsiders (Out), and the MCT accessory protein Basigin (Bsg), sugar transporters (Tret1-1 and Tret1-2), and 17 enzymes involved in β-oxidation (Cai, 2021).

Glial MCTs promote lipid production in neurons and LD accumulation in glia by establishing a neuron and glia 'lactate shuttle'. To test a potential role for STAT signalling in influencing this shuttle at the AL, the accumulation of LDs was assessed at the AL in infected young flies using a combination of a neutral lipid probe (LipidTox, deep red) and a lipid peroxidation probe (C11-Bodipy, 581/591). A transient accumulation of LDs was observed 24 h after infection that decreased 4 days after infection, possibly owing to increased levels of β-oxidation. Overexpression of hop^tuml in the EG of young flies also promoted LD accumulation, whereas knocking down Dome or STAT rescued infection-induced accumulation. GLaz and Out were required for LD accumulation after infection, and overexpression of Upd2 and Upd3 in the gut induced LD accumulation at the AL, whereas knockdown of Upd2 or Upd3 alleviated LD accumulation in infected flies. Infection or JAK-STAT perturbation did not influence lipid peroxidation in LDs in young flies (Cai, 2021).

During neuronal stress, neurons can preferentially transfer fatty acids to glia, causing lipid accumulation and increasing fatty acid β-oxidation in glia. This study observed a significant induction of LDs specifically in EG at the AL in old flies, phenocopying hop^tuml overexpression. As fatty acid β-oxidation is a source of reactive oxygen species (ROS) that can result in lipid peroxidation, and lipid peroxidation in pigment cells (glia of the retina) promotes the demise of photoreceptors in the retina (whereas oxidative stress contributes to age-related dysfunction of cholinergic projection neurons within the olfactory circuit) it was reasoned that overall levels of ROS might increase in glia with age. Various genetically encoded ROS sensors were expressed in all glia (repo::Gal4) or in EG only (GMR56F03::Gal4) to measure levels of hydrogen peroxide (H2O2; measured by RoGFP2_Orp1) or the glutathione redox potential (measured by RoGFP2_Grx1) within the mitochondria or cytosol, respectively. Cytosolic levels of H2O2 were increased in the EG of old flies (both cytosolic and mitochondrial H2O2 levels were increased in all glia), whereas the cytosolic glutathione redox potential remained unchanged. In contrast to acute intestinal infection in young flies, lipids were peroxidated in LDs of old flies. Knocking down STAT specifically in EG, or knocking down Upd2 and Upd3 in gut enterocytes, inhibited LD accumulation and alleviated lipid peroxidation in old flies (Cai, 2021).

Olfactory discrimination was partially rescued in old and young infected flies after knockdown of GLaz and Out in EG. Knockdown of GLaz and Out also led to more Ecc15 food consumption, increased mortality after PE exposure, and reduced LD accumulation in the glia of old flies (Cai, 2021).

To confirm that infection or ageing-induced metabolic changes in EG affect neuron or glia metabolic coupling at the AL, the consequences of perturbing projection neurons directly were assessed using GH146::Gal4. Knocking down Out but not lactate dehydrogenase (Ldh) in projection neurons rescued olfactory discrimination of infected or aged flies, whereas food preference or mortality was not influenced. Overexpression of lipase 4 (Lip-4) in projection neurons, or knockdown of the neuronal lipid binding protein Neural lazarillo (NLaz), significantly improved olfactory discrimination in infected or old flies, and overexpression of Lip-4 increased Ecc15 food consumption and increased mortality after PE exposure (Cai, 2021).

This work suggests that gut-derived inflammatory cytokines modulate the metabolic coupling of glia and neurons in the brain of Drosophila to induce an adaptive temporary halt of olfactory discrimination after intestinal infection, but also contribute to age-related olfactory decline. It is proposed that gut-derived Upd2 and Upd3 reprogram lipid metabolism in EG, increasing lactate and lipid transport between glia and olfactory neurons, resulting in LD accumulation and upregulation of mitochondrial β-oxidation, potentially a source of increased ROS production. Chronic activation of this metabolic shift in old flies results in the accumulation of peroxidated lipids in EG, promoting their decay and contributing to the previously described functional decline of olfactory neurons. Detailed characterization of this metabolic reprogramming, and further exploration of the role of lipid synthesis in projection neurons for glial lipid accumulation and for olfactory discrimination are important avenues for further study (Cai, 2021).

These findings further determine the regulation of avoidance behaviour against enteropathogens in insects. In addition to gustatory bitter neurons and immune receptors in octopaminergic neurons, Upd proteins constitute a direct endocrine signal from the damaged intestinal epithelium in this complex but essential behaviour. It is proposed that Upd-mediated suppression of olfactory discrimination is required to prevent olfaction-mediated attraction to a food source after pathogenicity has been established and aversion is induced by gustatory neurons. It remains unclear, however, whether gustatory neurons are also affected by JAK-STAT signalling in EG. Whether similar mechanisms are conserved and control infection-induced loss of sensory perception in vertebrates including humans will be interesting to explore (Cai, 2021).

Drosophila Solute Carrier 5A5 Regulates Systemic Glucose Homeostasis by Mediating Glucose Absorption in the Midgut

The small intestine is the initial site of glucose absorption and thus represents the first of a continuum of events that modulate normal systemic glucose homeostasis. A better understanding of the regulation of intestinal glucose transporters is therefore pertinent to efforts in curbing metabolic disorders. Using molecular genetic approaches, this study investigated the role of Drosophila Solute Carrier 5A5 (dSLC5A5) in regulating glucose homeostasis by mediating glucose uptake in the fly midgut. By genetically knocking down dSLC5A5 in flies, it was found that systemic and circulating glucose and trehalose levels are significantly decreased, which correlates with an attenuation in glucose uptake in the enterocytes. Reciprocally, overexpression of dSLC5A5 significantly increases systemic and circulating glucose and trehalose levels and promotes glucose uptake in the enterocytes. dSLC5A5 was shown to undergo apical endocytosis in a dynamin-dependent manner, which is essential for glucose uptake in the enterocytes. Furthermore, this study showed that the dSLC5A5 level in the midgut is upregulated by glucose and that dSLC5A5 critically directs systemic glucose homeostasis on a high-sugar diet. Together, these studies have uncovered the first Drosophila glucose transporter in the midgut and revealed new mechanisms that regulate glucose transporter levels and activity in the enterocyte apical membrane (Li, 2021).

Drosophila transmembrane protein 214 (dTMEM214) regulates midgut glucose uptake and systemic glucose homeostasis

The availability of glucose transporter in the small intestine critically determines the capacity for glucose uptake and consequently systemic glucose homeostasis. Hence a better understanding of the physiological regulation of intestinal glucose transporter is pertinent. However, the molecular mechanisms that regulate sodium-glucose linked transporter 1 (SGLT1), the primary glucose transporter in the small intestine, remain incompletely understood. Recently, the Drosophila SLC5A5 (dSLC5A5) has been found to exhibit properties consistent with a dietary glucose transporter in the Drosophila midgut, the equivalence of the mammalian small intestine. Hence, the fly midgut could serve as a suitable model system for the study of the in vivo molecular underpinnings of SGLT1 function. This study reports the identification, through a genetic screen, of Drosophila transmembrane protein 214 (dTMEM214) that acts in the midgut enterocytes to regulate systemic glucose homeostasis and glucose uptake.dTMEM214 resides in the apical membrane and cytoplasm of the midgut enterocytes, and the proper subcellular distribution of dTMEM214 in the enterocytes is regulated by the Rab4 GTPase. As a corollary, Rab4 loss-of-function phenocopies dTMEM214 loss-of-function in the midgut as shown by a decrease in enterocyte glucose uptake and an alteration in systemic glucose homeostasis. It was further shown that dTMEM214 regulates the apical membrane localization of dSLC5A5 in the enterocytes, thereby revealing dTMEM214 as a molecular regulator of glucose transporter in the midgut (Li, 2023).

Microbiota alterations in proline metabolism impact depression

The microbiota-gut-brain axis has emerged as a novel target in depression, a disorder with low treatment efficacy. This study applied a multi-omics approach combining pre-clinical models with three human cohorts including patients with mild depression. Microbial functions and metabolites converging onto glutamate/GABA metabolism, particularly proline, were linked to depression. High proline consumption was the dietary factor with the strongest impact on depression. Whole-brain dynamics revealed rich club network disruptions associated with depression and circulating proline. Proline supplementation in mice exacerbated depression along with microbial translocation. Human microbiota transplantation induced an emotionally impaired phenotype in mice and alterations in GABA-, proline-, and extracellular matrix-related prefrontal cortex genes. RNAi-mediated knockdown of proline and GABA transporters in Drosophila and mono-association with L. plantarum, a high GABA producer, conferred protection against depression-like states. Targeting the microbiome and dietary proline may open new windows for efficient depression treatment (Mayneris-Perxachs, 2022).

Regulation of thermoregulatory behavior by commensal bacteria in Drosophila

Commensal bacteria affect many aspects of host physiology. This study focused on the role of commensal bacteria in the thermoregulatory behavior of Drosophila melanogaster. The elimination of commensal bacteria caused an increase in the preferred temperature of Drosophila third-instar larvae without affecting the activity of transient receptor potential ankyrin 1 (TRPA1)-expressing thermosensitive neurons. Eight bacterial strains were isolated from the gut and culture medium of conventionally reared larvae, and it was found that the preferred temperature of the larvae was decreased by mono-association with Lactobacillus plantarum or Corynebacterium nuruki. Mono-association with these bacteria did not affect the indices of energy metabolism such as ATP and glucose levels of larvae, which are closely linked to thermoregulation in animals. Thus, this study shows a novel role for commensal bacteria in host thermoregulation and identifies two bacterial species that affect thermoregulatory behavior in Drosophila (Suito, 2022).

Antibiotics increase aggression behavior and aggression-related pheromones and receptors in Drosophila melanogaster

Aggression is a behavior common in most species; it is controlled by internal and external drivers, including hormones, environmental cues, and social interactions, and underlying pathways are understood in a broad range of species. To date, though, effects of gut microbiota on aggression in the context of gut-brain communication and social behavior have not been completely elucidated. This study examined how manipulation of Drosophila melanogaster microbiota affects aggression as well as the pathways that underlie the behavior in this species. Male flies treated with antibiotics exhibited significantly more aggressive behaviors. Furthermore, they had higher levels of cVA and (Z)-9 Tricosene, pheromones associated with aggression in flies, as well as higher expression of the relevant pheromone receptors and transporters OR67d, OR83b, GR32a, and LUSH. These findings suggest that aggressive behavior is, at least in part, mediated by bacterial species in flies (Grinberg, 2022).

Bugs on Drugs: A Drosophila melanogaster Gut Model to Study In Vivo Antibiotic Tolerance of E. coli

With an antibiotic crisis upon us, antibiotic development and improve antibiotics' efficacy need to be boosted. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to the most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as flexible in vivo models are lacking. This study explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition (Van den Bergh, 2022).

Beneficial commensal bacteria promote Drosophila growth by downregulating the expression of peptidoglycan recognition proteins

Commensal bacteria are known to promote host growth. Such effect partly relies on the capacity of microbes to regulate the host's transcriptional response. However, these evidences mainly come from comparing the transcriptional response caused by commensal bacteria with that of axenic animals, making it difficult to identify the animal genes that are specifically regulated by beneficial microbes. This study employed Drosophila melanogaster associated with Lactiplantibacillus plantarum to understand the host genetic pathways regulated by beneficial bacteria and leading to improved host growth. Microbial benefit to the host relies on the downregulation of peptidoglycan-recognition proteins. Specifically, this paper reports that bacterial proliferation triggers the lower expression of PGRP-SC1 in larval midgut, which ultimately leads to improved host growth and development. This study helps elucidate the mechanisms underlying the beneficial effect exerted by commensal bacteria, defining the role of immune effectors in the relationship between Drosophila and its gut microbes (Gallo, 2022).

Enteric bacterial infection in Drosophila induces whole-body alterations in metabolic gene expression independently of the immune deficiency signaling pathway

When infected by intestinal pathogenic bacteria, animals initiate both local and systemic defence responses. These responses are required to reduce pathogen burden and also to alter host physiology and behavior to promote infection tolerance, and they are often mediated through alterations in host gene expression. This study used transcriptome profiling to examine gene expression changes induced by enteric infection with the Gram-negative bacteria Pseudomonas entomophila in adult female Drosophila. Infection was found to induce a strong upregulation of metabolic gene expression, including gut and fat body-enriched genes involved in lipid transport, lipolysis, and beta-oxidation, as well as glucose and amino acid metabolism genes. Furthermore, the classic innate immune deficiency (Imd)/Relish/NF-KappaB pathway were not required for, and in some cases limits, these infection-mediated increases in metabolic gene expression. Enteric infection with Pseudomonas entomophila downregulates the expression of many transcription factors and cell-cell signaling molecules, particularly those previously shown to be involved in gut-to-brain and neuronal signaling. Moreover, as with the metabolic genes, these changes occurred largely independent of the Imd pathway. Together, this study identifies many metabolic, signaling, and transcription factor gene expression changes that may contribute to organismal physiological and behavioral responses to enteric pathogen infection (Deshpande, 2022).

Microbiome derived acidity protects against microbial invasion in Drosophila

Microbial invasions underlie host-microbe interactions that result in microbial pathogenesis and probiotic colonization. While these processes are of broad interest, there are still gaps in understanding of the barriers to entry and how some microbes overcome them. This study explored the effects of the microbiome on invasions of foreign microbes in Drosophila melanogaster. Gut microbes Lactiplantibacillus plantarum and Acetobacter tropicalis improve survival during invasion of a lethal gut pathogen and lead to a reduction in microbial burden. Using a novel multi-organism interactions assay, it is reported that L. plantarum inhibits the growth of three invasive Gram-negative bacteria, while A. tropicalis prevents this inhibition. A series of in vitro and in vivo experiments revealed that inhibition by L. plantarum is linked to its ability to acidify both internal and external environments, including culture media, fly food, and the gut itself, while A. tropicalis diminishes the inhibition by quenching acids. It is proposed that acid produced by the microbiome serves as an important gatekeeper to microbial invasions, as only microbes capable of tolerating acidic environments can colonize the host. The methods described in this study will add to the growing breadth of tools to study microbe-microbe interactions in broad contexts (Barron, 2023).

The microbiome stabilizes circadian rhythms in the gut

The gut microbiome is well known to impact host physiology and health. Given widespread control of physiology by circadian clocks, it was asked how the microbiome interacts with circadian rhythms in the Drosophila gut. The microbiome did not cycle in flies fed ad libitum, and timed feeding (TF) drove limited cycling only in clockless per⁰¹ flies. However, TF and loss of the microbiome influenced the composition of the gut cycling transcriptome, independently and together. Moreover, both interventions increased the amplitude of rhythmic gene expression, with effects of TF at least partly due to changes in histone acetylation. Contrary to expectations, timed feeding rendered animals more sensitive to stress. Analysis of microbiome function in circadian physiology revealed that germ-free flies reset more rapidly with shifts in the light:dark cycle. It is proposed that the microbiome stabilizes cycling in the host gut to prevent rapid fluctuations with changing environmental conditions (Zhang, 2023).

Recognition of commensal bacterial peptidoglycans defines Drosophila gut homeostasis and lifespan

Commensal microbes in animals have a profound impact on tissue homeostasis, stress resistance, and ageing. Previous work has shown in Drosophila melanogaster that Acetobacter persici is a member of the gut microbiota that promotes ageing and shortens fly lifespan. However, the molecular mechanism by which this specific bacterial species changes lifespan and physiology remains unclear. The difficulty in studying longevity using gnotobiotic flies is the high risk of contamination during ageing. To overcome this technical challenge, a bacteria-conditioned diet was used, enriched with bacterial products and cell wall components. This study demonstrated that an A. persici-conditioned diet shortens lifespan and increases intestinal stem cell (ISC) proliferation. Feeding adult flies a diet conditioned with A. persici, but not with Lactiplantibacillus plantarum, can decrease lifespan but increase resistance to paraquat or oral infection of Pseudomonas entomophila, indicating that the bacterium alters the trade-off between lifespan and host defence. A transcriptomic analysis using fly intestine revealed that A. persici preferably induces antimicrobial peptides (AMPs), while L. plantarum upregulates amidase peptidoglycan recognition proteins (PGRPs). The specific induction of these Imd target genes by peptidoglycans from two bacterial species is due to the stimulation of the receptor PGRP-LC in the anterior midgut for AMPs or PGRP-LE from the posterior midgut for amidase PGRPs. Heat-killed A. persici also shortens lifespan and increases ISC proliferation via PGRP-LC, but it is not sufficient to alter the stress resistance. this study emphasizes the significance of peptidoglycan specificity in determining the gut bacterial impact on healthspan. It also unveils the postbiotic effect of specific gut bacterial species, which turns flies into a "live fast, die young" lifestyle (Onuma, 2023).

Structure-function analysis of Lactiplantibacillus plantarum DltE reveals D-alanylated lipoteichoic acids as direct cues supporting Drosophila juvenile growth

Metazoans establish mutually beneficial interactions with their resident microorganisms. However, understanding of the microbial cues contributing to host physiology remains elusive. Previously, a bacterial machinery was identified that is encoded by the dlt operon involved in Drosophila melanogaster's juvenile growth promotion by Lactiplantibacillus plantarum. Using crystallography combined with biochemical and cellular approaches, the physiological role was investigated of an uncharacterized protein (DltE) encoded by this operon. Lipoteichoic acids (LTAs) but not wall teichoic acids are D-alanylated in Lactiplantibacillus plantarum(NC8) cell envelope, and it was demonstrated that DltE is a D-Ala carboxyesterase removing D-Ala from LTA. Using the mutualistic association of L. plantarum(NC8) and Drosophila melanogaster as a symbiosis model, it was establish that D-alanylated LTAs (D-Ala-LTAs) are direct cues supporting intestinal peptidase expression and juvenile growth in Drosophila. These results pave the way to probing the contribution of D-Ala-LTAs to host physiology in other symbiotic models (Nikolopoulos, 2023).

Microbiota acquisition and transmission in Drosophila flies

Understanding the ecological and evolutionary dynamics of host-microbiota associations notably involves exploring how members of the microbiota assemble and whether they are transmitted along host generations. This study investigated the larval acquisition of facultative bacterial and yeast symbionts of Drosophila melanogaster and Drosophila suzukii in ecologically realistic setups. Fly mothers and fruit were major sources of symbionts. Microorganisms associated with adult males also contributed to larval microbiota, mostly in D. melanogaster. Yeasts acquired at the larval stage maintained through metamorphosis, adult life, and were transmitted to offspring. All these observations varied widely among microbial strains, suggesting they have different transmission strategies among fruits and insects. This approach shows microbiota members of insects can be acquired from a diversity of sources and highlights the compound nature of microbiotas. Such microbial transmission events along generations should favor the evolution of mutualistic interactions and enable microbiota-mediated local adaptation of the insect host (Guilhot, 2023).

Bacterial and fungal components of the gut microbiome have distinct, sex-specific roles in Hawaiian Drosophila reproduction

Gut microbiomes provide numerous physiological benefits for host animals. The role of bacterial members of microbiomes in host physiology is well-documented. However, much less is known about the contributions and interactions of fungal members of the microbiome even though fungi are significant components of many microbiomes, including those of humans and insects. This study used antibacterial and antifungal drugs to manipulate the gut microbiome of a Hawaiian picture-wing Drosophila species, D. grimshawi, and identified distinct, sex-specific roles for the bacteria and fungi in microbiome community stability and reproduction. Female oogenesis, fecundity and mating drive were significantly diminished when fungal communities were suppressed. By contrast, male fecundity was more strongly affected by bacterial but not fungal populations. For males and females, suppression of both bacteria and fungi severely reduced fecundity and altered fatty acid levels and composition, implicating the importance of interkingdom interactions on reproduction and lipid metabolism. Overall, these results reveal that bacteria and fungi have distinct, sexually-dimorphic effects on host physiology and interkingdom dynamics in the gut help to maintain microbiome community stability and enhance reproduction (Medeiros, 2023).

Gut microbial genetic variation modulates host lifespan, sleep, and motor performance

Recent studies have shown that gut microorganisms can modulate host lifespan and activities, including sleep quality and motor performance. However, the role of gut microbial genetic variation in regulating host phenotypes remains unclear. This study investigated the links between gut microbial genetic variation and host phenotypes using Saccharomyces cerevisiae and Drosophila melanogaster as research models. The results suggested a novel role for peroxisome-related genes in yeast in regulating host lifespan and activities by modulating gut oxidative stress. Specifically, it was found that deficiency in catalase A (CTA1) in yeast reduced both the sleep duration and lifespan of fruit flies significantly. Furthermore, this research also expanded understanding of the relationship between sleep and longevity. Using a large sample size and excluding individual genetic background differences, this study found that lifespan is associated with sleep duration, but not sleep fragmentation or motor performance. Overall, this study provides novel insights into the role of gut microbial genetic variation in regulating host phenotypes and offers potential new avenues for improving health and longevity (Li, 2023).

Intestinal GCN2 controls Drosophila systemic growth in response to Lactiplantibacillus plantarum symbiotic cues encoded by r/tRNA operons

Symbiotic bacteria interact with their host through symbiotic cues. This study took advantage of the mutualism between Drosophila and Lactiplantibacillus plantarum (Lp) to investigate a novel mechanism of host-symbiont interaction. Using chemically defined diets, it was found that association with Lp improves the growth of larvae-fed amino acid-imbalanced diets, even though Lp cannot produce the limiting amino acid. In this context Lp supports its host's growth through a molecular dialogue that requires functional operons encoding ribosomal and transfer RNAs (r/tRNAs) in Lp and the general control nonderepressible 2 (GCN2) kinase in Drosophila's enterocytes. These data indicate that Lp's r/tRNAs are packaged in extracellular vesicles and activate GCN2 in a subset of larval enterocytes, a mechanism necessary to remodel the intestinal transcriptome and ultimately to support anabolic growth. Based on these findings, a novel beneficial molecular dialogue is proposed between host and microbes, which relies on a non-canonical role of GCN2 as a mediator of non-nutritional symbiotic cues encoded by r/tRNA operons (Grenier, 2023).

Role of Rab5 early endosomes in regulating Drosophila gut antibacterial response

Interactions between prokaryotes and eukaryotes require a dialogue between microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs). In Drosophila, bacterial peptidoglycan is detected by PGRP receptors. While the components of the signaling cascades activated upon PGN/PGRP interactions are well characterized, little is known about the subcellular events that translate these early signaling steps into target gene transcription. Using a Drosophila enteric infection model, this study showed that gut-associated bacteria can induce the formation of intracellular PGRP-LE aggregates which colocalized with the early endosome marker Rab5. Combining microscopic and RNA-seq analysis, it was demonstrated that RNAi inactivation of the endocytosis pathway in the Drosophila gut affects the expression of essential regulators of the NF-κB response leading not only to a disruption of the immune response locally in the gut but also at the systemic level. This work sheds new light on the involvement of the endocytosis pathway in the control of the gut response to intestinal bacterial infection (Joshi, 2023).

Gut microbes predominantly act as living beneficial partners rather than raw nutrients

Animals and their gut microbes mutually benefit their health. Nutrition plays a central role in this, directly influencing both host and microbial fitness and the nature of their interactions. This makes nutritional symbioses a complex and dynamic tri-system of diet-microbiota-host. Despite recent discoveries on this field, full control over the interplay among these partners is challenging and hinders the resolution of fundamental questions, such as how to parse the gut microbes' effect as raw nutrition or as symbiotic partners? To tackle this, use was made of the well-characterized Drosophila melanogaster/Lactiplantibacillus plantarum experimental model of nutritional symbiosis to generate a quantitative framework of gut microbes' effect on the host. By coupling experimental assays and Random Forest analysis, it was shown that the beneficial effect of L. plantarum strains primarily results from the active relationship as symbionts rather than raw nutrients, regardless of the bacterial strain. Metabolomic analysis of both active and inactive bacterial cells further demonstrated the crucial role of the production of beneficial bacterial metabolites, such as N-acetylated-amino-acids, as result of active bacterial growth and function. Altogether, these results provide a ranking and quantification of the main bacterial features contributing to sustain animal growth.This study has demonstrated that bacterial activity is the predominant and necessary variable involved in bacteria-mediated benefit, followed by strain-specific properties and the nutritional potential of the bacterial cells. This contributes to elucidate the role of beneficial bacteria and probiotics, creating a broad quantitative framework for host-gut microbiome that can be expanded to other model systems (da Silva Soares, 2023).

A symbiotic physical niche in Drosophila melanogaster regulates stable association of a multi-species gut microbiota

The gut is continuously invaded by diverse bacteria from the diet and the environment, yet microbiome composition is relatively stable over time for host species ranging from mammals to insects, suggesting host-specific factors may selectively maintain key species of bacteria. To investigate host specificity, gnotobiotic Drosophila, microbial pulse-chase protocols, and microscopy were used to investigate the stability of different strains of bacteria in the fly gut. A host-constructed physical niche in the foregut was shown to selectively binds bacteria with strain-level specificity, stabilizing their colonization. Primary colonizers saturate the niche and exclude secondary colonizers of the same strain, but initial colonization by Lactobacillus species physically remodels the niche through production of a glycan-rich secretion to favor secondary colonization by unrelated commensals in the Acetobacter genus. These results provide a mechanistic framework for understanding the establishment and stability of a multi-species intestinal microbiome (Dodge, 2023).

Eucommia Polysaccharides Ameliorate Aging-Associated Gut Dysbiosis: A Potential Mechanism for Life Extension in Drosophila

The gut microbiota is increasingly considered to play a key role in human immunity and health. The aging process alters the microbiota composition, which is associated with inflammation, reactive oxygen species (ROS), decreased tissue function, and increased susceptibility to age-related diseases. It has been demonstrated that plant polysaccharides have beneficial effects on the gut microbiota, particularly in reducing pathogenic bacteria abundance and increasing beneficial bacteria populations. However, there is limited evidence of the effect of plant polysaccharides on age-related gut microbiota dysbiosis and ROS accumulation during the aging process. To explore the effect of Eucommiae polysaccharides (EPs) on age-related gut microbiota dysbiosis and ROS accumulation during the aging process of Drosophila, a series of behavioral and life span assays of Drosophila with the same genetic background in standard medium and a medium supplemented with EPs were performed. Next, the gut microbiota composition and protein composition of Drosophila in standard medium and the medium supplemented with EPs were detected using 16S rRNA gene sequencing analysis and quantitative proteomic analysis. This study shows that supplementation of Eucommiae polysaccharides (EPs) during development leads to the life span extension of Drosophila. Furthermore, EPs decreased age-related ROS accumulation and suppressed Gluconobacter, Providencia, and Enterobacteriaceae in aged Drosophila. Increased Gluconobacter, Providencia, and Enterobacteriaceae in the indigenous microbiota might induce age-related gut dysfunction in Drosophila and shortens their life span. This study demonstrates that EPs can be used as prebiotic agents to prevent aging-associated gut dysbiosis and reactive oxidative stress (Wei, 2023).

Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila

Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. This study shows that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions (Jneid, 2023).

The Drosophila AWP1 ortholog Doctor No regulates JAK/STAT signaling for left-right asymmetry in the gut by promoting receptor endocytosis

Many organs of Drosophila show stereotypical left-right (LR) asymmetry; however, the underlying mechanisms remain elusive. This study has identified an evolutionarily conserved ubiquitin-binding protein, AWP1/Doctor No (Drn), as a factor required for LR asymmetry in the embryonic anterior gut. drn is essential in the circular visceral muscle cells of the midgut for JAK/STAT signaling, which contributes to the first known cue for anterior gut lateralization via LR asymmetric nuclear rearrangement. Embryos homozygous for drn and lacking its maternal contribution showed phenotypes similar to those with depleted JAK/STAT signaling, suggesting that Drn is a general component of JAK/STAT signaling. Absence of Drn resulted in specific accumulation of Domeless (Dome), the receptor for ligands in the JAK/STAT signaling pathway, in intracellular compartments, including ubiquitylated cargos. Dome colocalized with Drn in wild-type Drosophila. These results suggest that Drn is required for the endocytic trafficking of Dome, which is a crucial step for activation of JAK/STAT signaling and the subsequent degradation of Dome. The roles of AWP1/Drn in activating JAK/STAT signaling and in LR asymmetric development may be conserved in various organisms (Lai, 2023).

Many animals show directional left-right (LR) asymmetry in their body structure and function. Several mechanisms, such as cilia-generated flow, contribute to LR axis formation in vertebrates. However, the mechanisms underlying LR asymmetric development in invertebrates are relatively obscure and remain an elemental question in biology (Lai, 2023).

Stereotypical LR asymmetry is present in several organs of Drosophila, including the gut, testis, male genitalia, and brain. Of these organs, the embryonic gut is the first to show LR asymmetry during development. Intriguingly, the anterior and posterior parts of the embryonic gut are controlled by two distinct gene groups. The LR asymmetry of the posterior part is regulated by Myo1D, which determines cell chirality. An entirely different mechanism governs the LR asymmetry of the anterior part. The anterior gut of the embryo consists of the foregut (FG) and midgut (MG), which are complex structures with directional and stereotypical LR asymmetry. Various genetic pathways, including the JNK and Wnt pathways, play important roles in LR asymmetric development. A genetic screen suggested that the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway is also involved in LR asymmetric development. The present study reveals the involvement of JAK/STAT signaling in LR asymmetric development of the anterior gut (Lai, 2023).

JAK/STAT signaling, which is evolutionarily conserved from Drosophila to humans, is essential for morphogenesis, cell proliferation, cell differentiation, cell death, immunity and other biological events. In JAK/STAT signaling in Drosophila, the ligands are encoded by unpaired (upd; upd1), upd2 and upd3 and the receptor is encoded by domeless (dome). JAK is encoded by hopscotch (hop) in Drosophila. Moreover, similar to the mammalian JAK/STAT system, Hop constitutively associates with the intracellular domain of Dome in Drosophila. Ligand binding induces conformational changes in Dome that lead to the phosphorylation of JAK, which phosphorylates Dome to create docking sites for STATs, particularly Stat92E in Drosophila. JAK then tyrosine phosphorylates STATs, which are subsequently dimerized and translocated to the nucleus to bind to enhancers of target genes and activate transcription (Lai, 2023).

In mammals, endocytosis regulates JAK/STAT signaling through various mechanisms. Endocytosis and the regulation of JAK/STAT signaling activity are also closely connected in Drosophila. In the classic working model of endocytosis, membrane receptors are internalized into endosomal compartments, where they are degraded and recycled. This reduces the number of receptors available to transduce signaling. Moreover, receptor activity requires endocytic trafficking in some signaling pathways. Studies on the relationship between endocytosis and JAK/STAT signaling in Drosophila have provided contradictory results regarding whether endocytosis upregulates or downregulates JAK/STAT activity. To clarify these conflicting results, it would be helpful to identify and examine a factor that specifically regulates the endocytosis of Dome (Lai, 2023).

This study found that the Drosophila ortholog of AWP1 (associated with PRK1, also known as ZFAND6), which is referred to as Doctor No (Drn), positively regulates JAK/STAT signaling by facilitating endocytic trafficking of the Dome receptor, which is required for normal LR asymmetric development of the embryonic gut. Drosophila Drn was named after Ian Fleming's fictional character, whose heart was located on the right side of his chest. Drn protein contains an A20-type zinc finger at the N terminus and an AN1-type zinc finger at the C terminus; this is similar to the mammalian ortholog, which was first identified in humans and mice. In vertebrates, AWP1 proteins bind to ubiquitin, regulate NF-κB activity, and stimulate the export of Pex5 from the peroxisome, among other roles. During Xenopus development, AWP1 modifies Wnt and FGF signaling to specify neural crest cells. However, the molecular mechanisms by which AWP1/Drn proteins influence these various cell signaling pathways are not well understood. This study found that AWP1/Drn plays a crucial role in internalizing the Dome receptor, and a mechanism is proposed by which AWP1/Drn positively modulates JAK/STAT signaling (Lai, 2023).

This study demonstrates that drn, which encodes a Drosophila ortholog of AWP1, plays crucial roles in the LR asymmetric development of the FG and AMG by positively regulating JAK/STAT signaling. In this process, wild-type drn is required for the normal endocytic trafficking of Dome, which is the Drosophila JAK/STAT signaling receptor. Whether the internalization as well as endocytic trafficking of Dome is required to activate JAK/STAT signaling in Drosophila has been a topic of debate. Analyses of mutations affecting Dome endocytosis have revealed that Dome must be internalized into early endosomes to activate JAK/STAT signaling. In contrast, the RNAi-mediated knockdown of genes required for Dome endocytosis was found to enhance JAK/STAT signaling activity, demonstrating that Dome endocytosis negatively regulates JAK/STAT signaling. This discrepancy can be explained by the multiple ramifications and parallelism of the endocytic pathway, as described in recent studies. In particular, blocking a particular step along the pathway can divert endocytic trafficking into various positive or negative regulatory cascades of cell signaling pathways. The relative contributions of endocytic regulators can also change the course of endocytic trafficking, as is well documented in Notch signaling. Moreover, a change in the balance of regulators may change how endocytosis regulates cell signaling pathways according to the context. Hence, to understand comprehensively how endocytosis contributes to the activation of Dome, it is necessary to examine the point at which endocytosis is disrupted and the altered course of endocytic trafficking when normal endocytosis fails (Lai, 2023).

A study using RNAi of cultured Drosophila cells identified drn as a negative regulator of JAK/STAT signaling; this finding appears to be opposite to the current model. However, as other genes required for Dome endocytosis were also identified as negative regulators of JAK/STAT signaling in that analysis, it is likely that this discordance regarding the role of Drn in JAK/STAT signaling results from a discrepancy in the contribution of Dome endocytosis to JAK/STAT signaling, as discussed above. Regarding this discrepancy, it has been proposed that long-term, loss-of-function analyses, including analyses of mutants in which Dome endocytosis is disrupted, may reflect unexpected cell fate changes induced by altered endocytic pathways and their influence on JAK/STAT signaling. However, in this study, three developmental contexts were examined in which JAK/STAT signaling is reduced in drn mutants. One of these involves the regulation of eve expression, which occurs before cell fate specification and during a short period in early embryogenesis; thus, it does not fit well with a mechanism involving unexpected cell fate changes. Therefore, a model is proposed in which Drn is required in some steps of the endocytic trafficking of Dome, which plays a role in the subsequent activation of JAK/STAT signaling. This model is consistent with that proposed by in another study in which the endocytic trafficking of Dome activates JAK/STAT signaling and also provides a mechanism to regulate it quantitatively. However, as where and how Drn can control the endocytic trafficking of Dome remain unclear, the coincidence between previous and current results should be interpreted with caution (Lai, 2023).

Drn is the Drosophila ortholog of AWP1, which binds to ubiquitin and modulates the functions of ubiquitylated proteins in mammals and Xenopus species. It is known that ubiquitylation of the receptors involved in JAK/STAT signaling is important for regulating signaling activities in mammals. The sorting of ubiquitylated membrane proteins into intraluminal vesicles relies on protein complexes in the ESCRT family. ESCRT-0, ESCRT-I, and ESCRT-II include multiple ubiquitin-binding proteins and interpret ubiquitin as a signal to sort membrane proteins. In Drosophila mutants of the ESCRT-0 complex components Hrs and Stam, ubiquitylated membrane proteins, such as Notch and Dome, aggregate at the cell cortex and in intracellular compartments. As such intracellular compartments, including aggregated Dome, were stained with an anti-ubiquitin antibody, it has been proposed that Drosophila Dome is ubiquitylated and its endosomal sorting is controlled by ubiquitylation; however, the ubiquitylation of Dome has not been confirmed biochemically. A loss of Drn caused Dome to accumulate in large clumps that frequently colocalized with ubiquitin but were not labeled by markers of typical intracellular compartments. In addition, Drn occasionally colocalized with markers of various endocytic compartments, demonstrating that Drn is an endocytic protein. Hence, it is speculated that Drn plays a role in the ubiquitin-dependent internalization or sorting of Dome through its potential ubiquitin-binding activity. This idea is consistent with the observation that Drn often colocalizes with Dome in some intracellular vesicles in wild-type Drosophila. Thus, in this model, Dome may be misrouted to atypical endocytic compartments, where it fails to be phosphorylated in the absence of Drn. Conversely, differences in Dome trafficking routes between wild-type and drn mutant embryos should help identify the endocytic compartment where Dome is activated by phosphorylation. However, it is difficult to delineate incorrect Dome trafficking routes in the drn mutant because Dome did not specifically colocalize with typical markers of endocytic compartments under this condition. The model also predicts that such misrouting can consequently prevent the degradation of Dome in lysosomes and leave it to accumulate, as observed in drn mutants (Lai, 2023).

These analyses revealed that drn mutations induce marked accumulation of Dome but not of Notch or Fz2. The intracellular distribution of Notch and Fz2 in the drn mutant appeared to be similar to that in the wild type. Thus, Drn is not a general component of endosomal protein sorting but is specific to Dome; however, how such specificity is achieved remains unclear (Lai, 2023).

This study found that JAK/STAT signaling activity must be maintained at proper levels for normal LR asymmetric development of the FG and AMG. A similar phenomenon was previously observed in Wnt or JNK signaling activity. However, LR asymmetry was bit detectuve in the activity or the distribution of molecules involved in the JAK/STAT, Wnt and JNK signaling pathways. As these three signaling pathways are required for LR asymmetric rearrangement of nuclei in the visceral muscles of the MG, they may play permissive roles in rearranging these nuclei upon a common cue, which is yet unknown, of LR polarity (Lai, 2023).

AWP1/Drn is highly conserved from Drosophila to humans. As receptors in the mammalian JAK/STAT pathway are also ubiquitylated, it is suggested that the role of AWP1/Drn in JAK/STAT signaling and LR asymmetric development is evolutionarily conserved in various organisms (Lai, 2023).

Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila

The gut sets the immune and metabolic parameters for the survival of commensal bacteria. This study reports that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP (Thor), a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. These results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut (Bahuguna, 2022).

The animal gut accommodates a diverse array of bacteria, which assist in regulation of digestion, supply of nutrients and metabolites as well as in immune development. To reap benefits from these microbes, the host provides a symbiotic environment for sustaining them in the gut. The Drosophila gut and its bacteriome is used as a simpler model to study such host-microbe interactions. Although much less diverse compared to humans, the fly bacteriome is equally dynamic and changes with age and environmental conditions connected to reinfections during fly culture (Bahuguna, 2022).

The Drosophila intestinal epithelium is immunocompetent and upon enteric infection initiates innate immune responses via the NF-κB pathway IMD, mediating the production of antimicrobial peptides (AMPs) as well as the pathway centered on Dual Oxidase, the enzyme needed for the generation of Reactive Oxygen Species (ROS). However, it also preserves commensal bacteria, since transcription of AMPs is suppressed by Caudal and Nubbin, while bacterial-derived uracil is important for distinguishing pathogens from commensals (Bahuguna, 2022).

The evolutionary conserved Target of Rapamycin (TOR) pathway is a major pathway controlling cellular metabolism and growth. TOR balances lipid and glucose anabolism and catabolism in the cell through the activity of the TORC1 protein complex. TORC1 promotes protein synthesis primarily through phosphorylation of the eIF4E Binding Protein (4E-BP/Thor) and p70S6 Kinase 1 (S6K1). 4E-BP is a translational inhibitor, which binds and inhibits the activity of eIF4E an eukaryotic translation initiation factor responsible for the recruitment of 40s ribosomal subunit at the 5'-cap of mRNA. Phosphorylation of 4E-BP lowers its affinity towards eIF4E. This frees eIF4E, enabling it to promote cap-dependent translation. In the case of S6K1, activated S6K1 promotes protein synthesis by activating inducers of mRNA translation initiation whilst degrading inhibitors (Bahuguna, 2022).

Drosophila TOR has been extensively studied for its role in growth and development, using fly mutants or by treating flies with rapamycin. Rapamycin treatment in stress conditions, led to upregulation of 4E-BP activity resulting in an increase of whole-fly lipid reserves that could be used for the long-term survival of these stress conditions. In contrast, 4E-BP mutants were unable to preserve lipid stores and had thus compromised survival following starvation or oxidative stress. More broadly, the consensus is that in both Drosophila and mice, 4E-BP regulates fat levels in stress conditions like starvation and oxidative stress. During larval stages and when in food with poor nutritional value, the presence of the commensal Lactobacillus plantarum is important to sustain development through TOR, which is in turn crucial for sustaining this mutualistic relationship. The metabolic state of the gut is also influenced by dietary conditions and in its turn influences the bacteriome. Diet-dependent adaptations of the microbiota require NF-κB-dependent control of the translational regulator 4E-BP and this where TOR and NF-κB 'meet' (Bahuguna, 2022).

Drosophila has three NF-κB proteins namely, Relish, Dorsal and the Dorsal-related Immunity Factor (DIF). DIF is downstream of the Toll signalling pathway. Toll and Toll-like receptor (TLR) signalling is one of the most important evolutionary conserved mechanisms by which the innate immune system senses the invasion of pathogenic microorganisms. Unlike its mammalian counterparts however, Drosophila Toll is activated by an endogenous cytokine-like ligand, the Nerve Growth Factor homologue, Spz. Spz is processed to its active form by the Spz-Processing Enzyme (SPE). Two serine protease cascades converge on SPE: one triggered by bacterial or fungal serine proteases through the host serine protease Persephone and a second activated by host receptors that recognise bacterial or fungal cell wall. Prominent among these host receptors is the Peptidoglycan Recognition Protein-SA or PGRP-SA. PGRP-SA binds to peptidoglycan on the bacterial cell wall without structural preference but depending on accessibility and generates the downstream signal (Bahuguna, 2022).

When the recognition signal reaches the cell surface, it is communicated intracellularly via the Toll receptor and a membrane-bound receptor-adaptor complex including dMyd88, Tube (as an IRAK4 functional equivalent) and the Pelle kinase (as an IRAK1 functional homologue). Transduction of the signal culminates in the phosphorylation of the IκB homologue, Cactus probably by Pelle, leaving the NF-κB homologue DIF to move to the nucleus and regulate hundreds of target genes including antimicrobial peptides (AMPs) (Bahuguna, 2022).

In this study, evidence is presented that PGRP-SA is important for the preservation of commensal intestinal bacterial density. The results reveal that larvae and adults that are deficient in PGRP-SA or DIF have a significantly reduced commensal gut bacterial density. Inhibition of the activity of TOR by Rapamycin or TOR RNAi in enterocytes, restores bacterial density (but not diversity) in PGRP-SA or DIF mutant guts. However, flies mutants for PGRP-SA and deficient for 4EBP in enterocytes were unable to restore bacterial density upon Rapamycin treatment or TOR RNAi, demonstrating the important role of 4EBP. PGRP-SA mutants had increased intestinal fat stores that were restored to normal levels through Rapamycin or TOR-RNAi treatment in enterocytes. This restoration failed in PGRP-SA;4EBP double mutants indicating that 4EBP was crucial in regulating fat stores in the gut. Fat catabolism was important for gut bacterial restoration as flies deficient for PGRP-SA and treated with rapamycin were unable to restore bacterial density if the triglyceride lipase Brummer was knocked down in enterocytes. This mechanism gives an insight into how the host integrates immunity and metabolism to maintain commensal bacteria at the intestinal epithelium (Bahuguna, 2022).

In the absence of the bacterial receptor PGRP-SA from enterocytes, a reduction was observed in intestinal bacterial density. It was restored with the use of rapamycin, which targets TORC1 or by knocking-down TOR in enterocytes. This suggested that loss of the immune receptor PGRP-SA, generated a metabolic environment unfavourable for intestinal bacterial growth. The results indicated that at the centre of this relationship was 4E-BP, which is activated by Toll and suppressed by TORC1. In keeping with this, PGRP-SA^seml; NP1GAL4>4E-BPRNAi flies treated with rapamycin were unable to restore gut bacterial density. Intestinal lipid catabolism downstream of 4EBP was paramount for the maintenance of cultivable bacterial density because the loss of the lipase Bmm blocked restoration of gut bacteria after rapamycin treatment. Silencing of bmm in enterocytes caused intestinal lipid accumulation and prevented any restoration via rapamycin in PGRP-SA^seml flies (Bahuguna, 2022).

These results indicate that downstream of Toll, intestinal triglyceride levels were under 4E-BP control in enterocytes. Although the phenomenon of cultivable bacteriome reduction in PGRP-SA^seml flies was readily manifested in larvae and young flies, the results indicated that it was also there in older flies. Conventional fly rearing techniques ensure a steady stream of defaecation and re-introduction of bacteria over time. However, when food vials were changed rapidly re-infection was reduced and CFUs in 30-day old flies were significantly lower in PGRP-SA^seml than yw flies. Preservation of the bacteriome was dependent on PG recognition as the rescue of enteric CFUs in PGRP-SA^seml flies was only possible with PGRP-SA transgenes that had an intact PG binding ability. This indicated that bacterial sensing was the initial trigger point to activate the process (Bahuguna, 2022).

A working model is depicted A schematic model outlining the role of PGRP-SA/Toll/Dif in the retention of the gut bacteriome. PGRP-SA recognises components of the intestinal bacteriome and activates the Toll pathway in enterocytes. In turn, this keeps increased 4E-BP transcription/4E-BP protein phosphorylation in enterocytes, preserving a steady rate of intestinal lipid catabolism. The latter is important for maintaining normal density of commensal bacteria. It is hypothesised that Bmm-mediated lipid catabolism is regulated by 4E-BP and released triglycerides act as fuel for the maintenance of commensal bacteria. In keeping with this, stopping lipid catabolism by silencing the Bmm lipase in ECs resulted in accumulation of lipids and reduction of enteric CFUs. According to the model, bacteria should trigger lipid catabolism and 5-day old axenic flies showed a clear trend for lipid accumulation in their gut, but this was marginally not statistically significant. Studies with Vibrio cholera, have shown that intestinal acetate leads to deactivation of host insulin signalling and lipid accumulation in enterocytes, resulting in host lethality. Loss of PGRP-SA/Dif leads to a decrease in lifespan. Whether this is due to the long-term accumulation of lipids is an open question (Bahuguna, 2022).

PGRP-SA recognises components of the intestinal bacteriome and activates the Toll pathway in enterocytes. This increases 4E-BP transcription/4E-BP protein phosphorylation in enterocytes. 4EBP is important for maintaining normal density of commensal bacteria. It is hypothesized that Bmm-mediated lipid catabolism is regulated by 4E-BP and released triglycerides act as fuel for the maintenance of commensal bacteria (Bahuguna, 2022).

More work is needed to understand whether/how stored intestinal lipids maybe released to circulation, how commensal bacteria receive them and which component(s) of the bacteriome are recognised by PGRP-SA (Bahuguna, 2022).

Ergothioneine exhibits longevity-extension effect in Drosophila melanogaster via regulation of cholinergic neurotransmission, tyrosine metabolism, and fatty acid oxidation

Many studies have demonstrated the protective effect of ergothioneine (EGT), the unique sulfur-containing antioxidant found in mushrooms, on several aging-related diseases. Nevertheless, to date, no single study has explored the potential role of EGT in the lifespan of animal models. This study shows that EGT consistently extends fly lifespan in diverse genetic backgrounds and both sexes, as well as in a dose and gender-dependent manner. Additionally, EGT is shown to increases the climbing activity of flies, enhance acetylcholinesterase (AchE) activity, and maintain the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) of aged flies. The increase in lifespan by EGT is gut microorganism dependent. This study proposed potential mechanisms of lifespan extension in Drosophila by EGT through RNA-seq analysis: preservation of the normal status of the central nervous system via the coordination of cholinergic neurotransmission, tyrosine metabolism, and peroxisomal proteins, regulation of autophagic activity by altering the lysosomal protein CTSD, and the preservation of normal mitochondrial function through controlled substrate feeding into the tricarboxylic acid (TCA) cycle, the major energy-yielding metabolic process in cells (Pan, 2022).

VhaAC39-1 regulates gut homeostasis and affects the health span in Drosophila

Gut homeostasis is a dynamically balanced state to maintain intestinal health. Vacuolar ATPases (V-ATPases) are multi-subunit proton pumps that were driven by ATP hydrolysis. Several subunits of V-ATPases may be involved in the maintenance of intestinal pH and gut homeostasis in Drosophila. However, the specific role of each subunit in this process remains to be elucidated. This study knocked down the Drosophila gene VhaAC39-1 encoding the V0d1 subunit of V-ATPases to assess its function in gut homeostasis. Knockdown of VhaAC39-1 resulted in the loss of midgut acidity, the increase of the number of gut microbiota and the impairment of intestinal epithelial integrity in flies. The knockdown of VhaAC39-1 led to the hyperproliferation of intestinal stem cells, increasing the number of enteroendocrine cells, and activated IMD signaling pathway and JAK-STAT signaling pathway, inducing intestinal immune response of Drosophila. In addition, knockdown of VhaAC39-1 caused the disturbance of many physiological indicators such as food intake, triglyceride level and fecundity of flies, which ultimately led to the shortening of the life span of Drosophila. These results shed light on the gut homeostasis mechanisms which would help to identify interventions to promote healthy aging (Tian, 2022).

SUMOylation of Jun fine-tunes the Drosophila gut immune response

Post-translational modification by the small ubiquitin-like modifier, SUMO can modulate the activity of its conjugated proteins in a plethora of cellular contexts. The effect of SUMO conjugation of proteins during an immune response is poorly understood in Drosophila. Previous work found that the transcription factor Jra, the Drosophila Jun ortholog and a member of the AP-1 complex is one such SUMO target. This study found that Jra is a regulator of the Pseudomonas entomophila induced gut immune gene regulatory network, modulating the expression of a few thousand genes, as measured by quantitative RNA sequencing. Decrease in Jra in gut enterocytes is protective, suggesting that reduction of Jra signaling favors the host over the pathogen. In Jra, lysines 29 and 190 are SUMO conjugation targets, with the JraK29R+K190R double mutant being SUMO conjugation resistant (SCR). Interestingly, a JraSCR fly line, generated by CRISPR/Cas9 based genome editing, is more sensitive to infection, with adults showing a weakened host response and increased proliferation of Pseudomonas. Transcriptome analysis of the guts of JraSCR and JraWT flies suggests that lack of SUMOylation of Jra significantly changes core elements of the immune gene regulatory network, which include antimicrobial agents, secreted ligands, feedback regulators, and transcription factors. Mechanistically, SUMOylation attenuates Jra activity, with the TFs, forkhead, anterior open, activating transcription factor 3 and the master immune regulator Relish being important transcriptional targets. This study implicates Jra as a major immune regulator, with dynamic SUMO conjugation/deconjugation of Jra modulating the kinetics of the gut immune response (Soory, 2022).

Phosphorylation of Yun is required for stem cell proliferation and tumorigenesis

Stem cells maintain adult tissue homeostasis under physiological conditions. Uncontrolled stem cell proliferation will lead to tumorigenesis. How stem cell proliferation is precisely controlled is still not fully understood. Phosphorylation of Yun is essential for ISC proliferation. Yun is essential for the proliferation of normal and transformed intestinal stem cells. Mass spectrometry and biochemical data suggest that Yun can be phosphorylated at multiple residues in vivo. Interestingly, it was shown that the phosphorylation among these residues is likely interdependent. Furthermore, phosphorylation of each residue in Yun is important for its function in ISC proliferation regulation. Thus, this study unveils the important role of post-translational modification of Yun in stem cell proliferation (Ren, 2022).

Prediction of intestinal stem cell regulatory genes from Drosophila gut damage model created using multiple inducers: Differential gene expression-based protein-protein interaction network analysis

Intestinal tissue functions in innate immunity to prevent the entry of harmful substances, and to maintain homeostasis through the constant proliferation of intestinal stem cells (ISC). To understand the mechanisms which regulate ISC in response to gut damage, this study identified 81 differentially expressed genes (DEGs) through RNA-seq analysis after oral administration of three intestinal-damaging substances to Drosophila melanogaster. Through protein-protein interaction (PPI) and functional annotation studies, the top 22 DEGs ordered by the number of nodes in the PPI network were analyzed in relation to cell development. Through network topology analysis, 12 essential seed genes were identified. From this it can be confirmed that p53, RpL17, Fmr1, Stat92E, CG31343, Cnot4, CG9281, CG8184, Evi5, and to were essential for ISC proliferation during gut damage using knockdown RNAi Drosophila. This study presents a method for identifying candidate genes relating to intestinal damage that has scope for furthering understanding of gut disease (Lee, 2022).

Imp interacts with Lin28 to regulate adult stem cell proliferation in the Drosophila intestine

Stem cells are essential for the development and long-term maintenance of tissues and organisms. Preserving tissue homeostasis requires exquisite control of all aspects of stem cell function: cell potency, proliferation, fate decision and differentiation. RNA binding proteins (RBPs) are essential components of the regulatory network that control gene expression in stem cells to maintain self-renewal and long-term homeostasis in adult tissues. While the function of many RBPs may have been characterized in various stem cell populations, how these interact and are organized in genetic networks remains largely elusive. This report shows that the conserved RNA binding protein IGF2 mRNA binding protein (Imp) is expressed in intestinal stem cells (ISCs) and progenitors in the adult Drosophila midgut. Imp was demonstrated to be required cell autonomously to maintain stem cell proliferative activity under normal epithelial turnover and in response to tissue damage. Mechanistically,Imp cooperates and directly interacts with Lin28, another highly conserved RBP, to regulate ISC proliferation. Both proteins bind to and control the InR mRNA, a critical regulator of ISC self-renewal. Altogether, these data suggests that Imp and Lin28 are part of a larger gene regulatory network controlling gene expression in ISCs and required to maintain epithelial homeostasis (Sreejith, 2022).

A gut-derived hormone suppresses sugar appetite and regulates food choice in Drosophila

Animals must adapt their dietary choices to meet their nutritional needs. How these needs are detected and translated into nutrient-specific appetites that drive food-choice behaviours is poorly understood. This study shows that enteroendocrine cells of the adult female Drosophila midgut sense nutrients and in response release neuropeptide F (NPF), which is an ortholog of mammalian neuropeptide Y-family gut-brain hormones. Gut-derived NPF acts on glucagon-like adipokinetic hormone (AKH) signalling to induce sugar satiety and increase consumption of protein-rich food, and on adipose tissue to promote storage of ingested nutrients. Suppression of NPF-mediated gut signalling leads to overconsumption of dietary sugar while simultaneously decreasing intake of protein-rich yeast. Furthermore, gut-derived NPF has a female-specific function in promoting consumption of protein-containing food in mated females. Together, these findings suggest that gut NPF-to-AKH signalling modulates specific appetites and regulates food choice to ensure homeostatic consumption of nutrients, providing insight into the hormonal mechanisms that underlie nutrient-specific hungers (Malita, 2022).

To maintain nutritional homeostasis, animals need to match their ingestion of specific nutrients to their needs. This is achieved by modulating appetite towards the specific nutrients needed. A number of factors, including gut hormones, that regulate food consumption have been identified in both flies and mammals, and reports have also described central brain mechanisms that induce ingestion of protein food in response to amino-acid deprivation, that sense amino acids and promote food consumption and that reject food lacking essential amino acids. However, little is known about the hormonal mechanisms that regulate nutrient-specific appetite, and gut hormones that regulate selective food intake are completely unknown. The current findings indicate that, in mated female Drosophila, gut-derived NPF is a selective driver of sugar satiety and protein consumption, providing a basis for understanding these mechanisms. Hormone-based therapies that inhibit appetite offer promising new directions for weight-loss treatment. For example, Fibroblast growth factor 21 (FGF21) is a liver-derived hormone that promotes protein consumption, and it is emerging as a promising target for metabolic disorders. Uncovering appetite-regulatory hormones such as gut-derived NPF that specifically inhibit sugar consumption while promoting the intake of protein-rich foods could provide effective new weight-management strategies by promoting healthier food choices (Malita, 2022).

The SLC2-family sugar transporter Sut2 is the closest Drosophila homologue of human SLC2A7 (GLUT7), a transporter expressed mainly in the intestine whose function is poorly defined. In flies, GLUT1 is important for Bursicon secretion from the EECs, and Sut1, another SLC2-family sugar transporter protein, was recently shown to be involved in midgut NPF release in virgin females. The current results implicate Sut2 in the release of NPF from EECs in mated females and thus link it to the mechanism by which NPF-mediated gut signalling controls feeding decisions. This indicates that both Sut1 and Sut2 sugar transporters are involved in glucose-stimulated NPF secretion from the gut. In mammals, several mechanisms also regulate glucose-stimulated GLP-1 secretion from intestinal endocrine cells, which involves sodium-glucose cotransporter 1 (SGLT1), the glucose transporter GLUT2 and sweet taste receptors. Targeting of these intestinal glucose-sensing mechanisms therefore has become a focus of weight-management therapies because of its potential in regulating appetite and incretin effects. Future studies should investigate whether GLUT7, like its Drosophila homologue Sut2, affects appetite-regulatory mechanisms in the mammalian gut (Malita, 2022).

NPF is orthologous with the mammalian NPY family of gut-brain peptides, including peptide YY (PYY), pancreatic polypeptide and NPY itself, that regulate food-seeking behaviours and metabolism. Like mammalian NPY-family hormones, Drosophila NPF is expressed in both the nervous system and the gut. While NPY is abundant in the nervous system and, like brain NPF, promotes food intake, PYY is mainly produced by endocrine cells of the gut as a satiety factor. Gut-expressed PYY is homologous to NPY, and both act through specific G-protein coupled receptors, called NPY receptors (NPYRs), that are orthologous with Drosophila NPFR. Thus, in mammals, multiple NPY-family peptides from different tissues sources exert their functions on target organs through several related NPYRs, while in Drosophila, these functions may be regulated through the single peptide-receptor pair of NPF and NPFR (Malita, 2022).

The results indicate that gut-derived Drosophila NPF fulfills the function of mammalian PYY. PYY is produced by the endocrine L-cells of the gut, which, like the EECs of Drosophila, produce a context-dependent combination of multiple hormones49. The physiological role of PYY in feeding regulation has been difficult to clarify, but it is believed to act through different NPYRs on tissues including the hypothalamus and the pancreatic islets to suppress appetite. These findings show that, in flies, NPF injection strongly reduces the intake of sugar-containing food and promotes the ingestion of protein-rich food. In humans, PYY infusion also been shown to strongly reduce food intake. Although the satiety function of human PYY has made it a prime therapeutic target for potential weight management, it is not clear whether PYY regulates nutrient-specific appetite, which would be important from a therapeutic perspective. These results indicate that Drosophila gut NPF, perhaps filling the role of mammalian gut PYY, acts to mediate sugar-specific satiety, illustrating a key hormonal mechanism that underlies selective hunger by which animals adjust their intake of specific nutrients (Malita, 2022).

Feeding decisions are based on internal state and exhibit sexual dimorphism. In Drosophila, males and females differ in their preference for and intake of dietary sugar and protein6. The current findings define a complex interorgan communication system through which mating influences food choices in females. Midgut NPF was found to be involved in mediating SP-induced postmating responses in females, inhibiting sugar appetite and promoting the ingestion of protein-rich yeast food, and it was further shown that AKH is required for mediating the effects of NPF. When mated females consume dietary carbohydrates, NPF is released from the EECs and inhibits the AKH axis by directly suppressing AKH release from the AKH-producing cells (APCs) as well as by inhibiting the release of midgut AstC, a factor that stimulates AKH secretion. Furthermore, NPF acts directly on the fat body through NPFR to inhibit energy mobilization, thereby antagonizing AKH-mediated signalling in the adipose tissue. Likewise, mammalian NPY-family peptides also regulate metabolism by direct actions on adipose tissue via NPYR. Although a number of studies have demonstrated that AKH is a regulator of metabolism, the current findings uncover a key role of AKH in governing nutrient-specific feeding decisions. It is becoming clear that the APCs integrate many signals that affect AKH release, and these signals may therefore also affect food choice. The APCs therefore seem to function as a signal-integration hub, similar to the IPCs, which receive many different inputs to control insulin production and release. AstC, Bursicon and NPF from the gut control AKH expression and secretion, indicating that multiple signals, even from the same organ, converge on the APCs. These signals presumably convey different aspects of nutritional status and may act with different dynamics to regulate AKH production and/or release, or even in a redundant manner to regulate AKH signalling. Likewise, many signals released from the fat body convey similar and seemingly redundant nutritional information to the IPCs (Malita, 2022).

Recent work has also revealed a sex-specific role of AKH, with lower activity in females underlying differences in male and female metabolism. Consistent with this notion, the current results indicate that in mated females the midgut NPF system inhibits AKH signalling, suppressing intake of sugar-rich food. Furthermore, it was recently shown that in mated females, midgut-derived AstC acts in a sex-specific manner through AKH to coordinate metabolism and food intake under nutritional stress. The current work shows that NPF also works sex-specifically to sustain physiological requirements in mated females by signalling from the gut to control AKH, suggesting that the gut-AKH axis occupies a central link in the hormonal relays underlying sex-specific regulation of physiology. A recent report showed that female germline cells modulate sugar appetite, but this effect is not induced by mating and does not affect yeast feeding as this study has found for gut NPF and AKH, suggesting that it is an independent mechanism (Malita, 2022).

How nutrient signals from the gut modulate feeding is key to understanding how nutritional needs are translated into specific feeding actions to maintain balance. This study has identified a homeostatic circuit triggered by gut-derived NPF that limits sugar consumption. Similar mechanisms for sugar-induced satiety that promote protein consumption may also enable mammals to balance their intake of different nutrients with their metabolic needs. Explaining how nutrient-responsive gut hormones such as NPF affect dietary choice is important to better understand hunger and cravings for specific nutrients that may ultimately lead to obesity (Malita, 2022).

Visceral organ morphogenesis via calcium-patterned muscle constrictions

Organ architecture is often composed of multiple laminar tissues arranged in concentric layers. During morphogenesis, the initial geometry of visceral organs undergoes a sequence of folding, adopting a complex shape that is vital for function. Genetic signals are known to impact form, yet the dynamic and mechanical interplay of tissue layers giving rise to organs' complex shapes remains elusive. This study traced the dynamics and mechanical interactions of a developing visceral organ across tissue layers, from sub-cellular to organ scale in vivo. Combining deep tissue light-sheet microscopy for in toto live visualization with a novel computational framework for multilayer analysis of evolving complex shapes, this study found a dynamic mechanism for organ folding using the embryonic midgut of Drosophila as a model visceral organ. Hox genes, known regulators of organ shape, control the emergence of high-frequency calcium pulses. Spatiotemporally patterned calcium pulses trigger muscle contractions via myosin light chain kinase. Muscle contractions, in turn, induce cell shape change in the adjacent tissue layer. This cell shape change collectively drives a convergent extension pattern. Through tissue incompressibility and initial organ geometry, this in-plane shape change is linked to out-of-plane organ folding. This analysis follows tissue dynamics during organ shape change in vivo, tracing organ-scale folding to a high-frequency molecular mechanism. These findings offer a mechanical route for gene expression to induce organ shape change: genetic patterning in one layer triggers a physical process in the adjacent layer - revealing post-translational mechanisms that govern shape change (Mitchell, 2022).

Longitudinal visceral muscles in Drosophila fully dedifferentiate and fragment prior to their reestablishment during metamorphosis

Although the Drosophila longitudinal visceral muscles have been shown to undergo major morphological changes during the transition from larval to adult gut musculature, there have been conflicting views as to whether these muscles persist as such during metamorphosis or whether they are built anew. This study presents an independent analysis using HLH54Fb-eGFP as a cell type specific marker, which strengthens the proposition that the syncytial larval longitudinal gut muscles completely dedifferentiate and fragment into mononucleated myoblasts during pupariation before they fuse again and redifferentiate to form the adult longitudinal gut muscles (Schultheis, 2023).

Independently paced calcium oscillations in progenitor and differentiated cells in an ex vivo epithelial organ

Cytosolic calcium is a highly dynamic, tightly regulated, and broadly conserved cellular signal. Calcium dynamics have been studied widely in cellular monocultures, yet organs in vivo comprise heterogeneous populations of stem and differentiated cells. This study examined calcium dynamics in the adult Drosophila intestine, a self-renewing epithelial organ in which stem cells continuously produce daughters that differentiate into either enteroendocrine cells or enterocytes. Live imaging of whole organs ex vivo reveals that stem cell daughters adopt strikingly distinct patterns of calcium oscillations after differentiation: Enteroendocrine cells exhibit single-cell calcium oscillations, while enterocytes exhibit rhythmic, long-range calcium waves. These multicellular waves do not propagate through immature progenitors (stem cells and enteroblasts), whose oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates calcium oscillations in all cell types--even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. These findings establish that cells adopt fate-specific modes of calcium dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently (Kim, 2022).

Using tissue specific P450 expression in Drosophila melanogaster larvae to understand the spatial distribution of pesticide metabolism in feeding assays

Drug metabolizing enzymes such as cytochrome P450s have often been implicated in influencing levels of pesticide toxicology and resistance. Consequently, a variety of different P450 genes and variants have been linked to pesticide metabolism. Substantially less is known in regards to which tissues these P450s contribute to pesticide metabolism. This study isolated the effect of different tissues in pesticide toxicology by driving the model P450 Cyp6g1 in specific tissues of Drosophila melanogaster. Fluorescent and luminescent assays were used to compare the strength of GAL4 lines specific to the midgut (Mex-GAL4), Malpighian tubules (UO-GAL4) and the fat body (LSP2-GAL4) with the widely used HR-GAL4 line which drives GAL4 expression in all three tissues simultaneously. These data suggested that GAL4 drivers specific for the midgut and fat body were of approximately equal strength to the HR-GAL4 line, while the Malpighian tubule specific line was significantly weaker. Multiple toxicology assays using the pesticides bendiocarb, imidacloprid and malathion were then performed to assess which tissues provide the most chemoprotection. In the long-term feeding assay, transgenic expression of Cyp6g1 specifically in the midgut accounted for the majority of the resistance caused by Cyp6g1 overexpression with the HR-GAL4 driver. Real-time toxicology assays on third instar larvae were also performed and showed variable contributions of tissues to acute toxicology response depending on which pesticide was used. These data suggest a strong influence of bioassay parameters such as life stage and dosing method on outcome but suggest a prominent role for the midgut in larval toxicology (Luong, 2022).

Phenotypic analyses, protein localization, and bacteriostatic activity of Drosophila melanogaster transferrin-1

Transferrin-1 (Tsf1) is an extracellular insect protein with a high affinity for iron. The functions of Tsf1 are still poorly understood; however, Drosophila melanogaster Tsf1 has been shown to influence iron distribution in the fly body and to protect flies against some infections. The goal of this study was to better understand the physiological functions of Tsf1 in D. melanogaster by 1) investigating Tsf1 null phenotypes, 2) determining tissue-specific localization of Tsf1, 3) measuring the concentration of Tsf1 in hemolymph, 4) testing Tsf1 for bacteriostatic activity, and 5) evaluating the effect of metal and paraquat treatments on Tsf1 abundance. Flies lacking Tsf1 had more iron than wild-type flies in specialized midgut cells that take up iron from the diet; however, the absence of Tsf1 had no effect on the iron content of whole midguts, fat body, hemolymph, or heads. Thus, as previous studies have suggested, Tsf1 appears to have a minor role in iron transport. Tsf1 was abundant in hemolymph from larvae (0.4 μM), pupae (1.4 μM), adult females (4.4 μM) and adult males (22 μM). Apo-Tsf1 at 1 μM had bacteriostatic activity whereas holo-Tsf1 did not, suggesting that Tsf1 can inhibit microbial growth by sequestering iron in hemolymph and other extracellular environments. This hypothesis was supported by detection of secreted Tsf1 in tracheae, testes and seminal vesicles. Colocalization of Tsf1 with an endosome marker in oocytes suggested that Tsf1 may provide iron to developing eggs; however, eggs from mothers lacking Tsf1 had the same amount of iron as control eggs, and they hatched at a wild-type rate. Thus, the primary function of Tsf1 uptake by oocytes may be to defend against infection rather than to provide eggs with iron. In beetles, Tsf1 plays a role in protection against oxidative stress. In contrast, this study found that flies lacking Tsf1 had a typical life span and greater resistance to paraquat-induced oxidative stress. In addition, Tsf1 abundance remained unchanged in response to ingestion of iron, cadmium or paraquat or to injection of iron. These results suggest that Tsf1 has a limited role in protection against oxidative stress in D. melanogaster (Weber, 2022).

Integrin-ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium

Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. Diedel, a secreted protein/cytokine, has been identified as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. This study shows that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, a resilience mechanism was uncovered of epithelial tissues, mediated by Integrin-ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, It was found that resilient epithelial cells, enriched for Diedel-Integrin-ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin-ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin-ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues (Mlih, 2022).

d-Chiro-Inositol extends the lifespan of male Drosophila melanogaster better than d-Pinitol through insulin signaling and autophagy pathways

d-Pinitol (DP) is the methylated product of d-Chiro-Inositol (DCI), which is one of the nine isomers of inositol with optical activity. Both substances possess antioxidant activity. This study was conducted to investigate and compare the antioxidant and life-prolonging effects of DCI and DP on male Drosophila melanogaster. Results showed that DCI and DP prolonged the lifespan and improved the climbing, anti-stress, and antioxidant activities. After treatment with DCI and DP, intestinal homeostasis was improved and the abnormal proliferation of intestinal stem cells (ISCs) was attenuated. Furthermore, real-time PCR revealed downregulated expression levels of PI3K and Akt and upregulated expression levels of Dilp5 and FOXO, which consequently activated Atg1, Atg5, Atg8a, and Atg8b and increased the number of lysosomes. Altogether, DCI exerts a slightly better effect than DP based on various indicators. RNAi D. melanogaster lifespan and molecular docking results further suggested that DCI and DP could prolong longevity through insulin signaling (IIS) and autophagy pathways (Du, 2022).

Hedgehog-mediated gut-taste neuron axis controls sweet perception in Drosophila

Dietary composition affects food preference in animals. High sugar intake suppresses sweet sensation from insects to humans, but the molecular basis of this suppression is largely unknown. This study revealed that sugar intake in Drosophila induces the gut to express and secrete Hedgehog (Hh) into the circulation. The midgut secreted Hh localized to taste sensilla and suppresses sweet sensation, perception, and preference. It was further found that the midgut Hh inhibits Hh signalling in the sweet taste neurons of the labellum and antennae. Electrophysiology studies demonstrate that the midgut Hh signal also suppresses bitter taste and some odour responses, affecting overall food perception and preference. It was further shown that the level of sugar intake during a critical window early in life, sets the adult gut Hh expression and sugar perception. These results together reveal a bottom-up feedback mechanism involving a "gut-taste neuron axis" that regulates food sensation and preference (Zhao, 2023).

Piwi maintains homeostasis in the Drosophila adult intestine

PIWI genes are well known for their germline but not somatic functions. This study reports the function of the Drosophila piwi gene in the adult gut, where intestinal stem cells (ISCs) produce enteroendocrine cells and enteroblasts that generate enterocytes. piwi is expressed in ISCs and enteroblasts. Piwi deficiency reduced ISC number, compromised enteroblasts maintenance, and induced apoptosis in enterocytes, but did not affect ISC proliferation and its differentiation to enteroendocrine cells. In addition, deficiency of zygotic but not maternal piwi mildly de-silenced several retrotransposons in the adult gut. Importantly, either piwi mutations or piwi knockdown specifically in ISCs and enteroblasts shortened the Drosophila lifespan, indicating that intestinal piwi contributes to longevity. Finally, mRNA sequencing data implied that Piwi may achieve its intestinal function by regulating diverse molecular processes involved in metabolism and oxidation-reduction reaction (Tang, 2023).

The endogenous antioxidant ability of royal jelly in Drosophila is independent of Keap1/Nrf2 by activating oxidoreductase activity

Royal jelly (RJ) is a biologically active substance secreted by the hypopharyngeal and mandibular glands of worker honeybees. It is widely claimed that RJ reduces oxidative stress. However, the antioxidant activity of RJ has mostly been determined by in vitro chemical detection methods or by external administration drugs that cause oxidative stress. Whether RJ can clear the endogenous production of reactive oxygen species (ROS) in cells remains largely unknown. This study systematically investigated the antioxidant properties of RJ using several endogenous oxidative stress models of Drosophila. RJ was found to enhance sleep quality of aging Drosophila, which is decreased due to an increase of oxidative damage with age. RJ supplementation improved survival and suppressed ROS levels in gut cells of flies upon exposure to hydrogen peroxide or to the neurotoxic agent paraquat. Moreover, RJ supplementation moderated levels of ROS in endogenous gut cells and extended lifespan after exposure of flies to heat stress. Sleep deprivation leads to accumulation of ROS in the gut cells, and RJ attenuated the consequences of oxidative stress caused by sleep loss and prolonged lifespan. Mechanistically, RJ prevented cell oxidative damage caused by heat stress or sleep deprivation, with the antioxidant activity in vivo independent of Keap1/Nrf2 signaling. RJ supplementation activated oxidoreductase activity in the guts of flies, suggesting its ability to inhibit endogenous oxidative stress and maintain health, possibly in humans (Wen, 2023).

The Drosophila tumor necrosis factor receptor, Wengen, couples energy expenditure with gut immunity

It is well established that tumor necrosis factor (TNF) plays an instrumental role in orchestrating the metabolic disorders associated with late stages of cancers. However, it is not clear whether TNF/TNF receptor (TNFR) signaling controls energy homeostasis in healthy individuals. This study shows that the highly conserved Drosophila TNFR, Wengen (Wgn), is required in the enterocytes (ECs) of the adult gut to restrict lipid catabolism, suppress immune activity, and maintain tissue homeostasis. Wgn limits autophagy-dependent lipolysis by restricting cytoplasmic levels of the TNFR effector, TNFR-associated factor 3 (dTRAF3), while it suppresses immune processes through inhibition of the dTAK1/TAK1-Relish/NF-κB pathway in a dTRAF2-dependent manner. Knocking down dTRAF3 or overexpressing dTRAF2 is sufficient to suppress infection-induced lipid depletion and immune activation, respectively, showing that Wgn/TNFR functions as an intersection between metabolism and immunity allowing pathogen-induced metabolic reprogramming to fuel the energetically costly task of combatting an infection (Loudhaief, 2023).

Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster

Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. This report shows that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies, but lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Over-expression of wild type Atg1 in adult fly muscle or adipocytes reduces lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. These research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation (Bierlein, 2023).

Dietary compounds activate an insect gustatory receptor on enteroendocrine cells to elicit myosuppressin secretion

Sensing of midgut internal contents is important for ensuring appropriate hormonal response and digestion following the ingestion of dietary components. Studies in mammals have demonstrated that taste receptors (TRs), a subgroup of G protein-coupled receptors (GPCRs), are expressed in gut enteroendocrine cells (EECs) to sense dietary compounds and regulate the production and/or secretion of peptide hormones. Although progress has been made in identifying expression patterns of gustatory receptors (GRs) in gut EECs, it is currently unknown whether these receptors, which act as ligand-gated ion channels, serve similar functions as mammalian GPCR TRs to elicit hormone production and/or secretion. A Bombyx mori Gr, BmGr6, has been demonstrated to express in cells by oral sensory organs, midgut and nervous system; and to sense isoquercitrin and chlorogenic acid, which are non-nutritional secondary metabolites of host mulberry. This study shows that BmGr6 co-expresses with Bommo-myosuppressin (BMS) in midgut EECs, responds to dietary compounds and is involved in regulation of BMS secretion. The presence of dietary compounds in midgut lumen after food intake resulted in an increase of BMS secretions in hemolymph of both wild-type and BmGr9 knockout larvae, but BMS secretions in BmGr6 knockout larvae decreased relative to wild-type. In addition, loss of BmGr6 led to a significant decrease in weight gain, excrement, hemolymph carbohydrates levels and hemolymph lipid levels. Interestingly, although BMS is produced in both midgut EECs and brain neurosecretory cells (NSCs), BMS levels in tissue extracts suggested that the increase in hemolymph BMS during feeding conditions is primarily due to secretion from midgut EECs. These studies indicate that BmGr6 expressed in midgut EECs responds to the presence of dietary compounds in the lumen by eliciting BMS secretion in B. mori larvae (Mang, 2023).

Intestinal Immune Deficiency and Juvenile Hormone Signaling Mediate a Metabolic Trade-off in Adult Drosophila Females

A trade-off hypothesis pertains to the biased allocation of limited resources between two of the most important fitness traits, reproduction and survival to infection. This quid pro quo manifests itself within animals prioritizing their energetic needs according to genetic circuits balancing metabolism, germline activity and immune response. Key evidence supporting this hypothesis includes dipteran fecundity being compromised by systemic immunity, and female systemic immunity being compromised by mating. This study reveals a local trade-off taking place in the female Drosophila midgut upon immune challenge. Genetic manipulation of intestinal motility, permeability, regeneration and three key midgut immune pathways provides evidence of an antagonism between specific aspects of intestinal defense and fecundity. That is, juvenile hormone (JH)-controlled egg laying, lipid droplet utilization and insulin receptor expression are specifically compromised by the immune deficiency (Imd) and the dual oxidase (Duox) signaling in the midgut epithelium. Moreover, antimicrobial peptide (AMP) expression under the control of the Imd pathway is inhibited upon mating and JH signaling in the midgut. Local JH signaling is further implicated in midgut dysplasia, inducing stem cell-like clusters and gut permeability. Thus, midgut JH signaling compromises host defense to infection by reducing Imd-controlled AMP expression and by inducing dysplasia, while midgut signaling through the Imd and Duox pathways compromises JH-guided metabolism and fecundity (Shianiou, 2023).

Dietary Stimuli, Intestinal Bacteria and Peptide Hormones Regulate Female Drosophila Defecation Rate

Peptide hormones control Drosophila gut motility, but the intestinal stimuli and the gene networks coordinating this trait remain poorly defined. This study customized an assay to quantify female Drosophila defecation rate as a proxy of intestinal motility. Bacterial infection with the human opportunistic bacterial pathogen Pseudomonas aeruginosa (strain PA14) was found to increase defecation rate in wild-type female flies, and specific bacteria of the fly microbiota able to increase defecation rate were identified. In contrast, dietary stress, imposed by either water-only feeding or high ethanol consumption, decreased defecation rate and the expression of enteroendocrine-produced hormones in the fly midgut, such as Diuretic hormone 31 (Dh31). The decrease in defecation due to dietary stress was proportional to the impact of each stressor on fly survival. Furthermore, the Drosophila Genetic Reference Panel wild type strain collection was exploited, and strains displaying high and low defecation rates were identifed. The narrow-sense heritability of defecation rate was calculated to be 91%, indicating that the genetic variance observed using the current assay is mostly additive and polygenic in nature. Accordingly, a genome-wide association (GWA) analysis was performed revealing 17 candidate genes linked to defecation rate. Downregulation of four of them (Pmp70, CG11307, meso18E and mub) in either the midgut enteroendocrine cells or in neurons reduced defecation rate and altered the midgut expression of Dh31, that in turn regulates defecation rate via signaling to the visceral muscle. Hence, microbial and dietary stimuli, and Dh31-controlling genes, regulate defecation rate involving signaling within and among neuronal, enteroendocrine, and visceral muscle cells (Kotronarou, 2023).

A systematic analysis of Drosophila regulatory peptide expression in enteroendocrine cells

The digestive system is gaining interest as a major regulator of various functions including immune defense, nutrient accumulation, and regulation of feeding behavior. Intestinal stem cells constantly divide and differentiate into enterocytes that secrete digestive enzymes and absorb nutrients, or enteroendocrine cells that secrete regulatory peptides. This study systemically examined the expression of 45 regulatory peptide genes in the Drosophila midgut, and verified that at least 10 genes are expressed in the midgut enteroendocrine cells through RT-PCR, in situ hybridization, antisera, and 25 regulatory peptide-GAL transgenes. The Drosophila midgut is highly compartmentalized, and individual peptides in enteroendocrine cells were observed to express in specific regions of the midgut. It was also confirmed that some peptides expressed in the same region of the midgut are expressed in mutually exclusive enteroendocrine cells. These results indicate that the midgut enteroendocrine cells are functionally differentiated into different subgroups. Through this study, a basis has been established to study regulatory peptide functions in enteroendocrine cells as well as the complex organization of enteroendocrine cells in the Drosophila midgut (Chen, 2016b).

Spatiotemporal organization of enteroendocrine peptide expression in Drosophila

The digestion of food and absorption of nutrients occurs in the gut. The nutritional value of food and its nutrients is detected by enteroendocrine cells, and peptide hormones produced by the enteroendocrine cells are thought to be involved in metabolic homeostasis, but the specific mechanisms are still elusive. The enteroendocrine cells are scattered over the entire gastrointestinal tract and can be classified according to the hormones they produce. This study followed the changes in combinatorial expression of regulatory peptides in the enteroendocrine cells during metamorphosis from the larva to the adult fruit fly, and re-confirmed the diverse composition of enteroendocrine cell populations. Drosophila enteroendocrine cells appear to differentially regulate peptide expression spatially and temporally depending on midgut region and developmental stage. In the late pupa, Notch activity is known to determine which peptides are expressed in mature enteroendocrine cells of the posterior midgut; it was found that the loss of Notch activity in the anterior midgut results in classes of enteroendocrine cells distinct from the posterior midgut. These results suggest that enteroendocrine cells that populate the fly midgut can differentiate into distinct subtypes that express different combinations of peptides, which likely leads to functional variety depending on specific needs (Jang, 2021).

Expression of Hey marks a subset of enteroendocrine cells in the Drosophila embryonic and larval midgut

Hey is a conserved transcription factor of the bHLH-Orange family and it participates in the response to Notch signaling in certain tissues. Whereas three Hey paralogues exist in mammalian genomes, Drosophila possesses a single Hey gene. Fly Hey is expressed in the subset of newborn neurons that receive a Notch signal to differentiate them from their sibling cells after the asymmetric division of precursors called ganglion-mother-cells. A polyclonal anti-Hey antiserum and a GFP-tagged transgenic duplication of the Hey locus to examine its expression in tissues outside the nervous system were used in embryos and larvae. Robust Hey expression was detected in the embryonic midgut primordium at the time of birth of enteroendocrine cells, identified by expression of Prospero. About half of the Pros-positive cells were also Hey positive at mid-embryogenesis. By the end of embryogenesis, most enteroendocrine cells had downregulated Hey expression, although it was still detectable at low levels after hatching. Low levels of Hey were also detected in subsets of the epithelial enterocytes at different times. Embryo enteroendocrine Hey expression was found to be Notch dependent. In late third-instar larvae, when few new enteroendocrine cells are born, novel Hey expression was detected in one cell of each sibling pair. In conclusion, Hey is strongly expressed in one of each pair of newly-born enteroendocrine cells. This is consistent with a hypothesis that embryonic enteroendocrine cells are born by an asymmetric division of a precursor, where Notch/Hey probably distinguish between the subtypes of these cells upon their differentiation (Skafida, 2021).

A hierarchical transcription factor cascade regulates enteroendocrine cell diversity and plasticity in Drosophila

Enteroendocrine cells (EEs) represent a heterogeneous cell population in intestine and exert endocrine functions by secreting a diverse array of neuropeptides. Although many transcription factors (TFs) required for specification of EEs have been identified in both mammals and Drosophila, it is not understood how these TFs work together to generate this considerable subtype diversity. This study shows that EE diversity in adult Drosophila is generated via an 'additive hierarchical TF cascade'. Specifically, a combination of a master TF, a secondary-level TF and a tertiary-level TF constitute a "TF code" for generating EE diversity. A high degree of post-specification plasticity of EEs was found, as changes in the code-including as few as one distinct TF-allow efficient switching of subtype identities. This study thus reveals a hierarchically-organized TF code that underlies EE diversity and plasticity in Drosophila, which can guide investigations of EEs in mammals and inform their application in medicine (Guo, 2022).

A role for the Drosophila zinc transporter Zip88E in protecting against dietary zinc toxicity

Zinc absorption in animals is thought to be regulated in a local, cell autonomous manner with intestinal cells responding to dietary zinc content. The Drosophila zinc transporter Zip88E shows strong sequence similarity to Zips 42C.1, 42C.2 and 89B as well as mammalian Zips 1, 2 and 3, suggesting that it may act in concert with the apically-localised Drosophila zinc uptake transporters to facilitate dietary zinc absorption by importing ions into the midgut enterocytes. However, the functional characterisation of Zip88E presented in this study indicates that Zip88E may instead play a role in detecting and responding to zinc toxicity. Larvae homozygous for a null Zip88E allele are viable yet display heightened sensitivity to elevated levels of dietary zinc. This decreased zinc tolerance is accompanied by an overall decrease in Metallothionein B transcription throughout the larval midgut. A Zip88E reporter gene is expressed only in the salivary glands, a handful of enteroendocrine cells at the boundary between the anterior and middle midgut regions, and in two parallel strips of sensory cell projections connecting to the larval ventral ganglion. Zip88E expression solely in this restricted subset of cells is sufficient to rescue the Zip88E mutant phenotype. Together, these data suggest that Zip88E may be functioning in a small subset of cells to detect excessive zinc levels and induce a systemic response to reduce dietary zinc absorption and hence protect against toxicity (Richards, 2017).

Pleiotropic and novel phenotypes in the Drosophila gut caused by mutation of drop-dead

<{>Normal gut function is vital for animal survival. In Drosophila, mutation of the gene drop-dead (drd) results in defective gut function, as measured by enlargement of the crop and reduced food movement through the gut, and drd mutation also causes the unrelated phenotypes of neurodegeneration, early adult lethality and female sterility. This work shows that adult drd mutant flies lack the peritrophic matrix (PM), an extracellular barrier that lines the lumen of the midgut and is found in many insects including flies, mosquitos and termites. The use of a drd-gal4 construct to drive a GFP reporter in late pupae and adults revealed drd expression in the anterior cardia, which is the site of PM synthesis in Drosophila. Moreover, the ability of drd knockdown or rescue with several gal4 drivers to recapitulate or rescue the gut phenotypes (lack of a PM, reduced defecation, and reduced adult survival 10-40 days post-eclosion) was correlated to the level of expression of each driver in the anterior cardia. Surprisingly, however, knocking down drd expression only in adult flies, which has previously been shown not to affect survival, eliminated the PM without reducing defecation rate. These results demonstrate that drd mutant flies have a novel phenotype, the absence of a PM, which is functionally separable from the previously described gut dysfunction observed in these flies. As the first mutant Drosophila strain reported to lack a PM, drd mutants will be a useful tool for studying the synthesis of this structure (Conway, 2018).

The cis-regulatory dynamics of embryonic development at single-cell resolution

This study investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), chromatin accessibility was profiled in over 20,000 single nuclei from fixed Drosophila embryos spanning three embryonic stages: stage 5 blastoderm nuclei; 6-8 h after egg laying, to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12 h after egg laying, when cells are undergoing terminal differentiation. The results show that there is spatial heterogeneity in the accessibility of the regulatory genome before gastrulation, a feature that aligns with future cell fate, and that nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility while maintaining a signature of their germ layer of origin. Analysis of the data reveals overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program that is reminiscent of the mesendoderm lineage in other species. 30,075 distal regulatory elements were identified that exhibit tissue-specific accessibility. The germ-layer specificity of a subset of these predicted enhancers was validated in transgenic embryos, achieving an accuracy of 90%. Overall, these results demonstrate the power of shotgun single-cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types (Cusanovich, 2018).

Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster

At low temperatures, Drosophila, like most insects, lose the ability to regulate ion and water balance across the gut epithelia, which can lead to a lethal increase of [K(+)] in the hemolymph (hyperkalemia). Cold-acclimation, the physiological response to a prior low temperature exposure, can mitigate or entirely prevent these ion imbalances, but the physiological mechanisms that facilitate this process are not well understood. This study tested whether plasticity in the ionoregulatory physiology of the gut and Malpighian tubules of Drosophila may aid in preserving ion homeostasis in the cold. Upon adult emergence, D. melanogaster females were subjected to seven days at warm (25 ° C) or cold (10 ° C) acclimation conditions. The cold acclimated flies had a lower critical thermal minimum (CTmin), recovered from chill coma more quickly, and better maintained hemolymph K(+) balance in the cold. The improvements in chill tolerance coincided with increased Malpighian tubule fluid secretion and better maintenance of K(+) secretion rates in the cold, as well as reduced rectal K(+) reabsorption in cold-acclimated flies. To test whether modulation of ion-motive ATPases, the main drivers of epithelial transport in the alimentary canal, mediate these changes, the activities were measured of Na(+)-K(+)-ATPase and V-type H(+)-ATPase at the Malpighian tubules, midgut, and hindgut. Na(+)/K(+)-ATPase and V-type H(+)-ATPase activities were lower in the midgut and the Malpighian tubules of cold-acclimated flies, but unchanged in the hindgut of cold acclimated flies, and were not predictive of the observed alterations in K(+) transport. These results suggest that modification of Malpighian tubule and gut ion and water transport likely prevents cold-induced hyperkalemia in cold-acclimated flies and that this process is not directly related to the activities of the main drivers of ion transport in these organs, Na(+)/K(+)- and V-type H(+)-ATPases (Yerushalmi, 2018).

Yeasts affect tolerance of Drosophila melanogaster to food substrate with high NaCl concentration

The ability of model animal species, such as Drosophila melanogaster, to adapt quickly to various adverse conditions has been shown in many experimental evolution studies. It is usually assumed by default that such adaptation is due to changes in the gene pool of the studied population of macroorganisms. At the same time, it is known that microbiome can influence biological processes in macroorganisms. In order to assess the possible impact of microbiome on adaptation, an evolutionary experiment was performed in which some D. melanogaster lines were reared on a food substrate with high NaCl concentration while the others were reared on the standard (favourable) substrate. The reproductive efficiency of experimental lines was evaluated on the high salt substrate three years after the experiment started. These tests confirmed that the lines reared on the salty substrate became more tolerant to high NaCl concentration. Moreover, pre-inoculation of the high salt medium with homogenized salt-tolerant flies tended to improve reproductive efficiency of naive flies on this medium (compared to pre-inoculation with homogenized control flies). The analysis of yeast microbiome in fly homogenates revealed significant differences in number and species richness of yeasts between salt-tolerant and control lines. It was also found that some individual yeast lines extracted from the salt-tolerant flies improved reproductive efficiency of naive flies on salty substrate (compared to baker's yeast and no yeast controls), whereas the effect of the yeast lines extracted from the control flies tended to be smaller. The yeast Starmerella bacillaris extracted from the salt-tolerant flies showed the strongest positive effect. This yeast is abundant in all salt-tolerant lines, and very rare or absent in all control lines. The results are consistent with the hypothesis that some components of the yeast microbiome of D. melanogaster contribute to to flies' tolerance to food substrate with high NaCl concentration (Dmitrieva, 2019).

Drosophila-associated bacteria differentially shape the nutritional requirements of their host during juvenile growth

The interplay between nutrition and the microbial communities colonizing the gastrointestinal tract (i.e., gut microbiota) determines juvenile growth trajectory. Nutritional deficiencies trigger developmental delays, and an immature gut microbiota is a hallmark of pathologies related to childhood undernutrition. However, how host-associated bacteria modulate the impact of nutrition on juvenile growth remains elusive. Using gnotobiotic Drosophila melanogaster larvae independently associated with Acetobacter pomorumWJL (ApWJL) and Lactobacillus plantarumNC8 (LpNC8), 2 model Drosophila-associated bacteria, a large-scale, systematic nutritional screen was performed based on larval growth in 40 different and precisely controlled nutritional environments. These results were combined with genome-based metabolic network reconstruction to define the biosynthetic capacities of Drosophila germ-free (GF) larvae and its 2 bacterial partners. It was first established that ApWJL and LpNC8 differentially fulfill the nutritional requirements of the ex-GF larvae, and such difference was parsed down to individual amino acids, vitamins, other micronutrients, and trace metals. Drosophila-associated bacteria not only fortify the host's diet with essential nutrients but, in specific instances, functionally compensate for host auxotrophies by either providing a metabolic intermediate or nutrient derivative to the host or by uptaking, concentrating, and delivering contaminant traces of micronutrients. This systematic work reveals that beyond the molecular dialogue engaged between the host and its bacterial partners, Drosophila and its associated bacteria establish an integrated nutritional network relying on nutrient provision and utilization (Consuegra, 2020).

Functional traits of the gut microbiome correlated with host lipid content in a natural population of Drosophila melanogaster

Most research on the nutritional significance of the gut microbiome is conducted on laboratory animals, and its relevance to wild animals is largely unknown. This study investigated the microbiome correlates of lipid content in individual wild fruit flies, Drosophila melanogaster. Lipid content varied 3.6-fold among the flies and was significantly correlated with the abundance of gut-derived bacterial DNA sequences that were assigned to genes contributing to 16 KEGG pathways. These included genes encoding sugar transporters and enzymes in glycolysis/gluconeogenesis, potentially promoting sugar consumption by the gut microbiome and, thereby, a lean fly phenotype. Furthermore, the lipid content of wild flies was significantly lower than laboratory flies, indicating that, as for some mammalian models, certain laboratory protocols might be obesogenic for Drosophila. This study demonstrates the value of research on natural populations to identify candidate microbial genes that influence ecologically important host traits (Kang, 2020).

Gut Bacterial Species Distinctively Impact Host Purine Metabolites during Aging in Drosophila

Gut microbiota impacts the host metabolome and affects its health span. How bacterial species in the gut influence age-dependent metabolic alteration has not been elucidated. This study shows in Drosophila melanogaster that allantoin, an end product of purine metabolism, is increased during aging in a microbiota-dependent manner. Allantoin levels are low in young flies but are commonly elevated upon lifespan-shortening dietary manipulations such as high-purine, high-sugar, or high-yeast feeding. Removing Acetobacter persici in the Drosophila microbiome attenuated age-dependent allantoin increase. Mono-association with A. persici, but not with Lactobacillus plantarum, increased allantoin in aged flies. A. persici increased allantoin via activation of innate immune signaling IMD pathway in the renal tubules. On the other hand, analysis of bacteria-conditioned diets revealed that L. plantarum can decrease allantoin by reducing purines in the diet. These data together demonstrate species-specific regulations of host purine levels by the gut microbiome (Yamauchi, 2020).

Gut bacteria-derived peptidoglycan induces a metabolic syndrome-like phenotype via NF-kappaB-dependent insulin/PI3K signaling reduction in Drosophila renal system

Although microbiome-host interactions are usual at steady state, gut microbiota dysbiosis can unbalance the physiological and behavioral parameters of the host, mostly via yet not understood mechanisms. Using the Drosophila model, this study investigated the consequences of a gut chronic dysbiosis on the host physiology. The results show that adult flies chronically infected with the non-pathogenic Erwinia carotorova caotovora bacteria displayed organ degeneration resembling wasting-like phenotypes reminiscent of Metabolic Syndrome associated pathologies. Genetic manipulations demonstrate that a local reduction of insulin signaling consecutive to a peptidoglycan-dependent NF-κB (Relish) activation in the excretory system of the flies is responsible for several of the observed phenotypes. This work establishes a functional crosstalk between bacteria-derived peptidoglycan and the immune NF-κB cascade that contributes to the onset of metabolic disorders by reducing insulin signal transduction. Giving the high degree of evolutionary conservation of the mechanisms and pathways involved, this study is likely to provide a helpful model to elucidate the contribution of altered intestinal microbiota in triggering human chronic kidney diseases (Zugasti, 2020).

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, it was asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals (Jugder, 2021).

Interactions between the microbiome and mating influence the female's transcriptional profile in Drosophila melanogaster

Drosophila melanogaster females undergo a variety of post-mating changes that influence their activity, feeding behavior, metabolism, egg production and gene expression. These changes are induced either by mating itself or by sperm or seminal fluid proteins. In addition, studies have shown that axenic females-those lacking a microbiome-have altered fecundity compared to females with a microbiome, and that the microbiome of the female's mate can influence reproductive success. This study investigated fecundity and the post-mating transcript abundance profile of axenic or control females after mating with either axenic or control males. Interactions between the female's microbiome and her mating status were observed: transcripts of genes involved in reproduction and genes with neuronal functions were differentially abundant depending on the females' microbiome status, but only in mated females. In addition, immunity genes showed varied responses to either the microbiome, mating, or a combination of those two factors. It was further observed that the male's microbiome status influences the fecundity of both control and axenic females, while only influencing the transcriptional profile of axenic females. These results indicate that the microbiome plays a vital role in the post-mating switch of the female's transcriptome (Delbare, 2020).

Differential effects of commensal bacteria on progenitor cell adhesion, division symmetry and tumorigenesis in the Drosophila intestine

Microbial factors influence homeostatic and oncogenic growth in the intestinal epithelium. However, little is known about immediate effects of commensal bacteria on stem cell division programs. This study examined the effects of commensal Lactobacillus species on homeostatic and tumorigenic stem cell proliferation in the female Drosophila intestine. Lactobacillus brevis was identified as a potent stimulator of stem cell divisions. In a wild-type midgut, L. brevis activates growth regulatory pathways that drive stem cell divisions. In a Notch-deficient background, L. brevis-mediated proliferation causes rapid expansion of mutant progenitors, leading to accumulation of large, multi-layered tumors throughout the midgut. Mechanistically, this study showed that L. brevis disrupts expression and subcellular distribution of progenitor cell integrins, supporting symmetric divisions that expand intestinal stem cell populations. Collectively, these data emphasize the impact of commensal microbes on division and maintenance of the intestinal progenitor compartment (Ferguson, 2021).

The impact of the gut microbiome on memory and sleep in Drosophila

The gut microbiome has been proposed to influence diverse behavioral traits of animals, although the experimental evidence is limited and often contradictory. This study made use of the tractability of Drosophila melanogaster for both behavioral analyses and microbiome studies to test how elimination of microorganisms affects a number of behavioral traits. Relative to conventional flies (i.e., with unaltered microbiome), microbiologically-sterile (axenic) flies displayed a moderate reduction in memory performance in olfactory appetitive conditioning and courtship assays. The microbiological status of the flies had small or no effect on anxiety-like behavior (centrophobism) or circadian rhythmicity of locomotor activity, but axenic flies tended to sleep for longer and displayed reduced sleep rebound after sleep deprivation. The latter effects were robust for most tests conducted on both wildtype Canton S and w (1118) strains, as well for tests using an isogenized panel of flies with mutations in the period gene, which causes altered circadian rhythmicity. Interestingly, the effect of absence of microbiota on a few behavioral features, most notably instantaneous locomotor activity speed, varied among wild-type strains. Taken together, these findings demonstrate that the microbiome can have subtle but significant effects on specific aspects of Drosophila behavior, some of which are dependent on genetic background (Silva, 2020).

Systemic Regulation of Host Energy and Oogenesis by Microbiome-Derived Mitochondrial Coenzymes

Gut microbiota have been shown to promote oogenesis and fecundity, but the mechanistic basis of remote influence on oogenesis remained unknown. This study reports a systemic mechanism of influence mediated by bacterial-derived supply of mitochondrial coenzymes. Removal of microbiota decreased mitochondrial activity and ATP levels in the whole-body and ovary, resulting in repressed oogenesis. Similar repression was caused by RNA-based knockdown of mitochondrial function in ovarian follicle cells. Reduced mitochondrial function in germ-free (GF) females was reversed by bacterial recolonization or supplementation of riboflavin, a precursor of FAD and FMN. Metabolomics analysis of GF females revealed a decrease in oxidative phosphorylation and FAD levels and an increase in metabolites that are degraded by FAD-dependent enzymes (e.g., amino and fatty acids). Riboflavin supplementation opposed this effect, elevating mitochondrial function, ATP, and oogenesis. These findings uncover a bacterial-mitochondrial axis of influence, linking gut bacteria with systemic regulation of host energy and reproduction (Gnainsky, 2021).

Role of Commensal Microbes in the gamma-Ray Irradiation-Induced Physiological Changes in Drosophila melanogaster

Ionizing radiation induces biological/physiological changes and affects commensal microbes, but few studies have examined the relationship between the physiological changes induced by irradiation and commensal microbes. This study investigated the role of commensal microbes in the γ-ray irradiation-induced physiological changes in Drosophila melanogaster. The bacterial load was increased in 5 Gy irradiated flies, but irradiation decreased the number of operational taxonomic units. The mean lifespan of conventional flies showed no significant change by irradiation, whereas that of axenic flies was negatively correlated with the radiation dose. γ-Ray irradiation did not change the average number of eggs in both conventional and axenic flies. Locomotion of conventional flies was decreased after 5 Gy radiation exposure, whereas no significant change in locomotion activity was detected in axenic flies after irradiation. γ-Ray irradiation increased the generation of reactive oxygen species in both conventional and axenic flies, but the increase was higher in axenic flies. Similarly, the amounts of mitochondria were increased in irradiated axenic flies but not in conventional flies. These results suggest that axenic flies are more sensitive in their mitochondrial responses to radiation than conventional flies, and increased sensitivity leads to a reduced lifespan and other physiological changes in axenic flies (Lee, 2020).

Drice restrains Diap2-mediated inflammatory signalling and intestinal inflammation

The Drosophila IAP protein, Diap2, is a key mediator of NF-κB signalling and innate immune responses. Diap2 is required for both local immune activation, taking place in the epithelial cells of the gut and trachea, and for mounting systemic immune responses in the cells of the fat body. This study has found that transgenic expression of Diap2 leads to a spontaneous induction of NF-κB target genes, inducing chronic inflammation in the Drosophila midgut, but not in the fat body. Drice is a Drosophila effector caspase known to interact and form a stable complex with Diap2. This complex formation was found to induce its subsequent degradation, thereby regulating the amount of Diap2 driving NF-κB signalling in the intestine. Concordantly, loss of Drice activity leads to accumulation of Diap2 and to chronic intestinal inflammation. Interestingly, Drice does not interfere with pathogen-induced signalling, suggesting that it protects from immune responses induced by resident microbes. Accordingly, no inflammation was detected in transgenic Diap2 flies and Drice-mutant flies reared in axenic conditions. Hence, this study shows that Drice, by restraining Diap2, halts unwanted inflammatory signalling in the intestine (Kietz, 2021).

Social environment drives sex and age-specific variation in Drosophila melanogaster microbiome composition and predicted function

The composition of the microbiome (the assemblage of symbiotic microorganisms within a host) is determined by environmental factors and the host's immune, endocrine and neural systems. The social environment could alter host microbiomes extrinsically by affecting transmission between individuals. Alternatively, intrinsic effects arising from interactions between the microbiome and host physiology (the microbiota-gut-brain axis) could translate social stress into dysbiotic microbiomes, with consequences for host health. This study investigated how manipulating social environments during larval and adult life-stages altered the microbiome composition of Drosophila melanogaster fruit flies. Social contexts that particularly alter the development and lifespan of males were used, predicting that any intrinsic social effects on the microbiome would therefore be sex-specific. The presence of adult males during the larval stage significantly altered the microbiome of pupae of both sexes. In adults, same-sex grouping increased bacterial diversity in both sexes. Importantly, the microbiome community structure of males was more sensitive to social contact at older ages, an effect partially mitigated by housing focal males with young rather than coaged groups. Functional analyses suggest that these microbiome changes impact ageing and immune responses. This is consistent with the hypothesis that the substantial effects of the social environment on individual health are mediated through intrinsic effects on the microbiome, and provides a model for understanding the mechanistic basis of the microbiota-gut-brain axis (Leech, 2021).

Exposure to foreign gut microbiota can facilitate rapid dietary shifts

Dietary niche is fundamental for determining species ecology; thus, a detailed understanding of what drives variation in dietary niche is vital for predicting ecological shifts and could have implications for species management. Gut microbiota can be important for determining an organism's dietary preference, and therefore which food resources they are likely to exploit. Evidence for whether the composition of the gut microbiota is plastic in response to changes in diet is mixed. Also, the extent to which dietary preference can be changed following colonisation by new gut microbiota from different species is unknown. This study used Drosophila spp. to show that: (1) the composition of an individual's gut microbiota can change in response to dietary changes, and (2) ingestion of foreign gut microbes can cause individuals to be attracted to food types they previously had a strong aversion to. Thus, this study exposes a mechanism for facilitating rapid shifts in dietary niche over short evolutionary timescales (Heys, 2021).

Arc1 and the microbiota together modulate growth and metabolic traits in Drosophila

Perturbations to animal-associated microbial communities (the microbiota) have deleterious effects on various aspects of host fitness, but the molecular processes underlying these impacts are poorly understood. This study identified a connection between the microbiota and the neuronal factor Arc1 that affects growth and metabolism in Drosophila. Arc1 was found to exhibit tissue-specific microbiota-dependent expression changes, and germ-free flies bearing a null mutation of Arc1 exhibited delayed and stunted larval growth, along with a variety of molecular, cellular and organismal traits indicative of metabolic dysregulation. Remarkably, this study showed that the majority of these phenotypes can be fully suppressed by mono-association with a single Acetobacter sp. isolate, through mechanisms involving both bacterial diet modification and live bacteria. Additionally, evidence is provided that Arc1 functions in key neuroendocrine cells of the larval brain modulates growth and metabolic homeostasis under germ-free conditions. These results reveal a role for Arc1 in modulating physiological responses to the microbial environment, and highlight how host-microbe interactions can profoundly impact the phenotypic consequences of genetic mutations in an animal host (Keith, 2021).

Modeling Drosophila gut microbe interactions reveals metabolic interconnectivity

A lot is known about varying gut microbiome compositions. Yet, how the bacteria affect each other remains elusive. In mammals, this is largely based on the sheer complexity of the microbiome with at least hundreds of different species. Thus, model organisms such as Drosophila melanogaster are commonly used to investigate mechanistic questions as the microbiome consists of only about 10 leading bacterial species. This study isolated gut bacteria from laboratory-reared Drosophila, sequenced their respective genomes, and used this information to reconstruct genome-scale metabolic models. With these, growth was similated in mono- and co-culture conditions and different media including a synthetic diet designed to grow Drosophila melanogaster. These simulations reveal a synergistic growth of some but not all gut microbiome members, which stems on the exchange of distinct metabolites including tricarboxylic acid cycle intermediates. Culturing experiments confirmed these predictions. This study thus demonstrates the possibility to predict microbiome-derived growth-promoting cross-feeding (Schonborn, 2021).

TOR signaling is required for host lipid metabolic remodelling and survival following enteric infection in Drosophila

When infected by enteric pathogenic bacteria, animals need to initiate local and whole-body defence strategies. While most attention has focused on the role innate immune anti-bacterial responses, less is known about how changes in host metabolism contribute to host defence. Using Drosophila as a model system, this study identified induction of intestinal target-of-rapamycin (TOR) kinase signaling as a key adaptive metabolic response to enteric infection. Enteric infection induces both local and systemic induction of TOR independently of the IMD innate immune pathway, and TOR functions together with IMD signaling to promote infection survival. These protective effects of TOR signaling are associated with re-modelling of host lipid metabolism. Thus, TOR is required to limit excessive infection-mediated wasting of host lipid stores by promoting an increase in the levels of gut- and fat body-expressed lipid synthesis genes. These data supports a model in which induction of TOR represents a host tolerance response to counteract infection-mediated lipid wasting in order to promote survival (Deshpande, 2022).

Parasite reliance on its host gut microbiota for nutrition and survival

Studying the microbial symbionts of eukaryotic hosts has revealed a range of interactions that benefit host biology. Most eukaryotes are also infected by parasites that adversely affect host biology for their own benefit. However, it is largely unclear whether the ability of parasites to develop in hosts also depends on host-associated symbionts, e.g., the gut microbiota. The effects were studied of parasitic wasp Leptopilina boulardi (Lb) and its host Drosophila melanogaster. Results showed that Lb successfully develops in conventional hosts (CN) with a gut microbiota but fails to develop in axenic hosts (AX) without a gut microbiota. Developing Lb larvae consume fat body cells that store lipids. It was also determined that much larger amounts of lipid accumulate in fat body cells of parasitized CN hosts than parasitized AX hosts. CN hosts parasitized by Lb exhibited large increases in the abundance of the bacterium Acetobacter pomorum in the gut, but did not affect the abundance of Lactobacillus fructivorans which is another common member of the host gut microbiota. However, AX hosts inoculated with A. pomorum and/or L. fructivorans did not rescue development of Lb. In contrast, AX larvae inoculated with A. pomorum plus other identified gut community members including a Bacillus sp. substantially rescued Lb development. Rescue was further associated with increased lipid accumulation in host fat body cells. Insulin-like peptides increased in brain neurosecretory cells of parasitized CN larvae. Lipid accumulation in the fat body of CN hosts was further associated with reduced Bmm lipase activity mediated by insulin/insulin-like growth factor signaling (IIS). Altogether, these results identify a previously unknown role for the gut microbiota in defining host permissiveness for a parasite. These findings also identify a new paradigm for parasite manipulation of host metabolism that depends on insulin signaling and the gut microbiota (Zhou, 2022).

Dietary Utilization Drives the Differentiation of Gut Bacterial Communities between Specialist and Generalist Drosophilid Flies

Gut bacteria play vital roles in the dietary detoxification, digestion, and nutrient supplementation of hosts during dietary specialization. The roles of gut bacteria in the host can be unveiled by comparing communities of specialist and generalist bacterial species. However, these species usually have a long evolutionary history, making it difficult to determine whether bacterial community differentiation is due to host dietary adaptation or phylogenetic divergence. In this regard, this study investigated the bacterial communities from two Araceae-feeding Colocasiomyia species and further performed a meta-analysis by incorporating the published data from Drosophila bacterial community studies. The compositional and functional differentiation of bacterial communities was uncovered by comparing three (Araceae-feeding, mycophagous, and cactophilic) specialists with generalist flies. The compositional differentiation showed that Bacteroidetes and Firmicutes inhabited specialists, while more Proteobacteria lived in generalists. The functional prediction based on the bacterial community compositions suggested that amino acid metabolism and energy metabolism are overrepresented pathways in specialists and generalists, respectively. The differences were mainly associated with the higher utilization of structural complex carbohydrates, protein utilization, vitamin B(12) acquisition, and demand for detoxification in specialists than in generalists. The complementary roles of bacteria reveal a connection between gut bacterial communities and fly dietary specialization (Chen, 2022).

Microbiota aggravates the pathogenesis of Drosophila acutely exposed to vehicle exhaust

Vehicle exhaust (VE) is the primary cause of urban air pollution, which adversely affects the respiratory system, exacerbates lung diseases, and results in high mortality rates. However, the underlying mechanism of the pathogenesis is largely unclear. This study developed a Drosophila model to systematically investigate the effects of VE on their health and physiology. VE was found to significantly impaired life span and locomotion in Drosophila. Interestingly, there was an increase in bacterial load in the guts upon VE exposure, suggesting VE is able to induce dysbiosis in the guts. Microbiota depletion can ameliorate the impairment of life span and locomotion. VE causes permeability of intestinal epithelial cells and increases proliferation of intestinal cells, suggesting VE disrupts intestinal homeostasis. This study elucidated the underlying mechanism by which VE triggers Imd and DUOX gene expression. Taken together, this Drosophila model provides insight into the pathogenesis of Drosophila exposure to VE, enabling a better understanding of the specific role of microbiota (Li 2022).

An integrated host-microbiome response to atrazine exposure mediates toxicity in Drosophila

The gut microbiome produces vitamins, nutrients, and neurotransmitters, and helps to modulate the host immune system-and also plays a major role in the metabolism of many exogenous compounds, including drugs and chemical toxicants. However, the extent to which specific microbial species or communities modulate hazard upon exposure to chemicals remains largely opaque. Focusing on the effects of collateral dietary exposure to the widely used herbicide atrazine, integrated omics and phenotypic screening were applied to assess the role of the gut microbiome in modulating host resilience in Drosophila melanogaster. Sequencing the genomes of all abundant microbes in the fly gut revealed an enzymatic pathway responsible for atrazine detoxification unique to Acetobacter tropicalis. Acetobacter tropicalis alone, in gnotobiotic animals, wad found to be sufficient to rescue increased atrazine toxicity to wild-type, conventionally reared levels. This work points toward the derivation of biotic strategies to improve host resilience to environmental chemical exposures (Brown, 2021).

The Increased Abundance of Commensal Microbes Decreases Drosophila melanogaster Lifespan through an Age-Related Intestinal Barrier Dysfunction

Commensal microbiota live in their host with a symbiotic relationship that affects the host's health and physiology. Many studies showed that microbial load and composition were changed by aging and have observed that increasing the abundance and changing the composition of commensal microbes had detrimental effects on host lifespan. It was hypothesized that dysbiosis of the intestinal microbiota leads to systemic effects in aging flies as a result of the increased intestinal permeability. The fruit fly laboratory strain w(1118) was used as a model system. The incidence of intestinal dysfunction was increased with age, and intestinal dysfunction increased the permeability of the fly intestine to resident microbes. The lifespan of flies with an intestinal barrier dysfunction was increased by removal of the microbes. Interestingly, some bacteria were also found in the hemolymph of flies with intestinal barrier dysfunction. These findings suggest the possibility that, as the host ages, there is an increase in intestinal permeability, which leads to an increased intestinal microbial load and a reduction in the host lifespan. These data therefore indicate a connection between commensal microbes and host lifespan (Lee, 2022).

Microbiome-by-ethanol interactions impact Drosophila melanogaster fitness, physiology, and behavior

The gut microbiota can affect how animals respond to ingested toxins, such as ethanol, which is prevalent in the diets of diverse animals and often leads to negative health outcomes in humans. Ethanol is a complex dietary factor because it acts as a toxin, behavioral manipulator, and nutritional source, with both direct effects on the host as well as indirect ones through the microbiome. This study developed a model for chronic, non-intoxicating ethanol ingestion in the adult fruit fly, Drosophila melanogaster, and paired this with the tractability of the fly gut microbiota, which can be experimentally removed. Numerous physiological, behavioral, and transcriptional variables were linked to fly fitness, including a combination of intestinal barrier integrity, stored triglyceride levels, feeding behavior, and the immunodeficiency pathway. These results reveal a complex tradeoff between lifespan and fecundity that is microbiome-dependent and modulated by dietary ethanol and feeding behavior (Chandler, 2022).

Stochastic microbiome assembly depends on context

Observational studies reveal substantial variability in microbiome composition across individuals. Targeted studies in gnotobiotic animals underscore this variability by showing that some bacterial strains colonize deterministically, while others colonize stochastically. While some of this variability can be explained by external factors like environmental, dietary, and genetic differences between individuals, this paper shows that for the model organism Drosophila melanogaster, interactions between bacteria can affect the microbiome assembly process, contributing to a baseline level of microbiome variability even among isogenic organisms that are identically reared, housed, and fed. In germ-free flies fed known combinations of bacterial species, some species were found to colonize more frequently than others even when fed at the same high concentration. This study developed an ecological technique that infers the presence of interactions between bacterial species based on their colonization odds in different contexts, requiring only presence/absence data from two-species experiments. A progressive sequence of probabilistic models, in which the colonization of each bacterial species is treated as an independent stochastic process, was used to reproduce the empirical distributions of colonization outcomes across experiments. Incorporating context-dependent interactions substantially was found to improve the performance of the models. Stochastic, context-dependent microbiome assembly underlies clinical therapies like fecal microbiota transplantation and probiotic administration and should inform the design of synthetic fecal transplants and dosing regimes (Jones, 2022).

Enteric bacterial infection in Drosophila induces whole-body alterations in metabolic gene expression independently of the immune deficiency signaling pathway

When infected by intestinal pathogenic bacteria, animals initiate both local and systemic defence responses. These responses are required to reduce pathogen burden and also to alter host physiology and behavior to promote infection tolerance, and they are often mediated through alterations in host gene expression. This study used transcriptome profiling to examine gene expression changes induced by enteric infection with the Gram-negative bacteria Pseudomonas entomophila in adult female Drosophila. Infection was found to induce a strong upregulation of metabolic gene expression, including gut and fat body-enriched genes involved in lipid transport, lipolysis, and beta-oxidation, as well as glucose and amino acid metabolism genes. Furthermore, the classic innate immune deficiency (Imd)/Relish/NF-KappaB pathway were not required for, and in some cases limits, these infection-mediated increases in metabolic gene expression. Enteric infection with Pseudomonas entomophila downregulates the expression of many transcription factors and cell-cell signaling molecules, particularly those previously shown to be involved in gut-to-brain and neuronal signaling. Moreover, as with the metabolic genes, these changes occurred largely independent of the Imd pathway. Together, this study identifies many metabolic, signaling, and transcription factor gene expression changes that may contribute to organismal physiological and behavioral responses to enteric pathogen infection (Deshpande, 2022).

Age-associated loss of lamin-B leads to systemic inflammation and gut hyperplasia

Aging of immune organs, termed as immunosenescence, is suspected to promote systemic inflammation and age-associated disease. The cause of immunosenescence and how it promotes disease, however, has remained unclear. This study reports that the Drosophila fat body, a major immune organ, undergoes immunosenescence and mounts strong systemic inflammation that leads to deregulation of immune deficiency (IMD) signaling in the midgut of old animals. Inflamed old fat bodies secrete circulating peptidoglycan recognition proteins that repress IMD activity in the midgut, thereby promoting gut hyperplasia. Further, fat body immunosenecence is caused by age-associated lamin-B reduction specifically in fat body cells, which then contributes to heterochromatin loss and derepression of genes involved in immune responses. As lamin-associated heterochromatin domains are enriched for genes involved in immune response in both Drosophila and mammalian cells, these findings may provide insights into the cause and consequence of immunosenescence during mammalian aging (Chen, 2014).

By analyzing gene expression changes upon aging in fat bodies and midguts, it was shown that an increase of immune response in the fat body is accompanied by a striking reduction in the midgut. Specifically, it was demonstrate that the age-associated increase in Immune deficiency (IMD) signaling in fat bodies leads to reduction of IMD activity in the midgut, which in turn contributes to midgut hyperplasia. This fat body to midgut effect requires peptidoglycan recognition proteins (PGRPs) secreted from fat body cells and is mediated by both bacteria dependent and independent pathways. Therefore, fat body aging contributes to systemic inflammation, which contributes to the disruption of gut homeostasis. Importantly, it was shown that the age-associated lamin-B loss in fat body cells causes the derepression of a large number of immune responsive genes, thereby resulting in fat body-based systemic inflammation (Chen, 2014).

B-type lamins have long been suggested to have a role in maintaining heterochromatin and gene repression. Consistently, this study's global analyses of fat body depleted of lamin-B revealed a loss of heterochromatin and derepression of a large number of immune responsive genes. This is further supported by ChIP-qPCR analyses of H3K9me3 on specific IMD regulators. Recent studies in different cell types show that tethering genes to nuclear lamins do not always lead to their repression. Deleting B-type lamins or all lamins in mouse ES cells or trophectdoderm cells does not result in derepression of all genes in LADs. In light of these studies, it is suggested that the transcriptional repression function of lamin-B could be gene and cell type dependent. Interestingly, GO analyses revealed a significant enrichment of immune responsive genes in Lamin-associated domains (LADs) in four different mammalian cell types and Drosophila Kc cells. Since the large-scale pattern of LADs is conserved in different cell types in mammals, it is possible that the immune-responsive genes are also enriched in LADs in the fly fat body cells. Supporting this notion, the IKKγ, key, which is one of the two derepressed IMD regulators and was found to exhibit H3K9me3 reduction and gene activation, is localized to LADs in Kc cells. It is speculated that lamin-B might play an evolutionarily conserved role in repressing a subset of inflammatory genes in certain tissues, such as the immune organs, in the absence of infection or injury. Consistently, senescence-associated lamin-B1 loss in mammalian fibroblasts is correlated with senescence-associated secretory phenotype senescence-associated secretory phenotype (SASP). Although the in vivo relevance of fibroblast SASP in chronic inflammation and aging-associated diseases in mammals remains to be established, the findings in Drosophila provide insights and impetus to investigate the role of lamins in immunosenescence and systemic inflammation in mammals (Chen, 2014).

Lamin-B gradually decreases in fat body cells of aging flies, whereas lamin-C amount remains the same. Since it has been recently shown that the assembly of an even and dense nuclear lamina is dependent on the total lamin concentration, the age-associated appearance of lamin-B and lamin-C gaps around the nuclear periphery of fat body cells is likely caused by the drop of the lamin-B level. How aging triggers lamin-B loss is unknown, but it appears to be posttranscriptional, because lamin-B transcripts in fat bodies remain unchanged upon aging. Interestingly, among the tissues examined, no changes of lamin-B and lamin-C proteins were found in cells in the heart tube, oenocytes, or gut epithelia in old flies. Therefore, the age-associated lamin-B loss does not occur in all cell types in vivo. A systematic survey to establish the cell/tissue types that undergo age-associated reduction of lamins in both flies and mammals should provide clues to the cause of loss. Deciphering how advanced age leads to lamin loss should open the door to further investigate the cellular mechanism that contributes to chronic systemic inflammation and how it in turn promotes age-associated diseases in humans (Chen, 2014).

Old Drosophila gut is known to exhibit increased microbial load, which would cause increased stress response and activation of tissue repair, thereby leading to midgut hyperplasia. Systemic inflammation caused by lamin-B loss in fat body leads to repression of local midgut IMD signaling. The upregulation of targets of IMD in the aged whole gut has been recently reported, while a downregulation of target genes was observed in the current analyses of the midgut. However, the previous study found a similar upregulation of the genes when performing RNA-seq of the whole gut (Chen, 2014).

These studies reveal an involvement of bacteria in the repression of midgut IMD signaling by the PGRPs secreted from the fat body. How PGRPs from the fat body repress midgut IMD is still unknown. One possibility is that the body cavity bacteria contribute to the maintenance of midgut IMD activity, and the increased circulating PGRPs limit these bacteria. The circulating PGRPs may also reduce midgut IMD activity indirectly by affecting other tissues. The evidence suggests that lamin-B loss could also contribute to midgut hyperplasia independent of the IMD pathway. While it will be important to further address these possibilities, the findings have revealed a fat body mediated inflammatory pathway that can lead to reduced migut IMD, increased gut microbial accumulation, and midgut hyperplasia upon aging (Chen, 2014).

Interestingly, microbiota changes also occur in aging human intestine and have been linked to altered intestinal inflammatory states and diseases. Although, much effort has been devoted to understand how local changes in aging mammalian intestines affect gut microbial community, the cause remains unclear. The findings in Drosophila reveal the importance of understanding the impact of immunosenescence and systemic inflammation on gut microbial homeostasis. Indeed, if increased circulating inflammatory cytokines perturb the ability of local intestine epithelium and the gut-associated lymphoid tissue to maintain a balanced microbial community, the unfavorable microbiota in the old intestine would cause chronic stress response and tissue repair, thereby leading to uncontrolled cell growth as observed in age-associated cancers (Chen, 2014).

Genetic, molecular and physiological basis of variation in Drosophila gut immunocompetence

Gut immunocompetence involves immune, stress and regenerative processes. To investigate the determinants underlying inter-individual variation in gut immunocompetence, enteric infection was performed of 140 Drosophila lines with the entomopathogenic bacterium Pseudomonas entomophila, and extensive variation was observed in survival. Using genome-wide association analysis, several novel immune modulators were identified. Transcriptional profiling further shows that the intestinal molecular state differs between resistant and susceptible lines, already before infection, with one transcriptional module involving genes linked to reactive oxygen species (ROS) metabolism contributing to this difference. This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines. This study revealed how relatively minor, but systematic variation can mediate overt physiological differences that determine enteric infection susceptibility (Bou Sleiman, 2015).

Distinct shifts in microbiota composition during Drosophila aging impair intestinal function and drive mortality

Alterations in the composition of the intestinal microbiota have been correlated with aging and measures of frailty in the elderly. However, the relationships between microbial dynamics, age-related changes in intestinal physiology, and organismal health remain poorly understood. This study shows that dysbiosis of the intestinal microbiota, characterized by an expansion of the Gammaproteobacteria, is tightly linked to age-onset intestinal barrier dysfunction in Drosophila. Indeed, alterations in the microbiota precede and predict the onset of intestinal barrier dysfunction in aged flies. Changes in microbial composition occurring prior to intestinal barrier dysfunction contribute to changes in excretory function and immune gene activation in the aging intestine. In addition, a distinct shift in microbiota composition was shown to follow intestinal barrier dysfunction, leading to systemic immune activation and organismal death. These results indicate that alterations in microbiota dynamics could contribute to and also predict varying rates of health decline during aging in mammals (Clark, 2015).

Preventing age-related decline of gut compartmentalization limits microbiota dysbiosis and extends lifespan

Compartmentalization of the gastrointestinal (GI) tract of metazoans is critical for health. GI compartments contain specific microbiota, and microbiota dysbiosis is associated with intestinal dysfunction. Dysbiosis develops in aging intestines, yet how this relates to changes in GI compartmentalization remains unclear. The Drosophila GI tract is an accessible model to address this question. This study shows that the stomach-like copper cell region (CCR) in the middle midgut controls distribution and composition of the microbiota. It was found that chronic activation of JAK/Stat signaling in the aging gut induces a metaplasia of the gastric epithelium, CCR decline, and subsequent commensal dysbiosis and epithelial dysplasia along the GI tract. Accordingly, inhibition of JAK/Stat signaling in the CCR specifically prevents age-related metaplasia, commensal dysbiosis and functional decline in old guts, and extends lifespan. These results establish a mechanism by which age-related chronic inflammation causes the decline of intestinal compartmentalization and microbiota dysbiosis, limiting lifespan (Li, 2016).

Gut microbiota in Drosophila melanogaster interacts with Wolbachia but does not contribute to Wolbachia-mediated antiviral protection

Animals experience near constant infection with microorganisms. A significant proportion of these microbiota reside in the alimentary tract. There is a growing appreciation for the roles gut microbiota play in host biology. The gut microbiota of insects, for example, have been shown to help the host overcome pathogen infection either through direct competition or indirectly by stimulating host immunity. These defenses may also be supplemented by coinfecting maternally inherited microbes such as Wolbachia. The presence of Wolbachia in a host can delay and/or reduce death caused by RNA viruses. Whether the gut microbiota of the host interacts with Wolbachia, or vice versa, the precise role of Wolbachia in antiviral protection is not known. This study used 16S rDNA sequencing to characterize changes in gut microbiota composition in Drosophila melanogaster associated with Wolbachia infection and antibiotic treatment. Subsequently, it was tested whether changes in gut composition via antibiotic treatment alter Wolbachia-mediated antiviral properties. It was found that both antibiotics and Wolbachia significantly reduce the biodiversity of the gut microbiota without changing the total microbial load. Changing the gut microbiota composition with antibiotic treatment enhances Wolbachia density but does not confer greater antiviral protection against Drosophila C virus to the host. In conclusion, there are significant interactions between Wolbachia and gut microbiota, but changing gut microbiota composition is not likely to be a means through which Wolbachia conveys antiviral protection to its host (Ye, 2017).

Orally acquired cyclic dinucleotides drive dSTING-dependent antiviral immunity in enterocytes

Enteric pathogens overcome barrier immunity within the intestinal environment that includes the endogenous flora. The microbiota produces diverse ligands, and the full spectrum of microbial products that are sensed by the epithelium and prime protective immunity is unknown. Using Drosophila, this study found that the gut presents a high barrier to infection, which is partially due to signals from the microbiota, as loss of the microbiota enhances oral viral infection. Cclic dinucleotide (CDN) feeding is sufficient to protect microbiota-deficient flies from enhanced oral infection, suggesting that bacterial-derived CDNs induce immunity. Mechanistically, this study found CDN protection is dSTING- and dTBK1-dependent, leading to NF-kappaB-dependent gene expression. Furthermore, this study identified the apical nucleoside transporter, CNT2, as required for oral CDN protection. Altogether, these studies define a role for bacterial products in priming immune defenses in the gut (Segrist, 2022).

Consumption of dietary sugar by gut bacteria determines Drosophila lipid content

Gut microorganisms are essential for the nutritional health of many animals, but the underlying mechanisms are poorly understood. This study investigated how lipid accumulation by adult Drosophila melanogaster is reduced in flies associated with the bacterium Acetobacter tropicalis which displays oral-faecal cycling between the gut and food. It was demonstrated that the lower lipid content of A. tropicalis-colonized flies relative to bacteria-free flies is linked with a parallel bacterial-mediated reduction in food glucose content; and can be accounted for quantitatively by the amount of glucose acquired by the flies, as determined from the feeding rate and assimilation efficiency of bacteria-free and A. tropicalis-colonized flies. The study recommends that nutritional studies on Drosophila include empirical quantification of food nutrient content, to account for likely microbial-mediated effects on diet composition. More broadly, the study demonstrates that selective consumption of dietary constituents by microorganisms can alter the nutritional balance of food and, thereby, influence the nutritional status of the animal host (Huang, 2015).

Stable host gene expression in the gut of adult Drosophila melanogaster with different bacterial mono-associations

There is growing evidence that the microbes found in the digestive tracts of animals influence host biology, but how they accomplish this is still not understood. This study evaluated how different microbial species commonly associated with laboratory-reared Drosophila melanogaster impact host biology at the level of gene expression in the dissected adult gut and in the entire adult organism. It was observed that guts from animals associated from the embryonic stage with either zero, one or three bacterial species demonstrated indistinguishable transcriptional profiles. Additionally, it was found that the gut transcriptional profiles of animals reared in the presence of the yeast Saccharomyces cerevisiae alone or in combination with bacteria could recapitulate those of conventionally-reared animals. In contrast, whole body transcriptional profiles of conventionally-reared animals were distinct from all of the treatments tested. These data suggest that adult flies are insensitive to the ingestion of the bacteria found in their gut, but that prior to adulthood, different microbes impact the host in ways that lead to global transcriptional differences observable across the whole adult body (Elya, 2016).

Drosophila melanogaster establishes a species-specific mutualistic interaction with stable gut-colonizing bacteria

This study identified bacterial species from wild-caught D. melanogaster that stably associate with the host independently of continuous inoculation. Moreover, it was shown that specific Acetobacter wild isolates can proliferate in the gut. It was further demonstrated that the interaction between D. melanogaster and the wild isolated Acetobacter thailandicus is mutually beneficial and that the stability of the gut association is key to this mutualism. The stable population in the gut of D. melanogaster allows continuous bacterial spreading into the environment, which is advantageous to the bacterium itself. The bacterial dissemination is in turn advantageous to the host because the next generation of flies develops in the presence of this particularly beneficial bacterium. A. thailandicus leads to a faster host development and higher fertility of emerging adults when compared to other bacteria isolated from wild-caught flies. Furthermore, A. thailandicus is sufficient and advantageous when D. melanogaster develops in axenic or freshly collected figs, respectively. This isolate of A. thailandicus colonizes several genotypes of D. melanogaster but not the closely related D. simulans, indicating that the stable association is host specific. This work establishes a new conceptual model to understand D. melanogaster-gut microbiota interactions in an ecological context; stable interactions can be mutualistic through microbial farming, a common strategy in insects. Moreover, these results develop the use of D. melanogaster as a model to study gut microbiota proliferation and colonization (Pais, 2018).

The Drosophila microbiome has a limited influence on sleep, activity, and courtship behaviors

In animals, commensal microbes modulate various physiological functions, including behavior. While microbiota exposure is required for normal behavior in mammals, it is not known how widely this dependency is present in other animal species. The hypothesis is proposed that the microbiome has a major influence on the behavior of the vinegar fly (Drosophila melanogaster), a major invertebrate model organism. Several assays were used to test the contribution of the microbiome on some well-characterized behaviors: defensive behavior, sleep, locomotion, and courtship in microbe-bearing, control flies and two generations of germ-free animals. None of the behaviors were largely influenced by the absence of a microbiome, and the small or moderate effects were not generalizable between replicates and/or generations. These results refute the hypothesis, indicating that the Drosophila microbiome does not have a major influence over several behaviors fundamental to the animal's survival and reproduction. The impact of commensal microbes on animal behaviour may not be broadly conserved (Selkrig, 2018).

The Drosophila transcriptional network is structured by microbiota

This study investigated the impact of resident microorganisms (microbiota) on gene coexpression in Drosophila. Transcriptomic analysis, of 17 lines representative of the global genetic diversity of Drosophila, yielded a total of 11 transcriptional modules of co-expressed genes. For seven of these modules, the strength of the transcriptional network (defined as gene-gene coexpression) differed significantly between flies bearing a defined gut microbiota (gnotobiotic flies) and flies reared under microbiologically sterile conditions (axenic flies). Furthermore, gene coexpression was uniformly stronger in these microbiota-dependent modules than in both the microbiota-independent modules in gnotobiotic flies and all modules in axenic flies, indicating that the presence of the microbiota directs gene regulation in a subset of the transcriptome. The genes constituting the microbiota-dependent transcriptional modules include regulators of growth, metabolism and neurophysiology, previously implicated in mediating phenotypic effects of microbiota on Drosophila phenotype. Together these results provide the first evidence that the microbiota enhances the coexpression of specific and functionally-related genes. This system-wide analysis demonstrates that the presence of microbiota enhances gene coexpression. This finding has potentially major implications for understanding of the mechanisms by which microbiota affect host health and fitness, and the ways in which hosts and their resident microbiota coevolve (Dobson, 2016).

A Mesh-Duox pathway regulates homeostasis in the insect gut

The metazoan gut harbours complex communities of commensal and symbiotic bacterial microorganisms. The quantity and quality of these microorganisms fluctuate dynamically in response to physiological changes. The mechanisms that hosts have developed to respond to and manage such dynamic changes and maintain homeostasis remain largely unknown. This study identified a dual oxidase (Duox)-regulating pathway that contributes to maintaining homeostasis in the gut of both Aedes aegypti and Drosophila melanogaster. A gut-membrane-associated protein, named Mesh, plays an important role in controlling the proliferation of gut bacteria by regulating Duox expression through an Arrestin-mediated MAPK JNK/ERK phosphorylation cascade. Expression of both Mesh and Duox is correlated with the gut bacterial microbiome, which, in mosquitoes, increases dramatically soon after a blood meal. Ablation of Mesh abolishes Duox induction, leading to an increase of the gut microbiome load. This study reveals that the Mesh-mediated signalling pathway is a central homeostatic mechanism of the insect gut (Xiao, 2017).

Probabilistic invasion underlies natural gut microbiome stability

Species compositions of gut microbiomes impact host health, but the processes determining these compositions are largely unknown. An unexplained observation is that gut species composition varies widely between individuals but is largely stable over time within individuals. Stochastic factors during establishment may drive these alternative stable states (colonized versus non-colonized), which can influence susceptibility to pathogens, such as Clostridium difficile. A precise, high-throughput technique revealed stable between-host variation in colonization when individual germ-free flies were fed their own natural commensals (including the probiotic Lactobacillus plantarum). Some flies were colonized while others remained germ-free even at extremely high bacterial doses. Thus, alternative stable states of colonization exist even in this low-complexity model of host-microbe interactions. These alternative states are driven by a fundamental asymmetry between the inoculum population and the stably colonized population that is mediated by spatial localization and a population bottleneck, which makes stochastic effects important by lowering the effective population size. Prior colonization with other bacteria reduced the chances of subsequent colonization, thus increasing the stability of higher-diversity guts. Therefore, stable gut diversity may be driven by inherently stochastic processes, which has important implications for combatting infectious diseases and for stably establishing probiotics in the gut (Obadia, 2017).

Gut microbiota modifies olfactory-guided microbial preferences and foraging decisions in Drosophila

The gut microbiota affects a wide spectrum of host physiological traits, including development, germline, immunity, nutrition, and longevity. Association with microbes also influences fitness-related behaviors such as mating and social interactions. Although the gut microbiota is evidently important for host wellbeing, how hosts become associated with particular assemblages of microbes from the environment remains unclear. This study presents evidence that the gut microbiota can modify microbial and nutritional preferences of Drosophila melanogaster. By experimentally manipulating the gut microbiota of flies subjected to behavioral and chemosensory assays, fly-microbe attractions were found to be shaped by the identity of the host microbiota. Conventional flies exhibit preference for their associated Lactobacillus, a behavior also present in axenic flies as adults and marginally as larvae. By contrast, fly preference for Acetobacter is primed by early-life exposure and can override the innate preference. These microbial preferences are largely olfactory guided and have profound impact on host foraging, as flies continuously trade off between acquiring beneficial microbes and balancing nutrients from food. This study shows a role of animal microbiota in shaping host fitness-related behavior through their chemosensory responses, opening a research theme on the interrelationships between the microbiota, host sensory perception, and behavior (Wong, 2017).

How gut transcriptional function of Drosophila melanogaster varies with the presence and composition of the gut microbiota

Despite evidence from laboratory experiments that perturbation of the gut microbiota affects many traits of the animal host, understanding of the effect of variation in microbiota composition on animals in natural populations is very limited. This study used Drosophila to identify the impact of natural variation in the taxonomic composition of gut bacterial communities on host traits, with the gut transcriptome as a molecular index of microbiota-responsive host traits. Use of the gut transcriptome was validated by demonstrating significant transcriptional differences between the guts of laboratory flies colonized with bacteria and maintained under axenic conditions. Wild Drosophila from six field collections made over two years had gut bacterial communities of diverse composition, dominated to varying extents by Acetobacteraceae and Enterobacteriaceae. The gut transcriptomes also varied among collections and differed markedly from those of laboratory flies. However, no overall relationship between variation in the wild fly transcriptome and taxonomic composition of the gut microbiota was evident at all taxonomic scales of bacteria tested for both individual fly genes and functional categories in Gene Ontology. It is concluded that the interaction between microbiota composition and host functional traits may be confounded by uncontrolled variation in both ecological circumstance and host traits (e.g., genotype, age physiological condition) under natural conditions, and that microbiota effects on host traits identified in the laboratory should, therefore, be extrapolated to field population with great caution (Bost, 2017).

Microbiota-derived lactate activates production of reactive oxygen species by the intestinal NADPH Oxidase Nox and shortens Drosophila lifespan

Commensal microbes colonize the gut epithelia of virtually all animals and provide several benefits to their hosts. Changes in commensal populations can lead to dysbiosis, which is associated with numerous pathologies and decreased lifespan. Peptidoglycan recognition proteins (PGRPs) are important regulators of the commensal microbiota and intestinal homeostasis. This study found that a null mutation in Drosophila PGRP-SD was associated with overgrowth of Lactobacillus plantarum in the fly gut and a shortened lifespan. L. plantarum-derived lactic acid triggered the activation of the intestinal NADPH oxidase Nox and the generation of reactive oxygen species (ROS). In turn, ROS production promoted intestinal damage, increased proliferation of intestinal stem cells, and dysplasia. Nox-mediated ROS production required lactate oxidation by the host intestinal lactate dehydrogenase, revealing a host-commensal metabolic crosstalk that is probably broadly conserved. These findings outline a mechanism whereby host immune dysfunction leads to commensal dysbiosis that in turn promotes age-related pathologies (Iatsenko, 2018).

The epithelial surfaces of most metazoan organisms are inhabited by complex microbial communities. The composition of these microbial communities is determined by an intricate interplay of genetic and environmental factors. Changes in healthy microbiota composition, referred to as commensal dysbiosis, have been associated with pathologies like inflammatory bowel disease, obesity, diabetes, neurological disorders, chronic inflammation, and cancer. However, the vast diversity of mammalian microbiota and genetic complexity of the immune system are major obstacles to clearly establishing mechanistic links between host immune genotype, microbiota structure, and disease phenotype (Iatsenko, 2018).

Because of the simplicity of its microbiota and physiological similarity with the mammalian intestine, the Drosophila gut is a model of choice to study human intestinal pathophysiology. Studies using this model have provided insights into innate immunity signaling, host-commensal interactions, and epithelial homeostasisduring aging. Drosophila harbors a microbiota composed of 5 to 30 bacterial species, dominated by the genera Acetobacter and Lactobacillus. Although many Drosophila commensals inhabit the gut transiently and are constantly replenished from food, they affect various aspects of host physiology ranging from the promotion of larval growth to the defense against pathogens. As in humans, dysbiosis in flies is associated with disruption of gut homeostasis, inflammation, and reduced lifespan, highlighting the importance of maintaining healthy microbiota composition and abundance (Iatsenko, 2018).

Several host mechanisms restrict growth of both symbiotic and pathogenic bacteria in the Drosophila gut. Acid secretion by V-ATPases of the copper cell region in the middle midgut has been shown to eliminate most intestinal bacteria, while a chitinous barrier, the peritrophic matrix, shields epithelial cells from invading bacteria. Moreover, two inducible host defense mechanisms control both pathogens and microbiota in the gut: antimicrobial peptides (AMPs) and reactive oxygen species (ROS). Two ROS-producing enzymes, the NADPH oxidases Duox and Nox, have been implicated in the control of intestinal microbes in Drosophila. The dual oxidase Duox produces microbicidal ROS in response to uracil released by pathogenic bacteria. Nox produces ROS in response to commensal bacteria, such as L. plantarum, but how Nox is activated is not yet known. ROS not only eliminate ingested pathogens but also damage enterocytes, thereby promoting the compensatory proliferation of intestinal stem cells (Iatsenko, 2018).

In addition to triggering ROS, ingested bacteria activate the expression of several antimicrobial peptide genes in specific domains along the digestive tract. This response is initiated when DAP-type peptidoglycan from Gram-negative bacteria is sensed by the transmembrane recognition receptor PGRP-LC in the ectodermal parts of the gut or by the intracellular receptor PGRP-LE in the midgut. PGRP-LC and PGRP-LE then recruit the adaptor IMD to finally activate the NF-κB-like transcription factor Relish. The gut antibacterial response is kept in check by several negative regulators of the IMD pathway, notably by enzymatic PGRPs such as PGRP-LB and PGRP-SC that scavenge peptidoglycan. Flies lacking these negative regulators show excessive, deleterious local and systemic immune activation (Iatsenko, 2018).

The IMD pathway also shapes the commensal community structure in the intestine. For example, PGRP-LC and relish flies with defects in the IMD pathway are short lived and exhibit increased bacterial loads in their guts upon aging. Chronic over-activation of the IMD pathway is also associated with microbiota dysbiosis, characterized by the expansion of antimicrobial peptide-resistant pathobionts. However, the mechanism whereby immune dysfunction causes commensal dysbiosis and leads to age-related pathologies and lifespan reduction is not fully understood (Iatsenko, 2018).

Previously identified Drosophila PGRP-SD as a secreted pattern recognition receptor that functions upstream of PGRP-LC to enhance IMD pathway activation during systemic infection (Iatsenko, 2016). In contrast to canonical mutants of the IMD pathway, a null mutation in PGRP-SD reduces but does not abolish the immune response, providing a sensitive tool to study the IMD pathway. This study used the sensitized PGRP-SD background to investigate the role of the IMD pathway in the control of intestinal homeostasis during infection and aging. Specifically, it was asked whether PGRP-SD was required to maintain a stable commensal composition and whether loss of PGRP-SD would lead to intestinal dysbiosis and dysplasia (Iatsenko, 2018).

The mucosal immune system uses multiple complex mechanisms to maintain a balance between preserving a beneficial microbiota and eliminating pathogens. This dynamic between immune system and microbiota undergoes age-related changes in animals, including humans. In Drosophila, aging is associated with a series of hallmarks: a higher microbiota load, an increased immune response, elevated ROS, dysplasia, loss of compartmentalization, and rupture of barrier permeability. Interestingly, precocious intestinal senescence equally affects mutants with immune over-activation (e.g., Caudal, PGRP-LB) and immune deficiency (Relish, Foxo). However, causal relationships between immune dysfunction, dysbiosis, and dysplasia are not clearly established. This study used PGRP-SD^sk1 mutant flies with reduced immune reactivity as a tool to characterize a pathway linking immune deficiency to precocious intestinal aging. Immune-deficient flies were found to carry increased loads of the dominant microbiota member, Lp, which led to a higher release of lactate/lactic acid. This bacterial metabolite not only acidified the intestine, but also stimulated ROS production by NOX, which triggered precocious aging. This model is appealing for two reasons. First, it does not involve a change in the microbiota composition per se, but rather an overgrowth of a common, preexisting microbiota member. Second, it shows how a bacterial metabolite, lactate, when processed by the host epithelia, drives ROS production and intestinal senescence. This model is likely to apply to other contexts, including mammalian gut microbiota (Iatsenko, 2018).

Previously, PGRP-SD was identified as a major immune sensor implicated in systemic immunity. The present study uncovered the central function PGRP-SD plays in intestinal immunity, notably by inducing an efficient immune response to pathogens while promoting tolerance to microbiota. This differential response is due to its ability to control the expression of negative regulators that confer immune tolerance. By controlling the local expression of negative regulators of IMD pathway activity, PGRP-SD might prevent the systemic spread of immune activation, a function similar to that of PGRP-LE (Iatsenko, 2018).

Numerous studies have reported an increase in total microbiota load in the gut upon aging and have speculated that this change contributes to host mortality, as elimination of microbiota often prolongs lifespan. It remained unclear whether increased microbiota loads caused accelerated ageing directly or rather indirectly, by inducing chronic immune activation in the gut. This study answers this question by proving that the PGRP-SD^sk1 mutant, which has increased microbiota loads but is incapable of chronic immune activation, still displays hallmarks of accelerated aging. The findings unravel a direct causal mechanism linking increases in bacterial loads to gut senescence. Specifically, this study showed that aging PGRP-SD^sk1 mutants lose control over their microbial communities, resulting in increased microbiota loads dominated by Lactobacillus. The Lp-derived metabolite lactic acid/lactate stimulated ROS production by the NADPH oxidase Nox. This model is supported by the observation that a lactic acid-deficient Lp strain did not cause any precocious aging in PGRP-SD^sk1 mutants. Moreover, feeding flies with lactic acid was sufficient to recapitulate all the aging hallmarks caused by Lp. Importantly, lactate produced by the symbionts needed to enter and be processed in host cells to activate NOX. This was supported by the observation that inactivating the host's lactate dehydrogenase or the lactate transporters in enterocytes suppressed Lp- or lactic acid-mediated dysplasia and lifespan shortening (Iatsenko, 2018).

In other contexts, dysbiosis with a change in microbiota composition has been recognized as a contributing factor to epithelial dysplasia, immune senescence, and age-related mortality. For example, flies with reduced Caudalexpression and high IMD pathway activity favor the growth of the pathobiont Gluconobacter EW707, which drives host mortality. In contrast, no changes were found in microbiota composition between PGRP-SD^sk1 mutant and wild-type flies, excluding the possibility that immune defects in PGRP-SD^sk1 mutants favor the selection of pathobionts. Thus, excessively increased loads of an otherwise beneficial Lp symbiont in PGRP-SDsk1 flies seem to be solely responsible for the lifespan shortening (Iatsenko, 2018).

Of note, mammalian PGRPs have also been implicated in the regulation of microbiota composition. Mice deficient for any of the four PGRPs (Pglyrp1-4) are more sensitive to colitis than wild-type mice due to a more inflammatory microbiota. This points to a conserved role of PGRPs as modulators of host-microbe interactions in the gut (Iatsenko, 2018).

Moreover, the finding that a commensal bacterium becomes detrimental in the immunocompromised background holds true for mammals as well. For instance, Lactobacilli that are beneficial members of human gut microbiota were associated with diseases like D-lactic acidosis, bacteremia, endocarditis, and localized infections (Iatsenko, 2018).

Gut microbiota affects development and olfactory behavior in Drosophila melanogaster

It has been shown that gut microbes are very important for the behavior and development of Drosophila, as the beneficial microbes are involved in the identification of suitable feeding and oviposition places. However, in what way these associated gut microbes influence the fitness-related behaviors of Drosophila melanogaster remains unclear. This study shows that D. melanogaster exhibits different behavioral preferences towards gut microbes. Both adults and larvae were attracted by the headspace of Saccharomyces cerevisiae and Lactobacillus plantarum, but were repelled by Acetobacter malorum in behavioral assays, indicating an olfactory mechanism involved in these preference behaviors. While the attraction to yeast was governed by olfactory sensory neurons expressing the odorant co-receptor Orco, the observed behaviors towards the other microbes still remained in flies lacking this co-receptor. By experimentally manipulating the microbiota of the flies, flies were found to not strive for a diverse microbiome by e.g. increasing their preference towards gut microbes that they had not experienced previously. Instead, in some cases the flies even increased preference for the microbes they were reared on. Furthermore, exposing Drosophila larvae to all three microbes promoted Drosophila's development while only exposure to S. cerevisiae and A. malorum resulted in the development of larger ovaries and in increased egg numbers the flies laid in an oviposition assay. Thus this study provides a better understanding of how gut microbes affect insect behavior and development, and offers an ecological rationale for preferences of flies for different microbes in their natural environment (Qiao, 2019).

Commensal gut bacteria buffer the impact of host genetic variants on Drosophila developmental traits under nutritional stress

Eukaryotic genomes encode several buffering mechanisms that robustly maintain invariant phenotypic outcome despite fluctuating environmental conditions. This study show that the Drosophila gut-associated commensals, represented by a single facultative symbiont, Lactobacillus plantarum (Lp(WJL)), constitutes a so far unexpected buffer that masks the contribution of the host's cryptic genetic variation (CGV) to developmental traits while the host is under nutritional stress. During chronic under-nutrition, Lp(WJL) consistently reduces variation in different host phenotypic traits and ensures robust organ patterning during development; Lp(WJL) also decreases genotype-dependent expression variation, particularly for development-associated genes. Evidence is provided that Lp(WJL) buffers via reactive oxygen species (ROS) signaling whose inhibition impairs microbiota-mediated phenotypic robustness. This study thus identified a hitherto unappreciated contribution of the gut facultative symbionts to host fitness that, beyond supporting growth rates and maturation timing, confers developmental robustness and phenotypic homogeneity in times of nutritional stress (Ma, 2019).

Yeasts affect tolerance of Drosophila melanogaster to food substrate with high NaCl concentration

The ability of model animal species, such as Drosophila melanogaster, to adapt quickly to various adverse conditions has been shown in many experimental evolution studies. It is usually assumed by default that such adaptation is due to changes in the gene pool of the studied population of macroorganisms. At the same time, it is known that microbiome can influence biological processes in macroorganisms. In order to assess the possible impact of microbiome on adaptation, an evolutionary experiment was performed in which some D. melanogaster lines were reared on a food substrate with high NaCl concentration while the others were reared on the standard (favourable) substrate. The reproductive efficiency of experimental lines on the high salt substrate was evaluated three years after the experiment started. The tests confirmed that the lines reared on the salty substrate became more tolerant to high NaCl concentration. Moreover, pre-inoculation of the high salt medium with homogenized salt-tolerant flies tended to improve reproductive efficiency of naive flies on this medium (compared to pre-inoculation with homogenized control flies). The analysis of yeast microbiome in fly homogenates revealed significant differences in number and species richness of yeasts between salt-tolerant and control lines. Some individual yeast lines extracted from the salt-tolerant flies improved reproductive efficiency of naive flies on salty substrate (compared to baker's yeast and no yeast controls), whereas the effect of the yeast lines extracted from the control flies tended to be smaller. The yeast Starmerella bacillaris extracted from the salt-tolerant flies showed the strongest positive effect. This yeast is abundant in all salt-tolerant lines, and very rare or absent in all control lines. The results are consistent with the hypothesis that some components of the yeast microbiome of D. melanogaster contribute to to flies' tolerance to food substrate with high NaCl concentration (Dmitrieva, 2019).

Misato underlies visceral myopathy in Drosophila

Genetic mechanisms for the pathogenesis of visceral myopathy (VM) have been rarely demonstrated. This study reports the visceral role of misato (mst) in Drosophila and its implications for the pathogenesis of VM. Depletion of mst using three independent RNAi lines expressed by a pan-muscular driver elicited characteristic symptoms of VM, such as abnormal dilation of intestinal tracts, reduced gut motility, feeding defects, and decreased life span. By contrast, exaggerated expression of mst reduced intestine diameters, but increased intestinal motilities along with thickened muscle fibers, demonstrating a critical role of mst in the visceral muscle. Mst expression was detected in the adult intestine with its prominent localization to actin filaments and was required for maintenance of intestinal tubulin and actomyosin structures. Consistent with the subcellular localization of Mst, the intestinal defects induced by mst depletion were dramatically rescued by exogenous expression of an actin member. Upon ageing the intestinal defects were deteriorative with marked increase of apoptotic responses in the visceral muscle. Taken together, it is proposed that the impairment of actomyosin structures induced by mst depletion in the visceral muscle as a pathogenic mechanism for VM (Min, 2017).

Inflammation-modulated metabolic reprogramming is Required for DUOX-dependent gut immunity in Drosophila

DUOX, a member of the NADPH xidase family, acts as the first line of defense against enteric pathogens by producing microbicidal reactive oxygen species. DUOX is activated upon enteric infection, but the mechanisms regulating DUOX activity remain incompletely understood. Using Drosophila genetic tools, this study shows that enteric infection results in "pro-catabolic" signaling that initiates metabolic reprogramming of enterocytes toward lipid catabolism, which ultimately governs DUOX homeostasis. Infection induces signaling cascades involving TRAF3 and kinases AMPK and WTS, which regulate TOR kinase to control the balance of lipogenesis versus lipolysis. Enhancing lipogenesis blocks DUOX activity, whereas stimulating lipolysis via ATG1-dependent lipophagy is required for DUOX activation. Drosophila with altered activity in TRAF3-AMPK/WTS-ATG1 pathway components exhibit abolished infection-induced lipolysis, reduced DUOX activation, and enhanced susceptibility to enteric infection. Thus, this work uncovers signaling cascades governing inflammation-induced metabolic reprogramming and provides insight into the pathophysiology of immune-metabolic interactions in the microbe-laden gut epithelia (Lee, 2018).

Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila

Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells' environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. This study showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. Flies were generated with knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, and Chk2, which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly's survival. These data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. These findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging (Park, 2018).

A Non-stop identity complex (NIC) supervises enterocyte identity and protects from premature aging

A hallmark of aging is loss of differentiated cell identity. Aged Drosophila midgut differentiated enterocytes (ECs) lose their identity, impairing tissue homeostasis. To discover identity regulators, an RNAi screen targeting ubiquitin-related genes was performed in ECs. Seventeen genes were identified, including the deubiquitinase Non-stop (CG4166). Lineage tracing established that acute loss of Non-stop in young ECs phenocopies aged ECs at cellular and tissue levels. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) containing E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Non-stop ensured chromatin accessibility, maintaining the EC-gene signature, and protected NIC subunit stability. Upon aging, the levels of Non-stop and NIC subunits declined, distorting the unique organization of the EC nucleus. Maintaining youthful levels of Non-stop in wildtype aged ECs safeguards NIC subunits, nuclear organization, and suppressed aging phenotypes. Thus, Non-stop and NIC, supervise EC identity and protects from premature aging (Erez, 2021).

Differentiated cell states are actively established and maintained through action of 'identity supervisors'. Identity supervisors safeguard the expression of genes that enable differentiated cells to respond to environmental cues and perform required physiological tasks. Concomitantly, they ensure silencing/repression of previous fate and non-relevant gene programs and reduce transcriptional noise. Inability to safeguard cell identity is a hallmark of aging and results in diseases such as neurodegeneration, diabetes, and cancer. In many cases, transcription factors (TFs) together with chromatin regulators and architectural/scaffold proteins establish and maintain large-scale chromatin and nuclear organization that is unique to the differentiated state of the cell (Erez, 2021).

In adult Drosophila midgut epithelia, the transcription factor Hey [Hairy/E(spl)-related with YRPW motif], together with Drosophila nuclear type A lamin, Lamin C (LamC), co-supervise identity of fully differentiated enterocytes (ECs) (Monastirioti, 2010; Flint Brodsly, 2019). Highly similar to the vertebrate gut, Drosophila midgut epithelia intestinal stem cells (ISC) either self-renew or differentiate into progenitor cells that mature into enteroendocrine cells (EEs) or give rise to enteroblast (EB) progenitors. EBs mature into fully polyploid differentiated enterocytes (ECs) that carry out many critical physiological tasks of the intestine. Aging affects the entire midgut, and is associated with loss of EC identity, mis-differentiation of progenitors, pathological activation of the immune system, and loss of the physiological properties of the gut and its integrity. It also results in loss of intestinal compartmentalization, and microbiota-dysbiosis, all leading to reduced lifespan. During aging, the protein levels of identity supervisors such as Hey and LamC decline, resulting in inability to maintain EC-gene programs and ectopic expression of previous- and non-relevant gene programs. Indeed, continuous expression of Hey in aged ECs restores and protects EC identity, gut integrity, and tissue homeostasis (Flint Brodsly, 2019; Erez, 2021 and references therein).

Regulation of EC identity requires signaling to the nucleus to communicate physiological changes in the gut environment. An important mechanism involves changes in post-transcriptional modifications (PTMs) which may propagate, amplify, or conduct signals, ultimately leading to differential gene regulation. One type of PTM is the covalent attachment of ubiquitin or ubiquitin-like (Ub/UbL) molecules, that affect protein stability, function, localization, as well as modulatex chromatin structure. Recent works suggest an intimate link between ubiquitin, proteostasis, and aging. A RNAi screen was performed in this study to search for Ub/UbL-related genes within ECs that supervise identity. Screening of 362 genes, 17 were identified whose conditional elimination in fully differentiated ECs resulted in loss of EC identity. Further analysis revealed one of them, the deubiquitinating isopeptidase (DUB) Non-stop (Non-stop/dUSP22) is a key EC identity supervisor. Purification and proteomic analysis identified Non-stop as part of a CP190/Nup98/Sgf11/e(y)2/mdg4 protein complex, termed Non-stop identity complex (NIC), that is essential for maintenance of EC identity. In part, Non-stop protects NIC proteins from age-dependent decline, safeguarding the EC-gene expression signature, as well as large-scale nuclear organization in these cells, preventing premature aging. Over lifespan, Non-stop protein levels in ECs declined, leading to loss of NIC subunits. This decline is associated with loss of gut identity and physiology at the cellular and tissue levels. Maintaining youthful levels of Non-stop prevented loss of the NIC and prevented aging of the gut (Erez, 2021).

Based on cell-specific secondary tests, this study identified three categories of supervisors; 1. EC-specific identity regulators 2. Genes that are required for differentiated cell identity (both EEs and ECs) 3. Genes that are required for identity of all cell types (general identity regulators). Of specific interest were a group of genes (CG1490; CG2926; CG4080) whose elimination in EEs resulted in a loss of EC, but not EE, identity, acting as inductive identity regulators. Likely their effect on ECs involves cell~cell communication via diffusible factors. While loss of each individual gene identified in the screen resulted in loss of EC identity, the molecular and genetic connections between different identity regulators requires further studies (Erez, 2021).

The observation that Ub/UbL-related genes protect the differentiated identity is conserved across species. Screens in mammalian systems identified enzymes within the SUMO and ubiquitin pathways acting as a barrier against forced reprogramming of differentiated cell. Among these genes were Ubc9 (Drosophila Lesswright), the sole SUMO conjugating enzyme, that was also identified in this screen and the isopeptidase Psmd14. In addition to Non-stop, the screen identified the iso-isopeptidase UTO6-like (CG7857), Usp7, and Rpn11 as regulators of EC identity. Rpn11 is part of the lid particle of the 26S proteasome, involved in deubiquitinating proteins undergoing proteasomal degradation (Erez, 2021).

Genes regulating identity are likely to serve as a barrier to tumorigenesis having a tumor suppressive function. The human ortholog of Non-stop, USP22 has mixed oncogenic and tumor-suppressive functions. Relevant to the current study is the observation that USP22 has tumor suppressive functions in colon cancer by reducing mTor activity. Along this line it is interesting to note that 10/17 of human orthologs to genes discovered in the screen are either mutated or silenced in cancer. Thus, future studies of these human orthologs may identify potent tumor suppressors in cancer (Erez, 2021).

The proteomic, biochemical, and genetic analyses established that to maintain identity Non-stop acts as part of the NIC protein complex. Like SAGA, NIC contained the entire DUB module including Non-stop, E(y)two and Sgf11. Within SAGA, Sgf11 is critical for Non-stop activity. However, loss of Sgf11 did not result in loss of EC identity. While this may reflect intrinsic differences between the complexes it may also be possible that knock-down of Sgf11 transcripts in ECs did not reduce Sgf11 protein levels sufficiently enough to result in a phenotype (Erez, 2021).

The BTB domain of Mod(mdg4) interacts with the M domain of Cp190, and together they enable the recruitment of NIC to the promoter/enhancer regions. There are more than 30 Mod(mdg4) isoforms with different C-ends, each of which interacts with different DNA-binding TF. Cp190 can also be able to recruit different TFs and interacts with Chromo and Zinc-finger 4, that are co-localized with Nup98-96. The Mod(mdg4)/CP190 sub-complex is bound to promoters through interactions with many promoter specific C2H2 proteins including dCTCF, Su(Hw), and possibly via other TFs enriched in the ATAC-seq analysis (Erez, 2021).

Prominent phenotypes of loss of Non-stop were the reduced protein level of Nup98 and its miss-localization from the nuclear periphery to an intranuclear punctate distribution. Nup98 was shown to recruit Set1~COMPASS to enhance histone H3K4me2-3 methylations in hematopoietic progenitors. Thus, a possible function of the NIC may be the recruitment of the H3K4me2/3 COMPASS methylases to catalyze H3K9 di- and tri-methylation at enhancers and promotors, which are fundamental for gene activation. Members of the NIC complex were previously shown to form a Nuclear pore complex that enhances transcriptional memory upon exposure to ecdysone (Pascual-Garcia, 2017), raising the possibility that NIC is required for enhancing transcriptional memory promoting the transcription of EC-related genes and prevents Polycomb-dependent transcriptional repression (Erez, 2021).

In addition to NIC subunits the complex described by Pascual-Garcia (2017) contained Trithorax-like protein (Trl)/GAF and the boundary/architectural proteins CTCF, Suppressor of Hairy Wing (SuHw) and Mtor, but not Non-stop or E(y)2. Interestingly, the DNA binding site of Trl/GAF/ was enriched in EC genes that their expression required Non-stop for maintaining chromatin accessibility. However, Trl, CTCF, SuHw, or Mtor that interact with Cp190 were not detected as Non-stop bound proteins in the proteomic analysis, and RNAi-mediated elimination of Trl/GAF, or SuHw from ECs did not result in loss of EC identity. Thus, suggesting that these are likely separate regulatory complexes with shared subunits (Erez, 2021).

Non-stop was found to be required for the stability of NIC in ECs. RNA-seq analysis established that Non-stop did not regulate the mRNA level of NIC subunits. Suggesting that Nonstop acts by deubiquitinating NIC subunits in ECs. In this regard, it is possible that the stabilization of NIC subunits may also require the EC-related lamin, LamC. It was noticed that Lamin C protein levels (but not mRNA) decline upon loss of Non-stop. Moreover, LamC expression partially restored the protein levels of NIC subunits and their intranuclear localization, potentially by serving as a scaffold for NIC at the nuclear periphery. Therefore, additional experiments are required to establish whether NIC subunits are directly deubiquitinated and stabilized by Non-stop iso-peptidase activity and the potential contribution of LamC to stabilization of NIC subunits (Erez, 2021).

Thus, Non-stop may function at two levels; One is a direct role in transcription safeguarding chromatin accessibility at EC genes, while a second function is the stabilization of identity supervisors including NIC subunits and LamC (Erez, 2021).

Both Non-stop and the transcription factor Hey are bona-fide regulators of EC identity required for the expression of EC-related genes. A significant number of EC-related genes required both Non-stop and Hey for their expression, suggesting that Hey and Non-stop may co-regulate these genes. However, functional and epistatic tests suggest that Hey also acts upstream or in additional pathways to Non-stop. Hey binds to enhancers in lamin genes repressing the expression of the ISC-related lamin, LamDm0 and enhances the expression of LamC. In contrast, Non-stop does not regulate the accessibility, or expression of either LamDm0 or LamC at mRNA level. However, Non-stop is required for maintaining the levels of LamC protein. Therefore, loss of Non-stop results in a decline in LamC but not in the ectopic expression of LamDm0, which is observed upon acute loss of Hey or aging. This discrepancy may be due to the presence of Hey on its repressed targets in young ECs where Non-stop is targeted, and directly repressing their expression as maybe in the case of LamDm0. Moreover, EC-specific expression of Non-stop did not suppress the phenotypes associated with acute loss of Hey in young ECs further supporting for Hey-dependent, but Non-stop independent functions. However, the ECs-specific expression of either Non-stop or Hey in aging midguts restores expression of LamC and repressed ectopic LamDm0 expression (Erez, 2021).

Changes in large-scale nuclear organization are hallmarks of aging. Expression of identity supervisors can prevent age-related distortion of the nucleus EC identity and protect overall the epithelial tissue). However, to accomplish this, Non-stop or Hey were continuously expressed in ECs and temporal expression of Hey or Non-stop in already aged ECs was not sufficient to suppress aging phenotypes. Thus, if the levels of identity supervisors are kept at youthful levels, they can continue to maintain cell identity and prevent signs of aging, effectively keeping the gut organization and structure similar to young tissue (Erez, 2021).

Furthermore, it is not clear how expression of a single regulator like Non-stop has an extensive impact on the entire nucleus. Recent studies suggest that Non-stop functions in additional multiprotein complexes that may regulate large-scale cellular organization. For example, Non-stop is part of an Arp2/3 and WAVE regulatory (WRC) actin-cytoskeleton organization complex where it deubiquitinates the subunit SCAR (Cloud, 2019). In this regard, a nuclear actin organizing complex, WASH, interacted with nuclear Lamin and was required for large scale nuclear organization. Thus, it is tempting to suggest that such complexes are required to maintain cell identity, and that subunits within these complexes are deubiquitinated by Non-stop (Erez, 2021).

In this regard many nuclear proteins are extremely long-lived proteins (LLPs) among them are nuclear pore complex proteins (NPCs) and core histones. The extended stability of LLPs may originate from intrinsic properties of LLPs, or due to sequestration and evading degradation. However, increased stability may be also actively maintained by constitutive de-ubiquitination. Indeed, post-translational modification by ubiquitin and SUMO were shown to regulate lamin stability and their intranuclear localization. Specifically, type-A lamin and its splice variant Progerin, the cause of Hutchinson Gilford progeria syndrome (HGPS), a premature aging syndrome, are degraded by the HECT-type E3 ligase Smurf2 via ubiquitin-dependent autophagy. The elimination of Progerin by expression of Smurf2 in HGSP-fibroblasts reduced the deformation observed in these cells. Thus, it is possible that enhancing Progerin degradation by inhibiting the human ortholog of Non-stop, USP22, will restore nuclear architecture, and suppress the premature aging phenotypes observed in HGPS cells (Erez, 2021).

Derlin-1 and TER94/VCP/p97 are required for intestinal homeostasis

Adult stem cells are critical for the maintenance of residential tissue homeostasis and functions. However, the roles of cellular protein homeostasis maintenance in stem cell proliferation and tissue homeostasis are not fully understood. This study found that Derlin-1 and TER94/VCP/p97, components of the ER-associated degradation (ERAD) pathway, restrain intestinal stem cell proliferation to maintain intestinal homeostasis in adult Drosophila. Depleting any of them results in increased stem cell proliferation and midgut homeostasis disruption. Derlin-1 is specifically expressed in the ER of progenitors and its C-terminus is required for its function. Interestingly, it was found that increased stem cell proliferation is resulted from elevated ROS levels and activated JNK signaling in Derlin-1- or TER94-deficient progenitors. Further removal of ROS or inhibition of JNK signaling almost completely suppressed increased stem cell proliferation. Together, these data demonstrate that the ERAD pathway is critical for stem cell proliferation and tissue homeostasis. Thus this study provides insights into understanding of the mechanisms underlying cellular protein homeostasis maintenance (ER protein quality control) in tissue homeostasis and tumor development (Liu, 2021).

Rab21 in enterocytes participates in intestinal epithelium maintenance

Membrane trafficking is defined as the vesicular transport of proteins into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo system, this study investigated enterocyte-specific functions for the early endosomal machinery. Focus was placed on Rab21, which regulates specific steps in early endosomal trafficking. Depletion of Rab21 in enterocytes led to abnormalities in intestinal morphology, with deregulated cellular equilibrium associated with a gain in mitotic cells and increased cell death. Increases in apoptosis and Yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using an RNA interference screen, this study identified regulators of autophagy and membrane trafficking that phenocopied Rab21 knockdown. It was further shown that Rab21 knockdown-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling. Moreover, quantitative proteomics identified proteins affected by Rab21 depletion. Of these, changes were validated in apolipoprotein ApoLpp and the trehalose transporter Tret1-1, indicating roles for enterocyte Rab21 in lipid and carbohydrate homeostasis, respectively. These data shed light on an important role for early endosomal trafficking, and Rab21, in enterocyte-mediated intestinal epithelium maintenance (Nassari, 2022).

Cytosolic O-GlcNAcylation and PNG1 maintain Drosophila gut homeostasis by regulating proliferation and apoptosis

It remains unknown how intracellular glycosylation, O-GlcNAcylation, interfaces with cellular components of the extracellular glycosylation machinery, like the cytosolic N-glycanase NGLY1. This study utilized the Drosophila gut and uncovered a pathway in which O-GlcNAcylation cooperates with the NGLY1 homologue PNG1 to regulate proliferation in intestinal stem cells (ISCs) and apoptosis in differentiated enterocytes. Further, the CncC antioxidant signaling pathway and ENGase, an enzyme involved in the processing of free oligosaccharides in the cytosol, interact with O-GlcNAc and PNG1 through regulation of protein aggregates to contribute to gut maintenance. These findings reveal a complex coordinated regulation between O-GlcNAcylation and the cytosolic glycanase PNG1 critical to balancing proliferation and apoptosis to maintain gut homeostasis (Na, 2022).

Intestinal stem cells regulate tissue homeostasis by balancing self-renewal, proliferation, and differentiation all of which are supported by elevated flux through the hexosamine biosynthetic pathway (HBP). Both N-linked glycosylation and intracellular O-GlcNAc modifications are regulated by the HBP pathway in a nutrient-sensing manner. However, how NGLY1 is utilized to control stem cell homeostasis and differentiation in cells remains largely unknown. This is a critical question as patients with NGLY1-deficiency display global developmental delay, movement disorder and growth retardation. Elevation of NGLY1 was observed in patients' tumor samples, suggesting a function in oncogenic signaling. In Drosophila, PNG1 mutants had severe developmental defects and reduced viability, with the surviving adults frequently sterile (Funakoshi, 2010). This study has identified a pathway by which PNG1 regulates ISC homeostasis in vivo. This study shows that PNG1 levels increased in ISC/EBs concomitant with O-GlcNAc. This interaction between PNG1 and O-GlcNAcylation is critical for maintaining normal ISC proliferation and differentiation. Thus, through their mutual regulation, OGT and PNG1 have key roles in both progenitor (ISCs/EBs) and differentiated cells (ECs) contributing to tissue homeostasis. Previous reports indicated that PNG1 null larvae have specific developmental abnormalities in their midgut that contributes to their lethality. Further, intestinal inflammation in Crohn's disease is associated with increased O-GlcNAc modification. A previous study also showed that increased O-GlcNAc promotes gut dysplasia through regulation of DNA damage (Na, 2020). Thus, PNG1 or O-GlcNAc might still be associated with gut dysfunction in a disease context (Na, 2022).

The regulation of O-GlcNAc by PNG1 and the interaction between PNG1 and O-GlcNAc has been implicated previously. In fact, GlcNAc supplementation partially rescued lethality associated with PNG1 knockdown (Funakoshi, 2010). Although the mechanism by which GlcNAc supplementation rescued these mutant flies has not been fully worked out, Gfat1 transcript levels were downregulated in PNG1 knockdown flies (Funakoshi, 2010). Gfat1 is the enzyme that controls the rate limiting step in the HBP to produce UDP-GlcNAc. Thus, PNG1 through regulation of Gfat1 could impact levels of UDP-GlcNAc and ultimately O-GlcNAc. Additionally, it has been hypothesized that the loss of PNG1 could increase the presence of intracellular N-GlcNAc modification, potentially interfering with O-GlcNAc mediated signaling. Therefore, alterations in UDP-GlcNAc levels or presence of intracellular N-GlcNAc upon PNG1-deficiency can interact with O-GlcNAc to regulate stem cell homeostasis (Na, 2022).

Previous reports have shown that Nrf1 undergoes NGLY1-mediated deglycosylation, followed by proteolytic cleavage and translocation into the nucleus as an active transcription factor. Loss of NGLY1 caused Nrf1 dysfunction, as evidenced by an enrichment of deregulated genes encoding proteasome components and proteins involved in oxidation reduction. Proteasome activity can induce an apoptotic cascade that leads to growth arrest and, subsequently, cell death. The current data indicated that PNG1 or OGT knockdown suppressed ISC proliferation, which was rescued by Oltipraz (CncC activation) treatment in ISCs/EBs. Furthermore, there was increased apoptosis in PNG1 or OGT knockdown with treatment compared to non-treated groups. Interestingly, it was found O-GlcNAc-induced intestinal dysplasia was rescued by knockdown of PNG1 in ISCs/EBs through regulation of ROS levels. Similarly, increases in global O-GlcNAcylation in embryos of diabetic mice caused an overproduction of ROS and subsequent oxidative and ER stress. It is known that activation of SKN-1A/Nrf1 also requires deglycosylation by PNG-1/NGLY1 in C. elegans. Further, SKN-1 is O-GlcNAc modified and translocates to the nucleus in ogt-1(ok430)-null worms. Together, these studies all suggest conserved functional connections between O-GlcNAc and Nrf family transcription factors. This study also showed EC-specific OGT or PNG1 knockdown-induced hyperproliferation and cell death was decreased by CncC activation. This data indicated OGT or PNG1 can be regulated by CncC activity in ISCs/EBs and ECs. CncC has high activity within ISCs/EBs of unstressed as well young ISCs and quiescent ISCs but decreases with age and damage. These data indicated that CncC acts to properly balance between signaling and damage responses necessary for tissue homeostasis. CncC activation increased ISC proliferation in ISCs/EBs and decreased ISC proliferation in ECs of OGT or PNG1 knockdown contributing towards tissue homeostasis. Another study showed that inhibition of NGLY1 resulted in Nrf1 being misprocessed, mislocated, and inactive, thus indicating that functional NGLY1 is essential for Nrf1 processing, nuclear translocation, and transcription factor activity (Tomlin, 2017). Therefore, the data suggests that PNG1 and OGT modulated by CncC activation contribute to ISC proliferation and ultimately regulating tissue homeostasis. Nrf2 activation was able to rescue the developmental growth of NGLY1 deficiency in worm and fly models. In cancer-initiating cells, ER stress-dependent (ROS-independent) CncC induction is an event necessary to maintain stemness. The data showed that PNG1 knockdown-induced Poly-UB accumulation and 26S proteasome expression that was rescued by CncC overexpression and chemical activation. Through functioning as a sensor of cytosolic proteasome activity and an activator of aggresomal formation, Nrf2 alleviates cell damages caused by proteasomal stress. Expression of proteasome subunit genes and mitophagy-related genes were broadly enhanced after sulforaphane (Keap1 inhibitor) treatment and pharmacologically induction of Nrf2 promotes mitophagy and ameliorates mitochondrial defect in Ngly1-/- cells. Thus, it is believed that the sensitized background of the OGT or PNG1 mutant provides an environment where CncC activation promotes proliferation to the normal level through regulation of proteasome activity and protein aggregation (Na, 2022).

In a previously published paper (Ha, 2020), it was shown that OGT overexpression and OGA knockdown in ISCs/EBs both increased O-GlcNAc levels and induced hyperproliferation of the stem cells, whereas OGT knockdown decreased proliferation. However, in differentiated ECs, OGT overexpression and OGA knockdown phenotypes were similar to the normal gut, whereas OGT knockdown elevated proliferation and cell death. In general, EC death promotes proliferation in order to maintain gut homeostasis. Here, NGLY1 knockdown in ISCs/EBs decreased proliferation and clone size but NGLY1 knockdown in ECs induced hyperproliferation and cell death and importantly decreased O-GlcNAc levels. Thus, the phenotypes of OGT and NGLY1 were similar, demonstrating that maintenance of OGT and NGLY1 protein expression is highly interdependent for the maintenance of tissue homeostasis. It is interesting that the progenitor and differentiated cell types within the gut respond differently to changes in O-GlcNAc. It is possible that a certain level of O-GlcNAcylation is needed to maintain stem cells and promote proliferation and self-renewal, however, differentiated cells that do not have the same energy and growth requirements are not as reliant on high levels of O-GlcNAc. On the other hand, both ISC/EBs and ECs require some level of O-GlcNAc and without OGT there is decreased proliferation in progenitor cells and increased cell death of ECs. There are a few possibilities how NGLY1 and OGT can collaboratively work, however, it is unlikely that they share protein targets. First, a previous publication showed that additional deletion of ENGase, another N-deglycosylating enzyme that leaves a single GlcNAc residue, alleviates some of the lethality of Ngly1-deficient mice. Thus, it is possible with the accumulation of aggregation prone intracellular N-GlcNacylated proteins, there is disruption of normal O-GlcNac signaling. These data also showed increased protein aggregation in OGT or NLGY1 knockdown that was rescued by ENGase knockdown. In addition, MYC-OGT protein levels in OGT overexpression fly guts were decreased by PNG1 knockdown. It is possible that loss of NGLY1 disrupts normal OGT degradation and thus impacts levels global of O-GlcNAcylation (Na, 2022).

This study has shown that ENGase levels increased in PNG1 or OGT knockdown ISCs/EBs and ECs. PNGase is involved in the process of endoplasmic reticulum associated degradation (ERAD), acting as a deglycosylating enzyme that cleaves N-glycans attached to ERAD substrates. The small molecule ENGase inhibitors have potential to treat pathogenesis associated with NGLY1 deficiency. Rabeprazole, a proton pump inhibitor, was identified as a potential ENGase inhibitor. It was demonstrated that the consequences of knockdown of OGT or PNG1 on ISC proliferation and ENGase activity was rescued by Rabeprazole treatment in ISCs/EBs or ECs. The data showed that cell death was elevated in ISCs/EBs-specific PNG1/OGT knockdown with Rabeprazole treatment compared to non-treated groups concomitant with an increase in ISC proliferation. On the other hand, cell death decreased in EC-specific PNG1 knockdown treated with Rabeprazole resulting in a decrease in ISC proliferation. It is known that loss of PNG1 function in cells can cause the accumulation of aberrant proteins in the cytosol and the interruption of ERAD. Further, downregulation of ER stress-related genes has been reported in B-cell-specific OGT mutant mice. The protective effects of O-GlcNAc are not limited to mitochondrial function but also rescue injury caused by ER stress. Therefore, NGLY1/OGT seems to be functionally associated with the ERAD machinery. More recently, using a model ERAD substrate, it was reported that the ablation of Ngly1 causes a disruption in the ERAD process in mouse embryonic fibroblast (MEF) cells. Moreover, lethality of mice bearing a knockout of the Ngly1-gene was partially rescued by the additional deletion of the Engase gene. Interestingly, this study showed that OGA knockdown rescued ENGase levels of PNG1 knockdown ISCs/EBs. Hence, these findings suggest that there is a correlation between OGT/PNG1 and ENGase contributing to tissue maintenance (Na, 2022).

Taken together, these findings implicate O-GlcNAc and PNG1 as key regulators of tissue maintenance. PNG1 can impact stem cell homeostasis through regulation of O-GlcNAc both in ISCs/EBs or ECs. Of significance is the finding that PNG1 and OGT phenotypes are rescued by modulating CncC and ENGase activity in ISCs/EBs or ECs. Thus, these findings reveal that nutrient-driven glycosylation contribute towards control of ISC and progenitor cell proliferation and EC cell death via regulation of CncC and ENGase. This study provides a platform for future designs of interventions in which changes in O-GlcNAc can be utilized as a therapeutic for stem-cell-derived diseases like cancer. This study also presents a molecular mechanism and unexpected pathway that can be targeted for treating NGLY1-deificient patients (Na, 2022).

A bacteria-regulated gut peptide determines host dependence on specific bacteria to support host juvenile development and survival

Commensal microorganisms have a significant impact on the physiology of host animals, including Drosophila. Lactobacillus and Acetobacter, the two most common commensal bacteria in Drosophila, stimulate fly development and growth, but the mechanisms underlying their functional interactions remain elusive. This study found that imaginal morphogenesis protein-Late 2 (Imp-L2), a Drosophila homolog of insulin-like growth factor binding protein 7, is expressed in gut enterocytes in a bacteria-dependent manner, determining host dependence on specific bacteria for host development. Imp-L2 mutation abolished the stimulatory effects of Lactobacillus, but not of Acetobacter, on fly larval development. The lethality of the Imp-L2 mutant markedly increased under axenic conditions, which was reversed by Acetobacter, but not Lactobacillus, re-association. The host dependence on specific bacteria was determined by Imp-L2 expressed in enterocytes, which was repressed by Acetobacter, but not Lactobacillus. Mechanistically, Lactobacillus and Acetobacter differentially affected steroid hormone-mediated Imp-L2 expression and Imp-L2-specific FOXO regulation. These findings may provide a way how host switches dependence between different bacterial species when benefiting from varying microbiota (Lee, 2022).

Erebosis, a new cell death mechanism during homeostatic turnover of gut enterocytes

Many adult tissues are composed of differentiated cells and stem cells, each working in a coordinated manner to maintain tissue homeostasis during physiological cell turnover. Old differentiated cells are believed to typically die by apoptosis. This study discovered a previously uncharacterized, new phenomenon, which was name erebosis based on the ancient Greek word erebos ("complete darkness"), in the gut enterocytes of adult Drosophila. Cells that undergo erebosis lose cytoskeleton, cell adhesion, organelles and fluorescent proteins, but accumulate Angiotensin-converting enzyme (Ance). Their nuclei become flat and occasionally difficult to detect. Erebotic cells do not have characteristic features of apoptosis, necrosis, or autophagic cell death. Inhibition of apoptosis prevents neither the gut cell turnover nor erebosis. It is hypothesized that erebosis is a cell death mechanism for the enterocyte flux to mediate tissue homeostasis in the gut (Ciesielski, 2022).

Ets21c governs tissue renewal, stress tolerance, and aging in the Drosophila intestine

Homeostatic renewal and stress-related tissue regeneration rely on stem cell activity, which drives the replacement of damaged cells to maintain tissue integrity and function. The Jun N-terminal kinase (JNK) signaling pathway has been established as a critical regulator of tissue homeostasis both in intestinal stem cells (ISCs) and mature enterocytes (ECs), while its chronic activation has been linked to tissue degeneration and aging. This study shows that JNK signaling requires the stress-inducible transcription factor Ets21c to promote tissue renewal in Drosophila. Ets21c controls ISC proliferation as well as EC apoptosis through distinct sets of target genes that orchestrate cellular behaviors via intrinsic and non-autonomous signaling mechanisms. While its loss appears dispensable for development and prevents epithelial aging, ISCs and ECs demand Ets21c function to mount cellular responses to oxidative stress. Ets21c thus emerges as a vital regulator of proliferative homeostasis in the midgut and a determinant of the adult healthspan (Mundorf, 2019).

The intestinal epithelium undergoes continuous homeostatic and acute, stress-induced cellular turnover to ensure tissue integrity and function throughout an organism's lifetime. The replacement of damaged and aberrant cells is fueled by somatic stem cells, whose proliferation is tightly controlled and coordinated with differentiation to satisfy tissue needs while preventing organ degeneration or tumor formation. In the Drosophila adult midgut, which is a functional equivalent of the mammalian small intestine, the intestinal stem cells (ISCs) divide asymmetrically to self-renew and generate two different cell types: the transient enteroblasts (EBs) and the enteroendocrine (EE) lineage-determined cells. Following several rounds of endoreplication, the EBs mature into the large, polyploid, and polarized enterocytes (ECs), which represent the major building blocks of the midgut epithelium. While primarily involved in nutrient resorption, the ECs also serve as a physical and chemical barrier protecting the organism against toxins, pathogens, oxidative stress, and mechanical damage. The runaway stem cell activity and loss of intestinal integrity due to chronic inflammation and increased stress load have been recognized as the prime underlying causes of aging-associated tissue decline and lifespan shortening. How stress signals are transduced and integrated with the homeostatic maintenance mechanisms at the cellular level and the organ level is only partially understood (Mundorf, 2019).

The evolutionarily conserved Jun N-terminal kinase (JNK) signaling is among the key pathways that govern regenerative responses to stress, infection, and damage in the intestine. Its chronic activation has been linked to the breakdown of epithelial integrity and accelerated aging. JNK signaling affects both ISCs and differentiated ECs. In the ECs, JNK confers stress tolerance and promotes epithelial turnover by triggering the apoptosis of damaged ECs and compensatory ISC proliferation. At the same time, cell-autonomous JNK activation in ISCs accelerates ISC mitosis in cooperation with the epidermal growth factor receptor (EGFR/Ras/ERK) signaling pathway, which provides the permissive signal for division. In contrast, JNK suppression prevents age-associated ISC hyperproliferation, accumulation of mis-differentiated cells, and epithelial dysplasia, resulting in lifespan extension. The canonical response to JNK signaling culminates in the activation of transcription factors that orchestrate gene expression. The basic leucine zipper (bZIP) transcription factors Fos (kayak) and Jun (jun-related antigen) are the best-characterized JNK pathway transcriptional effectors during development. In the adult intestine, however, the relation between JNK, Jun, and Fos is less clear. Deficiency for either Fos or Jun interferes with ISC survival, a response that is not observed upon JNK inhibition. In addition, the transcription factor Foxo has been shown to orchestrate adaptive metabolic responses downstream of JNK in ECs. However, Foxo overexpression does not drive epithelial renewal as JNK activation does. These data strongly suggest that other transcription factors may play a role in mediating the pleiotropic, adaptive JNK responses in the different cell types of the intestine (Mundorf, 2019).

The transcription factors of the E-twenty six (ETS) family are functionally conserved in all metazoans and are implicated in a plethora of processes, including cell-cycle control, differentiation, proliferation, apoptosis, and tissue remodeling. Several genome-wide sequencing approaches have determined that Drosophila ets21c, the ortholog of human proto-oncogenes FLI1 and ERG, is transcriptionally induced by infections, wounding, tumorigenesis, and aging. In the case of epithelial tumors and lipopolysaccharide treatment, ets21c upregulation required JNK activity, thus making Ets21c a plausible candidate to act as a JNK effector in the adult intestine (Mundorf, 2019).

This study shows that Ets21c acts as a critical and specific regulator of ISC and EC functions in the adult fly intestine downstream of JNK signaling and in response to oxidative stress and aging. Ets21c is necessary and sufficient to coordinate epithelial turnover by controlling ISC proliferation and the removal of ECs. By regulating specific sets of target genes, Ets21c orchestrates distinct cellular behaviors of midgut cells via intrinsic and non-autonomous signaling mechanisms. While dispensable for normal development, Ets21c functions as a vital determinant of stress tolerance and lifespan (Mundorf, 2019).

By targeted manipulation of Ets21c in progenitor cells, this study shows that Ets21c levels affect the rate of ISC proliferation intrinsically while their maintenance and survival remain unaltered. The reduced ERK activation in ISCs as a consequence of ets21c deficiency in the stress-free context could provide a mechanism for the observed decline in ISC mitotic capacity. This would be consistent with a described dependency of the JNK-induced ISC hyperproliferation on the EGFR/Ras/ERK signaling pathway. The precise mechanism by which Ets21c regulates ERK activity and ISC proliferation remains to be determined. However, the identification of pvf1 as a direct transcriptional target of Ets21c implies that Ets21c could modulate mitotic Pvr/Ras signaling in progenitors through the ISC-derived autocrine and EC-specific paracrine production of Pvf1 (Mundorf, 2019).

Differentiated ECs also require intrinsic Ets21c activity for proper function. EC-specific Ets21c activation drives epithelial turnover, which involves the apoptotic removal of mature ECs and ISC proliferation to renew the pool of differentiated cells. The Ets21c-mediated EC removal and compensatory ISC proliferation response could be suppressed by both co-expression of the pan-caspase inhibitor p35 or knockdown of the Ets21c target eip93F that controls Dcp1 activity. Neither upd3 nor pvf1 silencing in ECs interfered with Dcp1 activation, although both were indispensable for the non-autonomous induction of ISC proliferation by ets21c-expressing ECs. Based on these data, it is concluded that Ets21c orchestrates epithelial turnover by promoting EC apoptosis and stimulating compensatory ISC proliferation by apoptosis-dependent and -independent mechanisms exploiting intercellular signaling molecules such as Pvf1 growth factor and the chief stress-inducible cytokine Upd3. Apoptosis-dependent and -independent induction of ISC proliferation has also been demonstrated for JNK signaling in ECs. The cell death-independent mechanism would also explain why the ISC mitotic rate remains high in paraquat-exposed flies despite Dcp1 inactivation due to EC-specific ets21c silencing. In this respect, it is important to note that other transcription factors besides Ets21c have been shown to regulate Upd3 expression under stress conditions -- for example, in infected ECs or upon oncogene activation in imaginal discs (Mundorf, 2019).

Of note, increased JNK activity has been associated with age- and oxidative stress-related changes in the posterior midgut. Consistent with its role as a JNK-dependent transcriptional effector, Ets21c levels build up in response to paraquat and during aging. ISC/EB- and EC-specific TaDa profiling revealed that only a small fraction of the Ets21c-associated genes was actively transcribed, indicating that in the unstressed state, Ets21c contributes to the fine-tuning of gene expression that supports the steady-state epithelial replenishment. Its seemingly 'unproductive' binding to DNA, however, likely primes a genetic program that can be rapidly executed in response to JNK activation upon challenge. This notion is supported by the significant enrichment for functional GO terms associated with stress-related JNK signaling. Furthermore, Ets21c was shown to regulate genes that have been functionally linked to JNK signaling, including the autophagy-related gene 1 (atg1) and the insulin signaling intersecting-target (Jafrac1), or identified as JNK- and paraquat-responsive genes, such as the eukaryotic translation initiation factor 2α kinase (PEK), a thioredoxin-like protein (fax), or a glutathione S-transferase (gstD10). It is suggested that failure to accelerate intestinal regeneration and mount a robust cytoprotective response underlie the increased sensitivity of ets21c-deficient flies to oxidative stress. The capacity of Ets21c to confer cytoprotection but also trigger apoptosis is in line with the described roles of JNK signaling. It is proposed that the repertoire of Ets21c-regulated target genes and the biological outcome of Ets21c activation depends on the strength and duration of cellular stress and the signaling landscape in which Ets21c operates. Such a model would be in accordance with a showing that low stress levels can accelerate epithelial renewal in the absence of EC death due to moderate JNK induction that stimulates ISC division, while additional input from Hippo signaling accelerates apoptosis to prevent EC overcrowding (Mundorf, 2019).

In addition to its role in coordinating cellular behaviors within the intestine, Ets21c emerges as an important determinant of the adult intestinal healthspan and overall lifespan. Optimizing proliferative homeostasis in high-turnover tissues by, for example, moderate inhibition of insulin/IGF or JNK signaling activities has proven effective in prolonging the lifespan. As Ets21c represents a key effector of JNK in the adult gut and its knockdown reduced ISC proliferation, it is plausible that balanced intestinal function may contribute to the lifespan extension of unchallenged ets21c mutant flies. However, the use of a full-body ets21c mutant prevents drawing of a causal relation between the gut-specific role of Ets21c and longevity. The tissue- and cell-type-specific contribution of Ets21c to adult lifespan remains a question for future studies to address (Mundorf, 2019).

Finally, ets21c has been repeatedly picked up by gene expression profiling studies to be markedly increased in response to immune challenge, injury, oncogene activation, or aging. While functionally linked to JNK signaling in epithelial tumor models, Ets21c has also been classified as an effector of EGFR/ERK signaling in the intestine based on the binding of Capicua, the EGFR/Ras/ERK-regulated transcriptional repressor, to the ets21c locus and upregulation of ets21c expression following Capicua loss. However, the functional evidence placing Ets21c downstream of EGFR/Ras/ERK signaling has been missing. The current data show that while knockdown of ets21c completely rescues the phenotypic consequences of JNK activation in the ISCs or ECs, it fails to mitigate the effects of activated EGFR/Ras/ERK signaling. Therefore, it is proposed that the regulation of ets21c levels results from an integration of positive and negative signaling inputs. This regulatory network includes Capicua, which acts as a gatekeeper of ets21c transcription. Such regulatory mechanisms ensure that JNK-Ets21c-mediated responses are fast but transient in supporting efficient tissue renewal while preventing chronic or excessive Ets21c activation, which drives tissue dysplasia and epithelial degeneration (Mundorf, 2019).

EGFR/Ras signaling controls Drosophila intestinal stem cell proliferation via Capicua-regulated genes

Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. This study demonstrates that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs (Jin 2015).

It is well established that EGFR signaling is essential to drive ISC growth and division in the fly midgut. However, the precise mechanism via which this signal transduction pathway activates ISCs has remained a matter of inference from experiments with other cell types. Moreover, despite a vast literature on the pathway and ubiquitous coverage in molecular biology textbooks, the mechanisms of action of the pathway downstream of the MAPK are not well understood for any cell type. From this study, a model is proposed (see Model for Cic control of Drosophila ISC proliferation). Multiple EGFR ligands and Rhomboid proteases are induced in the midgut upon epithelial damage, which results in the activation of the EGFR, RAS, RAF, MEK, and MAPK in ISCs. MAPK phosphorylates Cic in the nucleus, which causes it to dissociate from regulatory sites on its target genes and also translocate to the cytoplasm. This allows the de-repression of target genes, which may then be activated for transcription by factors already present in the ISCs. The critical Cic target genes identified in this study include the cell cycle regulators stg (Cdc25) and Cyclin E, which in combination are sufficient to drive dormant ISCs through S and M phases, and pnt and Ets21C, ETS-type transcriptional activators that are required and sufficient for ISC activation (Jin 2015).

Upon damage, activated EGFR signaling mediates activation of ERK, which phosphorylates Cic, and relocates it to the cytoplasm. As a result, stg, CycE, Ets21C and pnt transcription are relieved from Cic repression, and induce ISC proliferation (Jin 2015).

Although this study found more than 2000 Cic binding sites in the ISC genome, not all of the genes associated with these sites were significantly upregulated, as assayed by RNA-Seq, upon Cic depletion. One possible explanation for this is that Cic binding sites from DamID-Seq were also associated with other types of transcription units (miRNAs, snRNAs, tRNAs, rRNAs, lncRNAs) that were not scored for activation by the RNA-Seq analysis. Indeed a survey of the Cic binding site distributions suggests this. This might classify some binding sites as non-mRNA-associated. However, it is also possible that many Cic target genes may require activating transcription factors that are not expressed in ISCs. Such genes might not be strongly de-repressed in the gut upon Cic depletion (Jin 2015).

In other Drosophila cells MAPK phosphorylation is thought to directly inactivate the ETS domain repressor Yan, and to directly activate the ETS domain transcriptional activator Pointed P2 (PNTP2). In fact Pnt and Yan have been shown to compete for common DNA binding sites on their target genes. Thus, previous studies proposed a model of transcriptional control by MAPK based solely on post-translational control of the activity of these ETS factors. However, this study found that Pnt and Ets21C were transcriptionally induced by MAPK signaling, and could activate ISCs if overexpressed, and that depleting yan or pntP2 had no detectable proliferation phenotype. In addition, overexpression of PNTP2 was sufficient to trigger ISC proliferation, suggesting either that basal MAPK activity is sufficient for its post-translational activation, or that PNTP2 phosphorylation is not obligatory for activity. Moreover, pntP2 loss of function mutant ISC clones had no deficiency in growth even after inducing proliferation by P.e. infection, which increases MAPK signaling. These observations indicate that the direct MAPK→PNTP2 phospho-activation pathway is not uniquely or specifically required for ISC proliferation. These results suggest instead that transcriptional activation of pnt and Ets21c via MAPK-dependent loss of Cic-mediated repression is the predominant mode of downstream regulation by MAPK in midgut ISCs (Jin 2015).

In addition to activating ISCs for division, EGFR signaling activates them for growth. Previous studies showed loss of EGFR signaling prevented ISC growth and division, and that ectopic RasV12 expression could accelerate the growth not only of ISCs but also post-mitotic enteroblasts. Similarly, this study shows that loss of cic caused ISC clones to grow faster than controls, by increasing cell number as well as cell size. For instance, increased size of GFP+ ISCs and EBs was observed when cic-RNAi was induced by the esg^ts or esg^tsF/O systems. Therefore, in a search for Cic target genes probable growth regulatory genes such as Myc, Cyclin D, the Insulin/TOR components InR, PI3K, S6K and Rheb, Hpo pathway components, and the loci encoding rRNA, tRNAs and snRNAs were specifically checked. It was found that Cic bound to the InR, Akt1, Rheb, Src42A and Yki loci. However, of these only InR mRNA was significantly upregulated in Cic-depleted progenitor cells. In surveying the non-protein coding genome, it was found that Cic had binding sites in many loci encoding tRNA, snRNA, snoRNA and other non-coding RNAs, though not in the 28S rRNA or 5S rRNA genes. Due to the method used for RNA-Seq library production, RNA expression profiling experiments could not detect expression of these loci, and so it remains to be tested whether Cic may regulate some of those non-coding RNA's transcription to control cell growth. It is also possible that Cic controls cell growth regulatory target genes indirectly, for instance via its targets Ets21C and Pnt, which are also strong growth promoters in the midgut. But given that no conclusive model can be drawn from the data regarding how Cic restrains growth, one must consider the possibility that ERK signaling stimulates cell growth via non-transcriptional mechanisms, as proposed by several studies (Jin 2015).

The critical role of Cic as a negative regulator of cell proliferation in the fly midgut is consistent with its tumor suppressor function in mammalian cancer development. Also consistent with the current findings are the observations that the ETS transcription factors ETV1 and ETV5 are upregulated in sarcomas that express CIC-DUX, an oncogenic fusion protein that functions as a transcriptional activator, and that knockdown of CIC induces the transcription of ETV1, ETV4 and ETV5 in melanoma cells. Moreover the transcriptional regulation by ETS transcription factors is important in human cancer development. Their expression is induced in many tumors and cancer cell lines. For example, ERG, ETV1, and ETV4 can be upregulated in prostrate cancers, and ETV1 is upregulated in post gastrointestinal stromal tumors and in more than 40% of melanomas. In addition, the mRNA expression of these ETS genes was correlated with ERK activity in melanoma and colon cancer cell lines with activating mutations in BRAF (V600E), such that their expression decreased upon MEK inhibitor treatment. Furthermore, overexpression of the oncogenic ETS proteins ERG or ETV1 in normal prostate cells can activate a Ras/MAPK-dependent gene expression program in the absence of ERK activation. These cancer studies imply that there is an unknown factor that links Ras/Mapk activity to the expression of ETS factors, and that some of the human ETS factors might act without MAPK phosphorylation, as does Drosophila PntP1. Combining the knowledge of Cic with what was previously known about CIC in tumor development, it is proposed that CIC is the unknown factor that regulates ETS transcription factors in Ras/MAKP-activated human tumors (Jin 2015).

In summary, this study has elucidated a mechanism wherein Cic controls the expression of the cell cycle regulators stg (Cdc25) and Cyclin E, along with the Ets transcription factor Pnt, and perhaps also Ets21C, by directly binding to regulatory sites in their promoters and introns. Using genetic tests it was shown that these interactions are meaningful for regulating stem cell proliferation. Therefore, it is suggested that human CIC may also lead to the transcriptional induction of cell cycle genes and ETS transcription factors in RAS/MAPK activated- or loss-of-function-CIC tumors such as brain or colorectal cancers (Jin 2015).

Loss of a proteostatic checkpoint in intestinal stem cells contributes to age-related epithelial dysfunction

A decline in protein homeostasis (proteostasis) has been proposed as a hallmark of aging. Somatic stem cells (SCs) uniquely maintain their proteostatic capacity through mechanisms that remain incompletely understood. This study describes and characterizes a 'proteostatic checkpoint' in Drosophila intestinal SCs (ISCs). Following a breakdown of proteostasis, ISCs coordinate cell cycle arrest with protein aggregate clearance by Atg8-mediated activation of the Nrf2-like transcription factor cap-n-collar C (CncC). CncC induces the cell cycle inhibitor Dacapo and proteolytic genes. The capacity to engage this checkpoint is lost in ISCs from aging flies, and it can be restored by treating flies with an Nrf2 activator, or by over-expression of CncC or Atg8a. This limits age-related intestinal barrier dysfunction and can result in lifespan extension. These findings identify a new mechanism by which somatic SCs preserve proteostasis, and highlight potential intervention strategies to maintain regenerative homeostasis (Rodriguez-Fernandez, 2019).

Protein Homeostasis (Proteostasis) encompasses the balance between protein synthesis, folding, re-folding and degradation, and is essential for the long-term preservation of cell and tissue function. It is achieved and regulated by a network of biological pathways that coordinate protein synthesis with degradation and cellular folding capacity in changing environmental conditions. This balance is perturbed in aging systems, likely as a consequence of elevated oxidative and metabolic stress, changes in protein turnover rates, decline in the protein degradation machinery, and changes in proteostatic control mechanisms. The resulting accumulation of misfolded and aggregated proteins is widely observed in aging tissues, and is characteristic of age-related diseases like Alzheimer's and Parkinson's disease. The age-related decline in proteostasis is especially pertinent in long-lived differentiated cells, which have to balance the turnover and production of long-lived aggregation-prone proteins over a timespan of years or decades. But it also affects the biology of somatic stem cells (SCs), whose unique quality-control mechanisms to preserve proteostasis are important for stemness and pluripotency (Rodriguez-Fernandez, 2019).

Common mechanisms to surveil, protect from, and respond to proteotoxic stress are the heat shock response (HSR) and the organelle-specific unfolded protein response (UPR). When activated, both stress pathways lead to the upregulation of molecular chaperones that are critical for the refolding of damaged proteins and for avoiding the accumulation of toxic aggregates. If changes to the proteome are irreversible, misfolded proteins are degraded by the proteasome or by autophagy. While all cells are capable of activating these stress response pathways, SCs deal with proteotoxic stress in a specific and state-dependent manner. Embryonic SCs (ESCs) exhibit a unique pattern of chaperone expression and elevated 19S proteasome activity, characteristics that decline upon differentiation. ESCs share elevated expression of specific chaperones (e.g., HspA5, HspA8) and co-chaperones (e.g., Hop) with mesenchymal SCs (MSCs) and neuronal SCs (NSCs), and elevated macroautophagy (hereafter referred to as autophagy) with hematopoietic SCs (HSCs), MSCs, dermal, and epidermal SCs. Defective autophagy contributes to HSC aging. It has further been proposed that SCs can resolve proteostatic stress by asymmetric segregation of damaged proteins, a concept first described in yeast (Rodriguez-Fernandez, 2019).

While these studies reveal unique proteostatic capacity and regulation in SCs, how the proteostatic machinery is linked to SC activity and regenerative capacity, and how specific proteostatic mechanisms in somatic SCs ensure that tissue homeostasis is preserved in the long term, remains to be established. Drosophila intestinal stem cells (ISCs) are an excellent model system to address these questions. ISCs constitute the vast majority of mitotically competent cells in the intestinal epithelium of the fly, regenerating all differentiated cell types in response to tissue damage. Advances made by numerous groups have uncovered many of the signaling pathways regulating ISC proliferation and self-renewal. In aging flies, the intestinal epithelium becomes dysfunctional, exhibiting hyperplasia and mis-differentiation of ISCs and daughter cells. This age-related loss of homeostasis is associated with inflammatory conditions that are characterized by commensal dysbiosis, chronic innate immune activation, and increased oxidative stress. It further seems to be associated with a loss of proteostatic capacity in ISCs, as illustrated by the constitutive activation of the unfolded protein response of the endoplasmic reticulum (UPR-ER), which results in elevated oxidative stress, and constitutive activation of JNK and PERK kinases. Accordingly, reducing PERK expression in ISCs is sufficient to promote homeostasis and extend lifespan (Rodriguez-Fernandez, 2019).

ISCs of old flies also exhibit chronic inactivation of the Nrf2 homologue CncC. CncC and Nrf2 are considered master regulators of the antioxidant response, and are negatively regulated by the ubiquitin ligase Keap1. In both flies and mice, this pathway controls SC proliferation and epithelial homeostasis. It is regulated in a complex and cell-type specific manner. Canonically, Nrf2 dissociates from Keap1 in response to oxidative stress and accumulates in the nucleus, inducing the expression of antioxidant genes. Drosophila ISCs, in turn, exhibit a 'reverse stress response' that results in CncC inactivation in response to oxidative stress. This response is required for stress-induced ISC proliferation, including in response to excessive ER stress, and is likely mediated by a JNK/Fos/Keap1 pathway (Rodriguez-Fernandez, 2019).

The Nrf2 pathway has also been linked to proteostatic control: 'Non-canonical' activation of Nrf2 by proteostatic stress as a consequence of an association between Keap1 and the autophagy scaffold protein p62 has been described in mammals. A similar non-canonical activation of Nrf2 has been described in Drosophila, where CncC activation is a consequence of the interaction of Keap1 with Atg8a, the fly homologue of the autophagy protein LC3. Nrf2/CncC activation induces proteostatic gene expression, including of p62 in mammalian cells and of p62/Ref2P and LC3/Atg8a in flies. Nrf2 is further a central transcriptional regulator of the proteasome in both Drosophila and mammals. Whether and how Nrf2 also influences proteostatic gene expression in somatic SCs remains unclear (Rodriguez-Fernandez, 2019).

This study shows that Drosophila CncC links cell cycle control with proteostatic responses in ISCs via the accumulation of dacapo, a p21 cell cycle inhibitor homologue, as well as the transcriptional activation of genes encoding proteases and proteasome subunits. This study establish that this program constitutes a transient 'proteostatic checkpoint', which allows clearance of protein aggregates before cell cycle activity is resumed. In old flies, this checkpoint is impaired and can be reactivated with a CncC activator (Rodriguez-Fernandez, 2019).

The central role of Nrf2/CncC in the proteostatic checkpoint is consistent with its previously described and evolutionarily conserved influence on longevity and tissue homeostasis, and is likely to be conserved in mammalian SC populations, as Nrf2 has for example been shown to influence proliferative activity, self-renewal and differentiation in tracheal basal cells. It may be unique to somatic SCs, however, as CncC or Nrf2-mediated inhibition of cell proliferation is not observed during development (such as in imaginal discs) or in other dividing cells. Assessing the existence of an Nrf2-induced proteostatic checkpoint in mammalian SC populations will be an important future endeavor (Rodriguez-Fernandez, 2019).

Mechanistically, the results support a model in which the presence of protein aggregates activates CncC through Atg8a-mediated sequestration of Keap1. In mammals, Nrf2 activation can also be achieved through the interaction of Keap1 with the Atg8a homologue LC3 and p62, and ref2p/p62 contributes to the degradation of polyQ aggregates in Drosophila, suggesting that a conserved Atg8a/p62/Keap1 interaction may be involved in the activation of the proteostatic checkpoint. The activation of CncC after cytosolic proteostatic stress described in this study thus differs mechanistically and in its consequence from the regulation of CncC after other types of protein stress in ISCs: in response to unfolded protein stress in the ER, CncC is specifically inactivated by a ROS/JNK-mediated signaling pathway. This mechanism allows ISC proliferation to be increased in response to tissue damage, but can also contribute to the loss of tissue homeostasis in aging conditions. The activation of CncC after cytosolic protein stress, in turn, allows arresting ISC proliferation during protein aggregate clearance. The distinct responses of ISCs to cytosolic or ER-localized proteostatic stress has interesting implications for understanding of the maintenance of tissue homeostasis. While the XBP1-mediated UPR-ER allows the expansion of the ER and the induction of ER chaperones to deal with a high load of unfolded proteins in the ER, it also stimulates ISC proliferation through oxidative stress and the activation of PERK and JNK. It is tempting to speculate that the sequestration of unfolded proteins within the ER allows ISCs to proceed through mitosis without the possibility of major misregulation, while the presence of cytosolic protein aggregates may be a unique danger to the viability of the cell and its daughters. It seems likely that constitutive activation of autophagy and proteasome pathways during the clearance of cytosolic aggregates is incompatible with the need for intricate regulation of these same pathways during the cell cycle in proliferating ISCs. It will be of interest to explore this hypothesis further in the future (Rodriguez-Fernandez, 2019).

The data suggest that the coordination of cell cycle arrest and aggregate clearance is achieved by the simultaneous induction of the cell cycle inhibitor Dacapo and a battery of genes encoding proteins involved in proteolysis. While it was possible to detect dacapo transcript expression in ISCs by fluorescent in situ hybridization at 24h after HttQ138 expression, it remains unclear whether Dacapo is induced directly by CncC or via the action of a CncC target gene. It is surprising that transcriptional induction of autophagy genes in was not seen in a RNAseq experiment, but it is possible that this is due to the fact that only one timepoint was sampled after induction of protein aggregates. Since the transcriptional response of autophagy genes is likely very dynamic, a more time-resolved transcriptome analysis during aggregate formation and clearance may have captured such a response (Rodriguez-Fernandez, 2019).

It is further notable that dap deficient ISC clones exhibit a significantly higher aggregate load in these experiments than wild-type ISC clones. This suggests that the induction of proteolytic genes and of cell cycle regulators is not only coincidentally linked by CncC, but that aggregate clearance and the cell cycle arrest mediated by Dacapo need to be tightly coordinated for effective ISC proteostasis. It will be interesting to explore the mechanism of this requirement in the future. It is tempting to speculate that, as the elimination of protein aggregates requires an increase in proteasome activity, and proteasome activity can influence cell cycle timing, cell cycle inhibition is a critical safeguard against de-regulation of normal cell cycle progression (Rodriguez-Fernandez, 2019).

The data suggest that Atg8a induction in ISCs experiencing proteostatic stress may serve a dual purpose: sustained activation of the proteostatic checkpoint as well as increased autophagy flux. This dual role is distinct from other autophagy components like Atg1, since Atg1 over-expression, an efficient way of promoting autophagy in Drosophila cells, counteracts the checkpoint rather than promoting it. Exploring the relative kinetics of Atg8a and Atg1 induction in ISCs after proteostatic stress is likely to provide deeper mechanistic insight into the regulation of the proteostatic checkpoint (Rodriguez-Fernandez, 2019).

Critically, the proteostatic checkpoint is reversible. Based on the current data and previous studies, it is proposed that upon clearance of aggregates, the Keap1/Atg8a interaction is decreased, thus releasing Keap1 to inhibit CncC. Lineage-tracing studies show that this allows re-activation of ISC proliferation and recovery of normal regenerative responses (Rodriguez-Fernandez, 2019).

The loss of proteostatic checkpoint efficiency in ISCs of old guts is likely a consequence of the age-related inactivation of CncC in these cells (possibly caused by chronic oxidative stres. Accordingly, reactivating Nrf2/CncC in the gut by overexpressing CncC is sufficient to restore epithelial homeostasis in the intestine of old flies, and this study found that exposing animals to Otipraz intermittently late in life promotes epithelial barrier function and extends lifespan (Rodriguez-Fernandez, 2019).

Since Nrf2/CncC and other components required for the proteostatic checkpoint are conserved across species, it is anticipated that the current findings will be relevant to homeostatic preservation of adult SCs in vertebrates. Supporting this view, mammalian Cdkn1a (p21) has been described as an Nrf2 target gene. Transient activation of Nrf2 may thus be a viable intervention strategy to improve proteostasis and maintain regenerative capacity in high-turnover tissues of aging individuals (Rodriguez-Fernandez, 2019).

Recruitment of adult precursor cells underlies limited repair of the infected larval midgut in Drosophila

Surviving infection requires immune and repair mechanisms. Developing organisms face the additional challenge of integrating these mechanisms with tightly controlled developmental processes. The larval Drosophila midgut lacks dedicated intestinal stem cells. This study shows that, upon infection, larvae perform limited repair using adult midgut precursors (AMPs). AMPs differentiate in response to damage to generate new enterocytes, transiently depleting their pool. Developmental delay allows for AMP reconstitution, ensuring the completion of metamorphosis. Notch signaling is required for the differentiation of AMPs into the encasing, niche-like peripheral cells (PCs), but not to differentiate PCs into enterocytes. Dpp (TGF-beta) signaling is sufficient, but not necessary, to induce PC differentiation into enterocytes. Infection-induced JAK-STAT pathway is both required and sufficient for differentiation of AMPs and PCs into new enterocytes. Altogether, this work highlights the constraints imposed by development on an organism's response to infection and demonstrates the transient use of adult precursors for tissue repair (Houtz, 2019).

Gilgamesh (Gish)/CK1gamma regulates tissue homeostasis and aging in adult Drosophila midgut

Adult tissues and organs rely on resident stem cells to generate new cells that replenish damaged cells. To maintain homeostasis, stem cell activity needs to be tightly controlled throughout the adult life. This study shows that the membrane-associated kinase Gilgamesh (Gish)/CK1gamma maintains Drosophila adult midgut homeostasis by restricting JNK pathway activity and that Gish is essential for intestinal stem cell (ISC) maintenance under stress conditions. Inactivation of Gish resulted in aberrant JNK pathway activation and excessive production of multiple cytokines and growth factors that drive ISC overproliferation. Mechanistically, Gish restricts JNK activation by phosphorylating and destabilizing a small GTPase, Rho1. Interestingly, this study found that Gish expression is down-regulated in aging guts and that increasing Gish activity in aging guts can restore tissue homeostasis. Hence, this study identifies Gish/CK1gamma as a novel regulator of Rho1 and gatekeeper of tissue homeostasis whose activity is compromised in aging guts (Li, 2020).

Adult stem cells are essential for tissue homeostasis and regeneration, and their activity needs to be tightly controlled in order to maintain the normal cellular architecture and physiological function of adult organs. The JNK pathway plays a pivotal role in the Drosophila midgut homeostasis and injury response. In addition, JNK pathway activity is elevated in aging guts, leading to aberrant stem cell proliferation and loss of tissue homeostasis. How the JNK pathway is kept in check during gut homeostasis and aging is not well understood. This study provides both genetic and biochemical evidence that the membrane-associated kinase Gish/CK1γ restricts JNK pathway activity by phosphorylating and destabilizing Rho1, an upstream activator of the JNK pathway. This regulatory pathway is critical for the maintenance of midgut homeostasis as loss of Gish results in elevated JNK pathway activity and ISC proliferation. Interestingly, Gish expression in the midgut declines with aging, which may contribute to the elevated JNK pathway activity and ISC overproliferation in old guts. In support of this notion, it was found that transgenic expression of Gish in old guts could restore JNK pathway activity and ISC proliferation to homeostatic levels. How Gish expression is down-regulated in aging guts remains an open question, but it appears to occur at the level of transcription, although the possibility cannot be ruled out that posttranscriptional regulation may also occur. The precise mechanism awaits further investigation (Li, 2020).

Prolonged activation of JNK can induce apoptosis in both Drosophila and mammalian cells. As a negative regulator of JNK pathway activity, Gish synergizes with Puc, another JNK pathway inhibitor, to protect ISCs from undergoing apoptosis; thus, it may play a role in stem cell maintenance under stress condition. The synergistic interaction between Gish and Puc is not restricted to ISCs but also occurs in other tissues. For example, in wing imaginal discs, depletion of Gish only resulted in low levels of apoptosis; however, removal of one copy of puc (puc^E96/+) in Gish-depleted wing discs (MS>GishRNAi, puc^E96/+) resulted in massive cell death, leading to the formation of small wings. The increased apoptosis in MS>GishRNAi, puc^E96/+ appeared to mirror the dramatic activation of the JNK pathway. Hence, the cell- and tissue-protective function of Gish could simply be attributed to its role in suppressing JNK signaling. However, it remains possible that other pathways downstream of Rho1 and/or Gish may contribute to the induction of apoptosis in Gish and Puc double-deleted cells in parallel to heightened JNK pathway activity (Li, 2020).

Although mainly regulated by GTP-GDP cycling, Rho GTPases can also be regulated by posttranslational modifications such as phosphorylation that control their subcellular localization, stability, and complex formation. For example, a previous study showed that Erk2-mediated phosphorylation of RhoA targeted it for ubiquitin/proteasome-mediated degradation in cultured epithelial cells. However, the phosphorylation site(s) on RhoA that regulates its stability remained unidentified, and the physiological role of Erk2-mediated phosphorylation of RhoA was not determined. This study provides both genetic and biochemical evidence that Gish phosphorylates a cluster of sites in the N-terminal region of Rho1, which targets Rho1 for ubiquitination, followed by proteasome-mediated degradation. Gish-mediated down-regulation of Rho1 restricts JNK pathway activity, which is critical for adult Drosophila midgut homeostasis. How Rho1 inhibits JNK pathway remains an open question. A previous study revealed that Rho1 physically interacts with Slpr/JNKKK regardless of its GDP/GTP binding state and that Rho1 promotes Slpr cortical localization. Therefore, it is possible that plasma membrane-associated Rho1 promotes JNK pathway activation by increasing the local concentration of Slpr/JNKKK at the plasma membrane. The precise biochemical mechanism by which Rho1 activates Slpr/JNKKK awaits further investigation (Li, 2020).

Rho GTPases shuttle between plasma membrane and cytoplasm with GTP-bound active forms associated with membrane. Gish/CK1γ belongs to the CK1 family kinases. Unlike other family members that are located mainly in the cytoplasm, Gish/CK1γ is associated with plasma membrane through its C-terminal lipid modification. This study has demonstrated that the function of Gish in restricting ISC overproliferation depended on its kinase activity and membrane association, suggesting that Gish may phosphorylate plasma membrane-associated Rho1 to prevent its aberrant accumulation at the plasma membrane where it can activate the JNK pathway. This may explain why the role of Gish/CK1γ cannot be replaced by other CK1 family members. Since the CK1 phosphorylation sites on Rho1 are also found in all members of mammalian Rho GTPase, it is speculated that CK1γ may play a conserved role in the regulation of Rho GTPase activity. Future study is needed to determine whether CK1γ regulates Rho GTPase activity and whether such regulation plays a role in development, tissue homeostasis, and aging in mammals (Li, 2020).

Taurine represses age-associated gut hyperplasia in Drosophila via counteracting endoplasmic reticulum stress

As they age, adult stem cells become more prone to functional decline, which is responsible for aging-associated tissue degeneration and diseases. One goal of aging research is to identify drugs that can repair age-associated tissue degeneration. Multiple organ development-related signaling pathways have recently been demonstrated to have functions in tissue homeostasis and aging process. Therefore, in this study, several chemicals that are essential for organ development were tested to assess their ability to delay intestinal stem cell (ISC) aging and promote gut function in adult Drosophila. Taurine, a free amino acid that supports neurological development and tissue metabolism in humans, was found to repress ISC hyperproliferation and restrain the intestinal functional decline seen in aged animals. Taurine was found to repress age-associated ISC hyperproliferation through a mechanism that eliminated endoplasmic reticulum (ER) stress by upregulation of the target genes of unfolded protein response in the ER (UPR(ER)) and inhibiting the c-Jun N-terminal kinase (JNK) signaling. These findings show that taurine plays a critical role in delaying the aging process in stem cells and suggest that it may be used as a natural compound for the treatment of age-associated, or damage-induced intestinal dysfunction in humans (Du, 2021).

Warburg-like metabolic reprogramming in aging intestinal stem cells contributes to tissue hyperplasia

In many tissues, stem cell (SC) proliferation is dynamically adjusted to regenerative needs. How SCs adapt their metabolism to meet the demands of proliferation and how changes in such adaptive mechanisms contribute to age-related dysfunction remain poorly understood. This study identified mitochondrial Ca(2+) uptake as a central coordinator of SC metabolism. Live imaging of genetically encodasensors in intestinal SCs (ISCs) of Drosophila reveals that mitochondrial Ca(2+) uptake transiently adapts electron transport chain flux to match energetic demand upon proliferative activation. This tight metabolic adaptation is lost in ISCs of old flies, as declines in mitochondrial Ca(2+) uptake promote a "Warburg-like" metabolic reprogramming toward aerobic glycolysis. This switch mimics metabolic reprogramming by the oncogene Ras(V12) and enhances ISC hyperplasia. These data identify a critical mechanism for metabolic adaptation of tissue SCs and reveal how its decline sets aging SCs on a metabolic trajectory reminiscent of that seen upon oncogenic transformation (Morris, 2020).

Low-protein diet applied as part of combination therapy or stand-alone normalizes lifespan and tumor proliferation in a model of intestinal cancer

Tumors of the intestinal tract are among the most common tumor diseases in humans, but, like many other tumor entities, show an unsatisfactory prognosis with a need for effective therapies. To test whether nutritional interventions and a combination with a targeted therapy can effectively cure these cancers, the fruit fly Drosophila was used as a model. In this system, tumors were introduced by EGFR overexpression in intestinal stem cells. Limiting the amount of protein in the diet restored life span to that of control animals. In combination with a specific EGFR inhibitor, all major tumor-associated phenotypes could be rescued. This form of treatment was also successful in a real treatment scenario, which means when they started after the full tumor phenotype was expressed. In conclusion, reduced protein administration can be a very promising form of adjuvant cancer therapy (Proske, 2021).

Ursolic Acid Protects Sodium Dodecyl Sulfate-Induced Drosophila Ulcerative Colitis Model by Inhibiting the JNK Signaling

Ursolic acid (UA) is a bioactive molecule widely distributed in various fruits and vegetables, which was reported to play a therapeutic role in ulcerative colitis (UC) induced by toxic chemicals. However, the underlying mechanism has not been well clarified in vivo. In this study, using a Drosophila UC model induced by sodium dodecyl sulfate (SDS), the defensive effect of UA on intestinal damage was investigated. The results showed that UA could significantly protect Drosophila from the damage caused by SDS exposure. Further, UA alleviated the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by SDS and upregulated the activities of total superoxide dismutase (T-SOD) and catalase (CAT). Moreover, the proliferation and differentiation of intestine stem cells (ISCs) as well as the excessive activation of the c-Jun N-terminal kinase (JNK)-dependent JAK/STAT signaling pathway induced by SDS were restored by UA. In conclusion, UA prevents intestine injury from toxic compounds by reducing the JNK/JAK/STAT signaling pathway. UA may provide a theoretical basis for functional food or natural medicine development (Wei, 2022).

The Drosophila CD36 Homologue croquemort Is Required to Maintain Immune and Gut Homeostasis during Development and Aging

Phagocytosis is an ancient mechanism central to both tissue homeostasis and immune defense. Both the identity of the receptors that mediate bacterial phagocytosis and the nature of the interactions between phagocytosis and other defense mechanisms remain elusive. This study reports that Croquemort (Crq), a Drosophila member of the CD36 family of scavenger receptors, is required for microbial phagocytosis and efficient bacterial clearance. Flies mutant for crq are susceptible to environmental microbes during development and succumb to a variety of microbial infections as adults. Crq acts parallel to the Toll and Imd pathways to eliminate bacteria via phagocytosis. crq mutant flies exhibit enhanced and prolonged immune and cytokine induction accompanied by premature gut dysplasia and decreased lifespan. The chronic state of immune activation in crq mutant flies is further regulated by negative regulators of the Imd pathway. Altogether, these data demonstrate that Crq plays a key role in maintaining immune and organismal homeostasis (Guillou, 2016).

This study shows that Crq is required for the engulfment of microbes by plasmatocytes and their clearance. The mild immune deficiency due to crq mutation is associated with increased susceptibility to infection, defects in immune homeostasis, gut hyperplasia, and decreased lifespan. This study also re-confirmed a role for crq in apoptotic cell clearance, although the phagocytosis defect of crq^ko plasmatocytes is less severe than what had been previously observed with two lethal crq deficiency mutants, Df(2L)al and Df(2L)XW88. A possible explanation is that these deficiencies may have deleted at least one other gene required for apoptotic cell clearance. Additionally, morphological defects associated with secondary mutations could have exacerbated the crq phagocytosis defect by preventing efficient plasmatocyte migration to apoptotic cells. These same deficiency mutants had been assessed qualitatively for phagocytosis of bacteria by injecting embryos with E. coli or S. aureus; their plasmatocytes had no obvious defect in their ability to engulf these bacteria. However, a role for crq in phagocytosis of S. aureus, but not that of E. coli, was subsequently proposed based on S2 cell phagocytosis assays following knock-down of crq by RNAi. The current study shows that crq is required in vivo for uptake and phagosome maturation of both S. aureus and E. coli. A simple explanation of this discrepancy with E. coli could be that knocking down crq by RNAi is not sufficient to affect its role in E. coli phagocytosis (but sufficient to affect its role in S. aureus phagocytosis) and that completely abrogating crq expression by in vivo knock-out leads to a stronger phenotype with both bacteria. The in vivo data in crq^ko flies further demonstrate that crq is required to resist multiple microbial infections, such as Ecc15, E. faecalis, B. bassiana, and C. albicans. These data therefore argue that crq plays a more general role in microbial phagocytosis than was previously anticipated. Previous experiments to test whether crq is required for bacterial phagocytosis in embryos were qualitative rather than quantitative and did not allow identification of a role for crq at that stage. In contrast, the current experiments in adult crq^ko flies are quantitative and allowed identification of a delay in phagocytosis, followed by a defect in bacterial clearance in crq^ko hemocytes. A possible explanation for this discrepancy would be that hemocytes may differ in their expression profile, behavior, and phagocytic ability at various developmental stages due to differences in their microenvironment and/or sensitivity to stimuli. Accordingly, it has recently been shown that the phagocytic activity of embryonic hemocytes acts as a priming mechanism, increasing the ability of primed cells to phagocytose bacteria at later stages. It is therefore possible that embryonic, larval and adult hemocytes display very different levels of priming and bacterial phagocytic activity, and that crq is required mostly in larval/adult bacterial phagocytosis. Alternatively, a potential defect in phagocytosis of bacteria by embryonic hemocytes of the crq deficiencies may have been suppressed by the deletion of (an)other gene(s) in that genomic region (Guillou, 2016).

Because the immune competence of hemocytes varies during development, the potential role for crq in innate immunity was examined by knocking it out. This study shows that Crq is a major plasmatocyte marker at all developmental stages of the fly. crq^ko flies are homozygous viable, but short-lived, and can hardly be maintained as a homozygous stock in a non-sterile environment; crq^ko pupae become susceptible to environmental bacteria and their microbiota during pupariation. In a recent study, Arefin (2015) induced the pro-apoptotic genes hid or Grim in plamatocytes and crystal cells using the Hml-Gal4 driver (Hml-apo). A similar pupal lethality was observed but also associated with an induction of lamellocyte differentiation and the apparition of melanotic tumors of hemocyte origin. It was therefore concluded that the death of hemocytes triggered lamellocyte accumulation and melanotic tumor phenotypes. In contrast, no obvious melanotic tumors were observed in crq^ko flies, despite observing a loss of hemocytes in aging crq^ko flies and crq^ko flies subjected to Ecc15 infection. One possible explanation is that hemocytes do not die of apoptosis in crq^ko flies, but of a distinct mechanism. Alternatively, crq mutation could affect more hemocytes than Hml-apo flies, as crq is expressed in all plasmatocytes while Hml is only expressed in 72.4% of all plasmatocytes expressing crq. Thus the 27.6% of non-Hml plasmatocytes (thus non induced for apoptosis, which is Hml-Gal4 dependent) may respond to the death of the other plasmatocytes by inducing a signal that triggers the induction of lamellocytes and the subsequent formation of melanotic tumors. Considering the role of crq in apoptotic cell clearance, this signal may require a functional crq, which could explain why crq^ko flies do not develop melanotic tumors. Strikingly, in the Arefin study, as well as in previous studies, targeted ablation of plasmatocytes also made resulting 'hemoless' pupae more susceptible to environmental microbes. Extensive tissue remodeling takes place at pupariation, and plasmatocytes are essential to remove dying cells, debris, and bacteria. Thus, it was argued that this increased susceptibility was likely due to environmental bacteria invading the body cavity after disruption of the gut. In addition, it was found that the gut microbiome of Hml-apo flies could influence pupal lethality, as the eclosure rate of Hml-apo flies varied depending on the quality of the food they were reared on. Accordingly, the rescue of the crq^ko pupal lethality with antibiotics demonstrates that their premature aging and death are indeed due to infection by normally innocuous environmental bacteria. Altogether, these data suggest that phagocytes and crq are important actors regulating the interaction between a host and its microbiome (Guillou, 2016).

Hosts use both resistance and tolerance mechanisms to withstand infection and survive a specific dose of microbes. crq^ko flies exhibit a shorter lifespan when compared to control flies, but they are equally tolerant to aseptic wounds and infections. The crq^ko flies are less resistant to infection, as crq is required to promote efficient microbial phagocytosis. crq^ko plasmatocytes can still engulf bacteria, albeit at a lower efficiency than their controls. The data also demonstrate that crq plays a major role in phagosome maturation during bacterial clearance. This is in agreement with a recent study showing that crq promotes phagosome maturation during the clearance of neuronal debris by epithelial cells (Han, 2014). Thus, crq is required at several stages of phagocytosis. Similar observations have been made for the C. elegans Ced-1 receptor and for Drpr, as both promote engulfment of apoptotic corpses and their degradation in mature phagosomes (Guillou, 2016).

'Hemoless', Hml-apo and crq^ko flies are all more susceptible to environmental microbes and their microbiota. While it is not known whether mutants of eater, which encodes a phagocytic receptor for bacteria but does not play a role in phagosome maturation, are more susceptible to environmental microbes during pupariation, both eater mutants and 'hemoless' flies showed either decreased or unaffected systemic responses. Hml-apo larvae however, showed an upregulation in Toll-dependent constitutive Drs mRNA levels whereas Dpt expression was suppresse. In contrast, crq^ko flies showed no significant difference in constitutive or infection induced expression of Drs, but showed an increased expression of Dpt with age, and infection induced an increased and chronic expression of Dpt. Altogether the results argue that phagosome maturation defects in crq^ko flies lead to persistence of bacteria and thus to an increased and persistent systemic immune response via the Imd pathway. Such defects in phagosome maturation are not present in hemocyte ablation experiments, which could explain different outcomes for the host immunity and survival (Guillou, 2016).

This study have found that Crq acts in parallel to the Toll and Imd pathways. In the mealworm Tenebrio molitor, hemocytes and cytotoxic enzymatic cascades eliminate most bacteria early during infection, and AMPs are required to eliminate persisting bacteria. These data suggest that AMPs act in parallel with hemocytes to fight infections. It was also found that crq^ko flies are more susceptible to infection with S. aureus than wild-type and Toll pathway-deficient flies. These results are consistent with S. aureus infection being mainly resolved via phagocytosis and Crq having a major role in this process. Surprisingly, the opposite was observed for infection with other Gram-negative or positive bacteria and fungi. Drosophila mutants for AMP production were more susceptible to infection than crq^ko flies, arguing that AMPs are critical to eliminate the bulk of pathogens. Indeed, crq (thus phagocytosis) is not essential for Ecc15 elimination, but accelerates bacterial clearance. The results also suggest that the defects in phagosome maturation may allow some bacteria to persist and grow within hemocytes, where they are hidden from systemic AMPs. Thus, depending on the microbe, humoral and cellular immune responses can act at distinct stages of infection. In this context, phagocytosis acts as a main defense mechanism against pathogens that may escape AMPs or modulate their production (Guillou, 2016).

Chronic activation of immune pathways can be detrimental to organismal health. In Drosophila, multiple negative regulators of the Imd pathway, including PGRP-LB, act in concert to maintain immune homeostasis. This study has observed that crq^ko flies sustain high production levels of the AMP Dpt and the cytokine Upd3, demonstrating that defects in phagocyte function can lead to chronic immune activation. Notably, the level of Dpt expression induced by activation of the Imd pathway in unchallenged conditions is stronger in crq^ko flies than was previously observed in mutants of three negative regulators of the Imd pathway, namely pirk^EY, PGRP-SC^Δ, and PGRP-^LBΔ, and over 1,000-fold higher in PGRP-LB^LBΔ, crq^ko double mutants. This is despite the persistence of only a few hundred bacteria in these mutants. This phenotype may be due solely to the accumulation of these persistent bacteria, or Crq may also function in plasmatocytes to remove immunostimulatory molecules from the hemolymph. Nonetheless, this study shows that plasmatocytes, Crq, and phagocytosis are all key factors in the immune response, and that losing crq induces a state of chronic immune induction (Guillou, 2016).

The ability of a host to control microbes decreases with age, a phenomenon called immune senescence. The causes of immune senescence remain elusive, but the loss of immune cells with age and a decline in their ability to phagocytose have been suggested. Recent studies have argued that microbial dysbiosis and disruption in gut homeostasis contribute to early aging. In addition, persistent activation of the JAK-STAT pathway in the gut has been linked to age-related decline in gut structure and function. Aging crq^ko flies lose a greater number of hemocytes than wild-type flies after infection, which may be the result of accumulating bacteria in these hemocytes in which phagosomes fail to mature. The premature death of crq^ko flies could be partially rescued by the presence of antibiotics. This demonstrates that phagocytosis, and phagosome maturation in particular, plays a crucial role in managing the response to environmental microbes and potentially, the gut microbiota directly to promote normal aging. This study also found that chronic upd3 expression in crq^ko flies triggers premature midgut hyperplasia, which is known to alter host physiology and promote premature aging. It has recently been proposed that plasmatocytes can influence gut homeostasis by secreting dpp ligands and modulating stem cell activity. These results reinforce the possibility of an interaction between plasmatocyte function and gut homeostasis, and suggests that cytokines derived from hemocytes can trigger cell responses in the gut. These results are also in agreement with a recent publication showing that Upd3 from hemocytes can trigger intestinal stem cell proliferation. Altogether, these results demonstrate that the interaction between hemocytes and the gut tissue are central to host health, and the data demonstrate that phagocytic defects can be associated with chronic gut inflammation and aberrant intestinal stem cell turn-over. As gut aging and barrier integrity are in turn important to maintain bodily immune homeostasis, the following model is proposed: in crq^ko flies, plasmatocyte-derived cytokines accelerate gut aging promoting loss of gut homeostasis and microbial dysbiosis, with immune and plasmatocyte activation acting in a positive feedback loop (Guillou, 2016).

Collectively, these data show that Crq is essential in development and aging to protect against environmental microbes. Interestingly, the impact of mutating crq on host physiology is strikingly different from previously reported phagocytic receptor mutations. It is speculated that this could be due to its dual role in uptake and phagosome maturation during phagocytosis. Crq is required for microbial elimination in parallel to the Toll and Imd pathways and acts to maintain immune homeostasis. This situation is surprisingly reminiscent of inflammatory disorders, such as Crohn's disease, that result from primary defects in bacterial elimination and trigger chronic immune activation and disruption of gut homeostasis. Further characterization of the crq mutation in Drosophila will provide an interesting conceptual framework to understand auto-inflammatory diseases and their repercussions on immune homeostasis and host health (Guillou, 2016).

Intestine-derived α-synuclein initiates and aggravates pathogenesis of Parkinson's disease in Drosophila

Aberrant aggregation of α-synuclein (α-syn) is a key pathological feature of Parkinson's disease (PD), but the precise role of intestinal α-syn in the progression of PD is unclear. In a number of genetic Drosophila models of PD, α-syn was frequently ectopically expressed in the neural system to investigate the pathobiology. This study investigated the potential role of intestinal α-syn in PD pathogenesis using a Drosophila model. Human α-syn was overexpressed in Drosophila guts, and life span, survival, immunofluorescence and climbing were evaluated. Immunofluorescence, Western blotting and reactive oxygen species (ROS) staining were performed to assess the effects of intestinal α-syn on intestinal dysplasia. High-throughput RNA and 16S rRNA gene sequencing, quantitative RT-PCR, immunofluorescence, and ROS staining were performed to determine the underlying molecular mechanism. It was found that the midgut α-syn alone recapitulated many phenotypic and pathological features of PD, including impaired life span, loss of dopaminergic neurons, and progressive motor defects. The intestine-derived α-syn disrupted intestinal homeostasis and accelerated the onset of intestinal ageing. Moreover, intestinal expression of α-syn induced dysbiosis, while microbiome depletion was efficient to restore intestinal homeostasis and ameliorate the progression of PD. Intestinal α-syn triggered ROS, and eventually led to the activation of the dual oxidase (DUOX)-ROS-Jun N-terminal Kinase (JNK) pathway. In addition, α-syn from both the gut and the brain synergized to accelerate the progression of PD. The intestinal expression of α-syn recapitulates many phenotypic and pathologic features of PD, and induces dysbiosis that aggravates the pathology through the DUOX-ROS-JNK pathway in Drosophila. These findings provide new insights into the role of intestinal α-syn in PD pathophysiology (Liu, 2022).

Hemocytes are critical for Drosophila melanogaster post-embryonic development, independent of control of the microbiota

Proven roles for hemocytes (blood cells) have expanded beyond the control of infections in Drosophila. Despite this, the critical role of hemocytes in post-embryonic development has long thought to be limited to control of microorganisms during metamorphosis. This has previously been shown by rescue of adult development in hemocyte-ablation models under germ-free conditions. This study shows that hemocytes have a critical role in post-embryonic development beyond their ability to control the microbiota. Using a newly generated, strong hemocyte-specific driver line for the GAL4/UAS system, it was shown that specific ablation of hemocytes is early pupal lethal, even under axenic conditions. Genetic rescue experiments prove that this is a hemocyte-specific phenomena. RNA-seq data suggests that dysregulation of the midgut is a prominent consequence of hemocyte ablation in larval stages, resulting in reduced gut lengths. Dissection suggests that multiple processes may be affected during metamorphosis. It is believed that this novel role for hemocytes during metamorphosis is a major finding for the field (Stephenson, 2022).

Kanamycin treatment in the pre-symptomatic stage of a Drosophila PD model prevents the onset of non-motor alterations

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor alterations, which is preceded by a prodromal stage where non-motor symptoms are observed. Over recent years, it has become evident that this disorder involves other organs that communicate with the brain like the gut. Importantly, the microbial community that lives in the gut plays a key role in this communication, the so-called microbiota-gut-brain axis. Alterations in this axis have been associated to several disorders including PD. This study proposed that the gut microbiota is different in the presymptomatic stage of a Drosophila model for PD, the Pink1^B9 mutant fly, as compared to that observed in control animals. The results show this is the case: there is basal dysbiosis in mutant animals evidenced by substantial difference in the composition of midgut microbiota in 8-9 days old Pink1^B9 mutant flies as compared with control animals. Further, young adult control and mutant flies were fed kanamycin, and motor and non-motor behavioral parameters were analyzed in these animals. Data show that kanamycin treatment induces the recovery of some of the non-motor parameters altered in the pre-motor stage of the PD fly model, while there is no substantial change in locomotor parameters recorded at this stage. On the other hand, the results show that feeding young animals the antibiotic, results in a long-lasting improvement of locomotion in control flies. These data support that manipulations of gut microbiota in young animals could have beneficial effects on PD progression and age-dependent motor impairments (Molina-Mateo, 2023).

The Drosophila melanogaster gut microbiota provisions thiamine to its host

The microbiota of Drosophila melanogaster has a substantial impact on host physiology and nutrition. Some effects may involve vitamin provisioning, but the relationships between microbe-derived vitamins, diet, and host health remain to be established systematically. This study explored the contribution of microbiota in supplying sufficient dietary thiamine (vitamin B1) to support D. melanogaster at different stages of its life cycle. Using chemically defined diets with different levels of available thiamine, it was found that the interaction of thiamine concentration and microbiota did not affect the longevity of adult D. melanogaster Likewise, this interplay did not have an impact on egg production. However, it was determined that thiamine availability has a large impact on offspring development, as axenic offspring were unable to develop on a thiamine-free diet. Offspring survived on the diet only when the microbiota was present or added back, demonstrating that the microbiota was able to provide enough thiamine to support host development. Through gnotobiotic studies, it was determined that Acetobacter pomorum, a common member of the microbiota, was able to rescue development of larvae raised on the no-thiamine diet. Further, it was the only microbiota member that produced measurable amounts of thiamine when grown on the thiamine-free fly medium. Its close relative Acetobacter pasteurianus also rescued larvae; however, a thiamine auxotrophic mutant strain was unable to support larval growth and development. The results demonstrate that the D. melanogaster microbiota functions to provision thiamine to its host in a low-thiamine environment (Sannino, 2018).

Microbial quantity impacts Drosophila nutrition, Development, and Lifespan

In Drosophila, microbial association can promote development or extend life. This study tested the impact of microbial association during malnutrition and shows that microbial quantity is a predictor of fly longevity. Although all tested microbes, when abundantly provided, can rescue lifespan on low-protein diet, the effect of a single inoculation seems linked to the ability of that microbial strain to thrive under experimental conditions. Microbes, dead or alive, phenocopy dietary protein, and the calculated dependence on microbial protein content is similar to the protein requirements determined from fly feeding studies, suggesting that microbes enhance host protein nutrition by serving as protein-rich food. Microbes that enhance larval growth are also associated with the ability to better thrive on fly culture medium. These results suggest an unanticipated range of microbial species that promote fly development and longevity and highlight microbial quantity as an important determinant of effects on physiology and lifespan during undernutrition (Keebaugh, 2018).

RNA polymerase III limits longevity downstream of TORC1

Three distinct RNA polymerases transcribe different classes of genes in the eukaryotic nucleus. RNA polymerase (Pol) III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including tRNAs and 5S rRNA. The historical focus on transcription of protein-coding genes has left the roles of Pol III in organismal physiology relatively unexplored. Target of rapamycin kinase complex 1 (TORC1) regulates Pol III activity, and is also an important determinant of longevity. This raises the possibility that Pol III is involved in ageing. This study shows that Pol III limits lifespan downstream of TORC1. A reduction in Pol III extends chronological lifespan in yeast and organismal lifespan in worms and flies. Inhibiting the activity of Pol III in the gut of adult worms or flies is sufficient to extend lifespan; in flies, longevity can be achieved by Pol III inhibition specifically in intestinal stem cells. The longevity phenotype is associated with amelioration of age-related gut pathology and functional decline, dampened protein synthesis and increased tolerance of proteostatic stress. Pol III acts on lifespan downstream of TORC1, and limiting Pol III activity in the adult gut achieves the full longevity benefit of systemic TORC1 inhibition. Hence, Pol III is a pivotal mediator of this key nutrient-signalling network for longevity; the growth-promoting anabolic activity of Pol III mediates the acceleration of ageing by TORC1. The evolutionary conservation of Pol III affirms its potential as a therapeutic target (Filer, 2017).

The task of carrying out transcription in the eukaryotic nucleus is divided among RNA Pol I, II and III. This specialization is evident in the biogenesis of the translation machinery, a task that requires the co-ordinated activity of all three polymerases: Pol I generates the 45S pre-rRNA that is subsequently processed into mature rRNAs, Pol II transcribes various RNAs including mRNAs encoding ribosomal proteins, while Pol III provides the tRNAs and 5S rRNA. This costly process of generating protein synthetic capacity is tightly regulated to match the extrinsic conditions and the intrinsic need for protein synthesis by the key driver of cellular anabolism, TORC1. The central position of TORC1 in the control of fundamental cellular processes is mirrored by the notable effect of its activity on organismal physiology: following its initial discovery in worms, inhibition of TORC1 has been demonstrated to extend lifespan in all tested organisms, from yeast to mice, with beneficial effects on a range of age-related diseases and dysfunctions. TORC1 strongly activates Pol III transcription and this relationship suggests the possibility that inhibition of Pol III promotes longevity (Filer, 2017).

In Saccharomyces cerevisiae, each of the 17 Pol III subunits is encoded by an essential gene. This study generated a yeast strain in which the largest Pol III subunit (C160, encoded by RPC160, also known as RPO31) is fused to the auxin-inducible degron (AID). The fusion protein can be targeted for degradation by the ectopically expressed E3 ubiquitin ligase (OsTir) in the presence of indole-3-acetic acid (IAA) to achieve conditional inhibition of Pol III. It was confirmed that IAA treatment triggered degradation of the fusion protein, and it was observed that IAA treatment also improved the survival of the RPC160-AID strain upon prolonged culture. In addition, IAA treatment of the control strain lacking the AID fusion reduced its survival relative to both the same strain in the absence of IAA and to the RPC160-AID strain in the presence of IAA. Hence, Pol III depletion appears to extend the chronological lifespan in yeast. While IAA had no substantial effect on the survival of a strain carrying the AID domain fused to the largest subunit of Pol II (RPB220, also known as RP021), this strain appeared to survive better than the control strain did in the presence of IAA, indicating that inhibition of Pol II may also extend chronological lifespan. Chronological lifespan of yeast is a measure of survival in a nutritionally limited, quiescent population, whereas replicative lifespan measures the number of daughters produced by a single mother cell in its lifetime. No evidence was found that inhibition of Pol III causes an increase in the replicative lifespan in yeast (Filer, 2017).

The observed increase in chronological lifespan may simply indicate increased stress resistance and hence be of limited relevance to organismal ageing. To examine the role of Pol III in organismal ageing directly, animal models were examined. RNA-mediated interference (RNAi) was initiated against rpc-1, the Caenorhabditis elegans orthologue of RPC160, in worms from the L4 stage, causing a partial knockdown of rpc-1 mRNA. This consistently extended the lifespan of worms at both 20°C and 25°C. To reduce Pol III activity in Drosophila melanogaster, a P-element insertion that deletes the transcriptional start site of the gene encoding the Pol III-specific subunit C53 (CG5147^EY22749, henceforth called dC53^EY, was backcrossed into a healthy, outbred population of flies. Homozygous dC53^EY/EY mutants were not viable, but heterozygous females had reduced dC53 mRNA levels and lived longer than controls. Taken together, these data strongly indicate that Pol III limits lifespan in multiple model organisms and conversely, that partial inhibition of its activity is an intervention that increases longevity in multiple species (Filer, 2017).

The longevity of an animal can be governed from a single organ. In the worm, this role is often played by the gut. To restrict the rpc-1 knockdown to the gut, worms were used that were deficient in rde-1, in which the RNAi machinery deficiency is restored in the gut by gut-specific rde-1 rescue. rpc-1 RNAi extended the lifespan of this strain, both at 20°C and 25°C. Similarly, in the adult fly, driving an RNAi construct targeting the RPC160 orthologue (CG17209, henceforth called dC160,with the mid-gut-specific, RU486-inducible driver TIGS extended the lifespan of females, while the presence of the inducer (RU486) did not affect survival of the control strains. The longevity phenotype could also be recapitulated with RNAi against dC53, another Pol III subunit, indicating that the phenotype was not subunit-specific or due to off-target effects. As well as the gut, longevity can also be associated with the fat body and neurons in flies. However, the longevity phenotype caused by dC160 RNAi appears to be specific to the gut, since no significant lifespan extension was observed upon induction of dC160 RNAi in the fat body of the adult fly, and only a modest, albeit significant, extension resulted from neuronal induction of dC160 RNAi (Filer, 2017).

The worm gut is composed of only post-mitotic cells. In flies, as in mammals, the adult gut epithelium contains mitotically active intestinal stem cells, and the mid-gut-specific driver TIGS appears to be active in at least some ISCs, prompting restricting of dC160 RNAi induction to this cell type. ISC-specific dC160 RNAi, achieved with the GS5961 driver, was sufficient to promote longevity. In summary, Pol III activity in the gut limits survival in worms and flies, and in the fly, Pol III can drive ageing specifically from the gut stem-cell compartment (Filer, 2017).

The consequences of Pol III inhibition in the fly gut was assessed. Pol III acts to generate precursor tRNAs (pre-tRNAs) that are processed rapidly to mature tRNAs. Owing to their short half-lives, pre-tRNAs are useful as readouts of in vivo Pol III activity. Profiling the levels of specific pre-tRNAs, pre-tRNA^His, pre-tRNA^Ala and pre-tRNA^Leu, relative to the levels of U3 (a small nucleolar RNA transcribed by Pol II) revealed a moderate but significant reduction in Pol III activity upon gut-specific induction of dC160 RNAi. The three polymerases can be directly coordinated to generate the translation machinery. Indeed, Pol III inhibition had knock-on effects on Pol I- but not Pol II-generated transcripts, revealing partial cross-talk. dC160 RNAi also reduced protein synthesis in the gut, consistent with reduced Pol III activity. These effects (reduction in pre-tRNAs or protein synthesis) were not observed after feeding RU486 to the driver-only control. The reduction in protein synthesis was not pathological: total protein content of the gut was unaltered; fecundity, a sensitive readout of a female's nutritional status, was unaffected; and the flies' weight, triacylglycerol and protein levels remained unchanged. Reduced protein synthesis can liberate protein-folding machinery from protein production and increase homeostatic capacity. Indeed, induction of dC160 RNAi in the gut increased the resistance of adult flies to proteostatic challenge with tunicamycin for TIGS-only control. Hence, Pol III can fine-tune the rate of protein synthesis in the adult fly gut without obvious detrimental outcomes, while increasing resistance to proteotoxic stress (Filer, 2017).

Having demonstrated the relevance of Pol III for ageing, whether it acts on lifespan downstream of TORC1 was investigated in Drosophila. Numerous observations in several organisms support the model in which TORC1 localizes on Pol III-transcribed loci and promotes phosphorylation of the components of the Pol III transcriptional machinery to activate transcription, in part by inhibition of the Pol III repressor, Maf1. Using chromatin immunoprecipitation (ChIP) with two independently generated antibodies against Drosophila TOR (target of rapamycin), TOR enrichment was observed on Pol III-target genes in the adult fly, relative to Pol II targets. Inhibition of TORC1 by feeding rapamycin to flies reduced the levels of pre-tRNAs in whole flies. Rapamycin also reduced pre-tRNA levels specifically in the gut relative to U3. Since rapamycin results in re-scaling of the gut, evidenced by the reduction in the total RNA content of the organ, it was also confirmed that the drug reduced pre-tRNA levels relative to total RNA. Interestingly, rapamycin did not cause a decrease in 45S pre-rRNA in the gut, suggesting a lack of sustained Pol I inhibition. Additionally, gut-specific overexpression of Maf1 reduced the levels of pre-tRNAs and extended lifespan, confirming that Maf1 acts on Pol III in the adult gut. These data are consistent with TORC1 driving systemic and gut-specific Pol III activity in the adult fruitfly (Filer, 2017).

To examine whether the lifespan effects of Pol III are downstream of TORC1, adult-onset Pol III inhibition was combined with rapamycin treatment. Rapamycin feeding or gut-specific dC160 RNAi resulted in the same magnitude of lifespan extension. The two treatments were not additive, consistent with their acting on the same longevity pathway. The same effect was observed with RNAi against dC53 in the gut, as well as when dC160 RNAi was restricted to the ISCs. Importantly, rapamycin feeding also inhibited phosphorylation of the TORC1 substrate, S6 kinase (S6K), in both the gut and the whole fly, and decreased fecundity, while gut-specific C160 RNAi did not have these effects. This confirms that Pol III inhibition does not impact TORC1 activity locally or systemically, and therefore, Pol III acts downstream of TORC1 in ageing (Filer, 2017).

TORC1 inhibition is known to ameliorate age-related pathology and functional decline of the gut. Whether inhibition of Pol III was sufficient to block the dysplasia resulting from hyperproliferation and aberrant differentiation of ISCs was examined by assessing the characteristic, age-dependent increase in dividing phospho-histone H3 (pH3)-positive cells. Inducing dC160 RNAi in the fly gut or solely in the ISCs ameliorated this pathology. These treatments also counteracted the age-related loss of gut barrier function, decreasing the number of flies displaying extra-intestinal accumulation of a blue food dye (the 'Smurf' phenotype). It as also found that rpc-1 RNAi reduced the severity of age-related loss of gut-barrier function in worms. In Drosophila, gut health and TORC1 inhibition are specifically linked to female survival. Indeed, induction of dC160 RNAi in the gut had a sexually dimorphic effect on lifespan, as the effect on males, although significant, was lower in magnitude relative to the effect on females. Overall, the data show that gut- or ISC-specific inhibition of Pol III, which extends lifespan, is sufficient to ameliorate age-related impairments in gut health, which may be causative of or correlate with this longevity (Filer, 2017).

This study demonstrates that the adult-onset decrease in the growth-promoting anabolic function mediated by Pol III in the gut, and specifically in the intestinal stem-cell compartment, is sufficient to recapitulate the longevity benefits of rapamycin treatment. Pol III activity is essential for growth; its detrimental effects on ageing suggest an antagonistic pleiotropy in which wild-type levels of Pol III activity are optimised for growth and reproductive fitness in early life but prove detrimental for later health. This study reveals a fundamental role for Pol III in adult physiology, implicating wild-type Pol III activity in age-related stem-cell dysfunction, declining gut health and organismal survival downstream of nutrient signalling pathways. The longevity resulting from partial Pol III inhibition in adulthood is likely to result from the reduced provision of protein synthetic machinery; however, differential regulation of tRNA genes or Pol III-mediated changes to chromatin organization may also be involved, as has been suggested in other contexts (Arimbasseri, 2016). The strong structural and functional conservation of Pol III in eukaryotes suggests that studies of its influence on mammalian ageing are warranted and could lead to important therapies (Filer, 2017).

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genes active in the midgut

Genes involved in tissue development

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