Gut Development

The ectodermal derivatives of the gut are the stomodeum and the proctodeum. The proctodeum is formed concurrently with germ-band elongation [Images] while formation of the foregut continues later and is associated with head involution. Adult foregut consists of the esophagus, pharynx, crop and the sucking pump or cibarium found at the base of the proboscis. Salivary gland cells, also of ectodermal origin, become part of the forgut and are derived independently from cells immediately behind the cephalic furrow.

The anterior endodermal anlage invaginates during gastrulation (stage 7) [Images] to produce the anterior midgut primordium, while the posterior midgut primordia sink into the embryo during stage 8 and enter the first postblastodermal division during stage 9. The anlagen for the hindgut form a ring encircling the posterior midgut primordia at the posterior pole. The whole of the hindgut is ectodermal in origin, including Malpighian tubules that form close to the junction between hindgut and posterior midgut.

The entire gut is remodeled during metamorphosis from imaginal cells at the base of the salivary glands (foregut), at the junction of the foregut and midgut, and in the hindgut and around the anus. Malpighian tubules are an exception to this remodeling process. Instead, they persist throughout metamorphosis unmodified.

Genes affecting ectodermal portions of the gut

Foregut is determined by the same genes that act in the gnathal (mouth) segments of head. Foregut is really an extention of mandible, maxillary and labial segments and is regulated by genes that are expressed in these segments including spalt, the head gap genes, and the ANTP-complex genes Deformed and Sex combs reduced.

Why is it that labial, the most anteriorly expressed ANTP-C gene, is not involved in foregut determination? The portion of the blastoderm that invaginates to form ectodermal gut derivatives is really ventral-anterior, resulting in the incorporation of cells of the gnathal segments into the forgut. The more dorsal anterior portions, including the pregnathal intercalary segment (expressing labial) form dorsal anterior structures such as the eyes and brain. tailless and forkhead are required for hindgut, including Malpighian tubules and anal plate, while huckebein is required for foregut only.

Cross regulation of intercellular gap junction communication and paracrine signaling pathways during organogenesis in Drosophila

The spatial and temporal coordination of patterning and morphogenesis is often achieved by paracrine morphogen signals or by the direct coupling of cells via gap junctions. How paracrine signals and gap junction communication cooperate to control the coordinated behavior of cells and tissues is mostly unknown. This study found that Hedgehog signaling is required for the expression of wingless and of Delta/Notch target genes in a single row of boundary cells in the foregut-associated proventriculus organ of the Drosophila embryo. These cells coordinate the movement and folding of proventricular cells to generate a multilayered organ. hedgehog and wingless regulate gap junction communication by transcriptionally activating the innexin2 gene, which encodes a member of the innexin family of gap junction proteins. In innexin2 mutants, gap junction-mediated cell-to-cell communication is strongly reduced and the proventricular cell layers fail to fold and invaginate, similarly as in hedgehog or wingless mutants. It was further found that innexin2 is required in a feedback loop for the transcriptional activation of the hedgehog and wingless morphogens and of Delta in the proventriculus primordium. It is proposed that the transcriptional cross regulation of paracrine and gap junction-mediated signaling is essential for organogenesis in Drosophila (Lechner, 2007).

In both vertebrates and invertebrates, the posterior foregut constitutes a center of organogenesis from which gut-associated organs such as the lung in vertebrates or the proventriculus in Drosophila develop. Proventriculus development involves the folding and invagination of epithelial cell layers to generate a multiply-folded organ. Two cell populations, the anterior and the posterior boundary cells, were shown previously to control cell movement and the folding of the proventriculus organ. In the posterior boundary cells, which organize the endoderm rim of the proventriculus, the JAK/STAT signaling cascade cooperates with Notch signaling to control the expression of the gene short stop encoding a cytoskeletal crosslinker protein of the spectraplakin superfamily. Thereby the Notch signaling pathway is connected to cytoskeletal organization in the posterior boundary cells, which have to provide a stiffness function to enable the invagination of the ectodermal foregut cells. The findings in this paper provide evidence that hedgehog is essential for the Notch signaling-dependent allocation of the anterior boundary cells. In amorphic hedgehog mutants, evagination and the formation of the constriction at the ectoderm/endoderm boundary are not affected, however, the inward movement of the anterior boundary cells is not initiated at the keyhole stage. The lack of cell movement of the ectodermal proventricular cells is consistent with the finding that hedgehog specifically controls Notch target gene activity in the anterior boundary cells. Genetic experiments further identify wingless as a target gene of hedgehog in the anterior boundary cells. wingless, in turn, controls the transcription of the innexin2 gene, which is expressed in the invaginating proventricular cells. When wingless is re-supplied in the genetic background of hedgehog mutants, innexin2 expression is rescued, providing further evidence that innexin2 is a target gene of wingless in the proventriculus primordium. Innexin2 encodes a member of the innexin family of gap junction proteins and is essential for the development of epithelial tissues. In the proventriculus, innexin2 mRNA is initially expressed in the early evagination stage in a broad domain covering both the ectodermal and endodermal precursor cells of the proventriculus primordium. When the ectodermal cells start to invaginate into the proventricular endoderm, innexin2 expression is upregulated in the ectodermal cell layer. Invagination of the ectodermal cells fails in hedgehog, wingless and kropf mutant proventriculi and dye tracer injection experiments demonstrate that hedgehog and kropf mutants show a strong reduction of gap junction communication. These data suggest that the direct coupling of cells via Innexin2-containing gap junctions, which are induced in response to hedgehog and wingless activities, is important for the coordinated movement of the ectodermal cell layer. It is known from extensive studies in mammals that the coupling of cells and tissues via gap junctions enables the diffusion of second messengers, such as Ca2+, inositol-trisphosphate (IP3) or cyclic nucleotides to allow the rapid coordination of cellular behavior during morphogenetic processes such as cell migration and growth control. Cell movement and folding involves a modulation of cell adhesion and of cytoskeletal architecture of the proventricular cells. A functional interaction of innexin2 with the cell adhesion regulator DE-cadherin, which is a core component of adherens junctions has been shown recently by co-immunoprecipitation, yeast two-hybrid studies, and genetic analysis. In mutants of DE-cadherin, Innexin2 is mislocalized and vice versa suggesting that the regulation of cell adhesion and gap junction-mediated communication may be linked. Similar evidence for a coordinated regulation of connexin activity and N-cadherin has been obtained in mammals during migration of neural crest cells (Lechner, 2007).

In kropf mutants or innexin2 knockdown animals, hedgehog, wingless and Delta transcription is strongly reduced as shown by in situ hybridization and by quantitative RT PCR experiments using mRNAs isolated from staged embryos. Furthermore, hedgehog, wingless and Delta are ectopically expressed and their mRNA is upregulated in embryos in which innexin2 is overexpressed. In summary, these experiments provide strong support that the gap junction protein Innexin2 plays an essential role enabling or promoting transcriptional activation of hedgehog, wingless and Delta. These data point towards an essential requirement of gap junction communication for the transcriptional activation of morphogen-encoding genes activating evolutionary conserved signaling cascades essential for patterning in animals. It is of note that gap junctions are established at very early stages of embryonic development, correlating with a maternal and zygotic expression of innexin2 and other innexin family members. kropf mutant animals, which are devoid of maternal and zygotic innexin2 expression are early embryonic lethal and develop no epithelia, consistent with a fundamental role of gap junctions in development, on top of which pattern formation of tissues and organs may occur. It has been shown previously that gap junctions are essential for C. elegans, Drosophila, and vertebrate embryogenesis from early stages onwards (Lechner, 2007 and references therein).

In the nematode C. elegans, a transient network formed by the innexin gap junction protein NSY-5 was recently shown to coordinate left-right asymmetry in the developing nervous system. Previous findings in chick and Xenopus laevis embryos have suggested an essential role of connexin43-mediated gap junction for the determination of the left-right asymmetry of the embryos. Treatment of cultured chick embryos with lindane, which results in a decreased gap junctional communication, frequently unbiased normal left-right asymmetry of Sonic hedgehog and Nodal gene expression, causing the normally left-sided program to be recapitulated. An important role of connexin43 (Cx43)-dependent gap junction communication for sonic hedgehog expression was also observed in limb patterning of the chick wing. Additionally, modulation of gap junctions in Xenopus embryos by pharmacological agents specifically induced heterotaxia involving mirror-image reversals of the heart, gut, and gall bladder. These data in combination with the current findings indicate that the transcriptional regulation of hedgehog and other morphogen-encoding genes by gap junction proteins may be evolutionary conserved between deuterostomes (vertebrates) and protostomes (Drosophila), although the Drosophila innexin gap junction genes share very little sequence homology with the connexin genes. The molecular mechanism underlying innexin2-mediated transcriptional regulation of hedgehog, wingless and Delta is not clear. It has been proposed that the nuclear localization of the carboxy-tail of connexin43 may exert effects on gene expression and growth in cardiomyocytes and HeLa cells. This would infer a cleavage of connexin43 to release the C-terminus, however, in vivo evidence for this event is still lacking. Sequence analysis reveals a nuclear receptor recognition motif within the C-terminus of Innexin2. It has been demonstrated that this recognition motif mediates the interaction of coactivators with nuclear receptors. However, there is no immunohistochemical evidence for a nuclear localization of Innexin2 or the Innexin2 C-terminus in Drosophila embryonic cells indicating that a direct involvement of Innexin2 in regulating transcription of target genes may not occur. The direct association of a transcription factor with gap junctions has been recently proposed for the mouse homolog of ZO-1-associated nucleic acid-binding protein (ZONAB). This transcription factor binds to ZO-1, which is associated with oligodendrocyte, astrocyte and retina gap junctions. It is possible that innexin2-dependent transcriptional regulation may involve a similar type of mechanism: a still unknown transcriptional regulator associated with the C-terminus of innexin2-containing gap junctions could be released upon modulation of gap junction composition thereby modulating the transcription of innexin2-dependent target genes (Lechner, 2007).

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).

The putative Na(+)/Cl(-)-dependent neurotransmitter/osmolyte transporter Inebriated in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis

Most organisms are able to maintain systemic water homeostasis over a wide range of external or dietary osmolarities. The excretory system, composed of the kidneys in mammals and the Malpighian tubules and hindgut in insects, can increase water conservation and absorption to maintain systemic water homeostasis, which enables organisms to tolerate external hypertonicity or desiccation. However, the mechanisms underlying the maintenance of systemic water homeostasis by the excretory system have not been fully characterized. The present study found that the putative Na(+)/Cl(-)-dependent neurotransmitter/osmolyte transporter inebriated (ine) is expressed in the basolateral membrane of anterior hindgut epithelial cells. This was confirmed by comparison with a known basolateral localized protein, the alpha subunit of Na(+)-K(+) ATPase (ATPalpha). Under external hypertonicity, loss of ine in the hindgut epithelium results in severe dehydration without damage to the hindgut epithelial cells, implicating a physiological failure of water conservation/absorption. It was also found that hindgut expression of ine is required for water conservation under desiccating conditions. Importantly, specific expression of ine in the hindgut epithelium can completely restore disrupted systemic water homeostasis in ine mutants under both conditions. Therefore, ine in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis (Luan 2015).

Water homeostasis is essential for the survival of all organisms. The mammalian kidney and the Malpighian tubule and hindgut of insects play indispensable roles in maintaining water homeostasis over a wide range of external or dietary osmolarities. These organs can increase water conservation and absorption to maintain systemic water homeostasis, which enables organisms to tolerate external hypertonicity or desiccation (Luan 2015).

The mammalian kidney regulates water balance mainly through the antidiuretic hormone (ADH), which enhances water absorption. Failure of antidiuretic mechanisms can result in disrupted systemic water homeostasis, causing pathological conditions like Diabetes Insipidus. Although antidiuretic factors for the enhancement of water absorption, such as Schgr-ITP and CAPA-related peptides, are also present in insects, the mechanisms of water conservation and absorption in the excretory system are not fully characterized, especially in Drosophila (Luan 2015).

Previous studies have shown that loss of the putative Na+/Cl--dependent neurotransmitter/osmolyte transporter ine causes hypersensitivity to dietary hypertonicity in Drosophila; however, the mechanism underlying this effect remains unknown. Ine is a member of the Na+/Cl--dependent neurotransmitter/osmolyte transporter family, which is conserved across invertebrates and vertebrates. Members of this family share several common structural features, including 12 transmembrane domains flanked by intracellular N and C termini, and an extracellular loop between the third and fourth transmembrane domains. These proteins play critical roles in neurotransmission, as well as cellular and systemic homeostasis, by transporting neurotransmitters, osmolytes, and energy metabolites across the plasma membrane. There is sequence similarity between ine and the betaine/GABA transporter (BGT1), a mammalian member of the Na+/Cl--dependent neurotransmitter/osmolyte transporter family. Both BGT1 and ine are expressed in the central nervous system (CNS), as well as organs that perform water absorption, and both are involved in the control of neuronal excitability and tolerance to hypertonicity. This suggests that these two proteins may function through a similar mechanism (Luan 2015).

Betaine, an active organic compound, is the substrate of BGT1 in renal medullary cells; however, the substrate of ine has yet to be identified. Betaine, like other intracellular organic osmolytes, can protect cells from external hypertonicity by balancing high extracellular osmolarity and preserving cell volume without interfering with cell function. However, no direct genetic evidence supports the osmoprotective function of the BGT1-mediated accumulation of betaine in renal medullary cells. Specifically, BGT1 knockout mice are healthy, and renal medullary cells appear to be normal in the hypertonic environment of the renal medulla. Therefore, the physiological function of the Na+/Cl--dependent neurotransmitter/osmolyte transporter in the excretory system remains to be elucidated (Luan 2015).

By investigating the function of ine in Drosophila, an excellent genetic model in which gene expression can be evaluated and manipulated in vivo, the physiological function will be better understood of Na+/Cl--dependent neurotransmitter/osmolyte transporters, including BGT1, in the excretory system. This study elucidates the role of ine in the Drosophila hindgut, and reveal a novel mechanism mediated by ine for the maintenance of systemic water homeostasis (Luan 2015).

This study has demonstrated that the mediation of water conservation/absorption by ine in the hindgut is essential for the maintenance of systemic water homeostasis in Drosophila. In insects, systemic water homeostasis is tightly regulated by the excretory system, including the Malpighian tubules and the hindgut, to ensure a constant internal environment. The dynamic balance between Malpighian tubule secretion and hindgut reabsorption, both of which are controlled by diuretic and antidiuretic hormones or factors, maintains water homeostasis in response to fluctuations in external osmotic conditions. However, in adult Drosophila, the water conservation/absorption mechanisms of the hindgut have not been elucidated. The current results demonstrate that ine is expressed in the basolateral membrane of the hindgut epithelium, suggesting that ine transports substrate from the hemolymph into hindgut epithelial cells. Surprisingly, under conditions of external hypertonicity, the systemic water homeostasis of ine mutant flies is disrupted, whereas that of WT flies is not disturbed. These results demonstrate that hindgut expression of ine mediates water conservation/absorption under external hypertonicity and maintains systemic water homeostasis. These results also suggest possible mechanism for ine function: transport of an osmolyte by ine into the hindgut epithelium increases intracellular molarity, which enhances water conservation/absorption from the hindgut lumen. Such a function would be particularly important in the condition of external hypertonicity, when increased molality in the hindgut lumen prevents osmotic flow of water into hindgut epithelium (Luan 2015).

It could be argued that ine functions through an osmoprotective mechanism, in which increased intracellular accumulation of osmolytes mediated by ine protects the hindgut epithelium from cellular death due to extracellular hypertonicity. However, this study demonstrates that anterior hindgut epithelial cells are not damaged by external hypertonicity in the absence of ine, suggesting that ine function in water conservation/absorption is not secondary to an osmoprotective effect. The existence of other osmolytes or transporters is proposed that function as osmoprotectors, and protect anterior hindgut epithelial cells against lethality under external hypertonicity. The expression of several genes, including some organic transporters, is up-regulated in the hindgut in response to external hypertonicity, supporting this possibility (Luan 2015).

Ine protein is expressed solely in the anterior hindgut. The anterior hindgut is an important site of water absorption, as demonstrated in insects other than Drosophila. In locusts, isosmotic fluid absorption in the anterior hindgut is driven by an apical membrane electrogenic Cl- pump. The antidiuretic hormone Schgr-ITP acts on the locust hindgut via cyclic AMP and GMP to increase the conductance of both K+ and Na+ and to stimulate the Cl- pump. As a result of the increased ion uptake, water absorption increases. It remains unknown, however, whether similar ion-uptake-coupled water absorption mechanisms are present in the Drosophila hindgut. This study found that loss of ine in the anterior hindgut epithelium causes severe dehydration in response to a hypertonic diet, and higher rates of body water loss under desiccation, which suggests the existence of a new mechanism of water conservation/absorption in the hindgut of Drosophila mediated by ine. It was proposed that ine transports osmolytes across the plasma membrane from the hemolymph and accumulates osmolytes within the hindgut epithelium, generating an osmotic driving force to conserve/absorb water from hindgut lumen against external hypertonicity. However, this theory lacks an explanation for how water is transferred into the hemolymph from epithelial cells, and to date, the transporter activity of ine has not been confirmed. The possibility thatine may improve water conservation/absorption through a different, unknown mechanism cannot be ruled out (Luan, 2015)

In addition to the anterior hindgut, the Malpighian tubules, rectum, and midgut also contribute to water absorption and conservation in insects under conditions of external hypertonicity or desiccation. During dehydration stress, the modulation of tyramine signaling in Drosophila Malpighian tubules enhances conservation of body water. Several anti-diuretic factors acting on the Malpighian tubules have been found. For example, CAPA-1 acts on Ncc69, the Na+-K+-2Cl- cotransporter, to increase water absorption through an ion uptake coupled mechanism. In addition, PKG, a cGMP-dependent kinase antagonizes the diuretic effects of tyramine and leukokinin. The rectum can also transport water from lumen to the hemolymph. In the locust, the chloride transport stimulating hormone (CTSH) acts to increase ion-dependent active transport of fluid from the rectum lumen. Finally, the antidiuretic hormone RhoprCAPA-2 inhibits fluid transport into the midgut lumen in Rhodnius prolixus to conserve water. Therefore, ine-mediated water conservation/absorption may not be the only mechanism by which systemic water homeostasis is maintained under external hypertonicity in Drosophila (Luan 2015).

Water is essential for the proper function of virtually all living cells. Organisms have developed mechanisms in the excretory system to maintain water hemostasis for a constant internal milieu under different external osmotic conditions, such as hypertonicity. This study reveals that hindgut expression of ine, a putative Na+/Cl-dependent neurotransmitter/osmolyte transporter, is indispensable for the maintenance of systemic water homeostasis in Drosophila. However, further investigation of the novel mechanism mediated by ine in the hindgut is necessary to fully understand the water conservation and absorption mechanisms of Drosophila hindgut, as well as the physiological functions of the members of the Na+/Cl-dependent neurotransmitter/osmolyte transporter family (Luan 2015)

Molecular and cellular organization of taste neurons in adult Drosophila pharynx

The Drosophila pharyngeal taste organs are poorly characterized despite their location at important sites for monitoring food quality. Functional analysis of pharyngeal neurons has been hindered by the paucity of molecular tools to manipulate them, as well as their relative inaccessibility for neurophysiological investigations. This study generated receptor-to-neuron maps of all three pharyngeal taste organs by performing a comprehensive chemoreceptor-GAL4/LexA expression analysis. The organization of pharyngeal neurons reveals similarities and distinctions in receptor repertoires and neuronal groupings compared to external taste neurons. The mapping results were validated by pinpointing a single pharyngeal neuron required for feeding avoidance of L-canavanine. Inducible activation of pharyngeal taste neurons reveals functional differences between external and internal taste neurons and functional subdivision within pharyngeal sweet neurons. These results provide roadmaps of pharyngeal taste organs in an insect model system for probing the role of these understudied neurons in controlling feeding behaviors (Chen, 2017).

In Drosophila, taste neurons located in sensilla in several body regions sense and distinguish nutritive substances such as sugars, amino acids, and low salt, and potentially harmful ones such as high salt, acids, and a diverse variety of bitter compounds. Hair-like sensilla on the labellum, distal segments of the legs (tarsi), anterior wing margins, and ovipositor have access to chemicals in external substrates. Pit-like sensilla (taste pegs) on the oral surface have access only once the fly extends its proboscis and opens the labellar palps; similar sensilla in the pharynx have access only when food intake is initiated. Based on its anatomical position, the pharynx is considered to act as a gatekeeper to control ingestion, promoting the intake of appetitive foods and blocking that of toxins (Chen, 2017).

Three distinct internal taste organs are present in the adult fly pharynx: the labral sense organ (LSO), the ventral cibarial sense organ (VCSO), and dorsal cibarial sense organ (DCSO). The VCSO and DCSO are paired on opposite sides of the rostrum, whereas the LSO is located in the haustellum. The organization and neuronal composition of all three organs, based on both light and electron microscopy data, have been described in detail. Nine separate sensilla are present in the LSO, of which 1-6 are innervated by a single mechanosensory neuron each. The remaining three, named 7-9, are uniporous sensilla, a feature that ascribes chemosensory function to them. Sensillum 7 is the largest one, with eight chemosensory neurons. Sensilla 8 and 9 have two neurons each (one mechanosensory and one chemosensory). Although one study reported two sensilla in the VCSO, this and other studies have observed three sensilla in the VCSO, innervated by a total of eight chemosensory neurons. The DCSO has two sensilla, each containing three chemosensory neurons. Notwithstanding the availability of detailed anatomical descriptions of pharyngeal taste organs, little is known about their function. The internal location of these organs poses challenges for electrophysiological analysis of taste neurons located within them. Additionally, few molecular tools are currently described to manipulate the function of selected pharyngeal taste neurons (Chen, 2017).

The expression and function of members of several chemosensory receptor gene families such as gustatory receptors (Grs), ionotropic receptors (Irs), Pickpocket (Ppk) channels, and transient receptor potential channels (Trps) have been found in external gustatory receptor neurons (GRNs) of the labellum and the tarsal segments. A number of Gr- and Ir-GAL4 drivers are also shown to label pharyngeal organs, but only a few, including Gr43a and members of sweet Gr clade, Gr2a, Ir60b, and TrpA1, have been mapped to specific taste neurons (Chen, 2017).

This study generated receptor-to-neuron maps for three pharyngeal taste organs by a systematic expression analysis of chemoreceptor reporter lines that represent Gr, Ir, and Ppk receptor families. The maps reveal a large and diverse chemoreceptor repertoire in the pharynx. Some receptors are expressed in combinations that are predictive of neuronal sweet or bitter taste function based on analysis of external GRNs. By contrast, some pharyngeal taste neurons express receptor combinations that are distinct from any that have been reported in other organs, leaving open questions about their functional roles. This study validated he receptor-to-neuron maps derived from reporter gene expression by assessing roles of pharyngeal GRNs predicted to detect L-canavanine, a bitter tastant for which a complete receptor repertoire has been reported. Interestingly, a systematic activation analysis of different classes of pharyngeal taste neurons reveals functional differences between external and internal taste neurons for bitter avoidance and functional subdivision within pharyngeal sweet neurons for sweet acceptance. Together, this study provides a molecular map of pharyngeal taste organs, which will serve as a resource for future studies of the roles of pharyngeal taste neurons in food evaluation (Chen, 2017).

Internal pharyngeal taste organs are the least explored taste organs, despite their obvious importance in insect feeding behaviors, which are crucial drivers for damaging crops and vectoring disease. The receptor-to-neuron maps of pharyngeal taste organs suggest a high degree of molecular complexity, with co-expression of different chemoreceptor family members in many pharyngeal GRNs. In particular, none of the pharyngeal GRNs were found to express Gr genes alone; rather, one or more Ir genes were always expressed in the same neurons. Gr and Ir genes are also co-expressed in some external sweet and bitter-sensing GRNs. Thus, both classes of receptors are likely to contribute to responses of Gr/Ir-expressing neurons in the LSO and VCSO, but whether they interact functionally or act independently remains to be determined. In the LSO, expression of sweet Grs and Ir76b overlaps in pharyngeal sweet GRNs, as observed in tarsi as well. In the pharynx, this study also found co-expression of ppk28 with Ir genes, which has not been described for external GRNs. These observations invite explorations of possible crosstalk, and its functional significance, between the two classes of receptors (Chen, 2017).

Pharyngeal GRNs also exhibit distinctive functional groupings. All external bitter GRNs have always been found grouped with sweet GRNs in taste hairs. By contrast, canonical sweet and bitter GRNs appear to segregate in different sensilla in the LSO, which is most well characterized for this perspective. L8 and L9 may be functionally identical and house only one Gr66a-expressing bitter GRN each, whereas L7 contains two sweet GRNs (L7-1 and L7-2). Moreover, external hairs typically have two to four GRNs, each of which has a distinct functional profile. In the LSO duplications are found (L7-1 and L7-2 are identical, as are L7-4 and L7-5), although differences between these pairs of GRNs may emerge as additional chemoreceptors are mapped in the pharynx. Finally, it is difficult to ascribe putative functions to most pharyngeal GRNs based on existing knowledge of receptor function in external counterparts. The L7-3 Gr-expressing neuron, for example, does not express members of the sweet clade, but neither does it express any of the common bitter Grs (Gr32a, Gr66a, and Gr89a) that would corroborate its role as a bitter GRN. Similarly, with the exception of salt neurons that may express Ir76b alone, there are few known functions for GRNs that solely express Ir genes. One possibility is that some of these GRNs possess novel chemoreceptor family ligand interactions. For example, L7-7 is involved in sensing sucrose but limiting sugar ingestion, representing an Ir neuron that operates in a negative circuit module for sugar intake. In addition, another recent study suggests that TRPA1 expression in L8 and L9 of the LSO is involved in feeding avoidance to bacterial endotoxins lipopolysaccharides (LPS). Alternatively, some pharyngeal GRNs may evaluate characteristics other than palatability, such as temperature or viscosity. Ir25a, which is broadly expressed in all 24 pharyngeal GRNs, is required for cool sensing and thermosensing. It will be worth investigating whether one or more pharyngeal GRNs act to integrate information about temperature and chemical quality of food substrates (Chen, 2017).

Expression analyses also hint at some functional subdivisions between pharyngeal taste organs. The LSO contains a smaller proportion of Gr-expressing neurons than the VCSO, which also expresses a larger number of Gr genes that are co-expressed with Gr66a. Thus, broader bitter taste function might be expected in the VCSO. By contrast, sweet taste function appears to be more dominant in the LSO; its sweet GRNs express more sweet Gr-GAL4 drivers than the ones in the VCSO, and their activation is sufficient to drive feeding preference. VCSO sweet GRNs fail to promote ingestion by themselves but may contribute to an increase in feeding preference when activated simultaneously with those in the LSO. Thus, there may be synergistic or hierarchical interactions between LSO and VCSO sweet taste circuits, with the latter coming into play only once the former is activated. The finding that Gr and Ir genes are expressed in the LSO and VCSO but only Ir genes in the DCSO is also striking and raises the possibility that the DCSO, which is present at the most internal location relative to the others, may serve a unique role in controlling ingestion (Chen, 2017).

Based on its molecular signature, the V5 neuron was identified as an L-canavanine-sensing neuron in the pharynx. As predicted, feeding avoidance of L-canavanine is dependent on V5. It was thus unexpected that capsaicin-mediated activation of bitter pharyngeal GRNs, which include V5, did not induce strong feeding avoidance either in the absence or presence of sugar. Because the strength and pattern of pharyngeal neuronal activation by bitter tastants or capsaicin is unknown, it is possible that capsaicin response may be weaker than that of canonical bitter tastants. Alternatively, sweet and bitter inputs from internal and external neurons may be summed differently. It is known that activation of one or few external sweet neurons can lead to proboscis extension, for example, but a larger number of bitter neurons may need to be activated for avoidance (Chen, 2017).

The afferents of pharyngeal GRNs target regions of the SEZ that are distinct from areas in which afferents from labellar and tarsal GRNs terminate. Interestingly, pharyngeal GRN projections between molecularly different classes of neurons, as well as between GRNs of the LSO and VCSO, are also distinct. Projections of sugar-sensing GRNs were found in separate ipsilateral regions, whereas those of neurons predicted to detect aversive tastants were found at the midline, suggesting the presence of contralateral termini. These observations may inform future functional studies of pharyngeal GRNs. L7-6 neurons, for example, would be predicted to sense aversive compounds based on the presence of their termini at the midline. Analysis of pharyngeal GRN projections also suggests distinct connectivity to higher order neuronal circuits. With the molecular tools described here, future investigations of pharyngeal GRNs and pharyngeal taste circuits will provide insight into how internal taste is integrated with external taste to control various aspects of feeding behavior (Chen, 2017).

A luminal glycoprotein drives dose-dependent diameter expansion of the Drosophila melanogaster hindgut tube

An important step in epithelial organ development is size maturation of the organ lumen to attain correct dimen/sions. This study shows that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein (Syed, 2012).

This study shows that the luminal glycoprotein Tnc promotes diameter expansion of the Drosophila hindgut in a dose-dependent manner. The domain organization of Tnc, its contribution to detectable O-glycans in the hindgut lumen and its ability to form a dense luminal matrix suggest that Tnc has mucin-like characteristics. A possible involvement of mucin-like molecules in tubulogenesis has previously been recognized. The Caenorhabditis elegans let-653 is a secreted protein with a PTS domain of around 90-200 amino acids, depending on the splice variant. In mutants for let-653, the single-celled excretory canals develop massively enlarged lumen by an as yet unknown mechanism. During cyst formation in Madin-Darby Canine Kidney (MDCK), it has been suggested that the initial separation of apical membranes involves de-adhesive properties conferred by large apically localized glycoproteins. Candidate molecules are mucin 1 (MUC1) and the sialomucin Podocalyxin, which localize to the nascent lumens in MDCK cysts and in vivo. Recently, it was indeed shown that Podocalyxin is required to separate apical membranes during initial lumen formation in developing blood vessels. Podocalyxin is membrane-bound, and its negatively charged sialic acids are thought to cause electrostatic repulsion of the apical surfaces. Tnc does however appear to function differently from these mucin-like molecules, since it is not required for lumen formation per se, but drives the subsequent step of tube diameter expansion (Syed, 2012).

The function of Tnc also differs from that of the chitinous matrix in the tracheal lumen, as the latter is not needed to increase the luminal volume during diameter expansion, but to shape a uniform diameter. A difference in action between the two luminal components is further supported by the slightly shorter tracheal tubes in tnc mutants, while loss of chitin causes too long tracheal tubes. It is proposed that Tnc-driven tube dilation represents a mechanism for shaping an epithelial tube, where the extent of tube wall extension and lumen volume expansion can be controlled by the intraluminal accumulation of a single protein (Syed, 2012).

During wing development, Tnc is found basal to the epithelium and is proposed to act as a ligand for PS2 integrin via RGD motifs in the vWC-like domains. It is therefore possibly that luminal Tnc might cause tube wall remodelling by signalling through an apical cognate receptor(s). However, the results do not indicate a signalling function for Tnc: First, over-expression of Tnc in the hindgut causes an increase in tube diameter according to the levels of Tnc expression. Thus, a signaling function of Tnc would imply that Tnc is the limiting factor in the pathway. This is unlikely, since Tnc is abundant and fills the lumen of the wild type hindgut. Second, when Tnc was expressed at one side of the tube wall, all cells at the lumen perimeter were similarly affected. If Tnc signals via an apical receptor, the effects should be higher at the site of its secretion, given its strictly dose-dependent function. Third, the observed lumen-dependent function of Tnc implies that a putative receptor would have to be present in many epithelia in which Tnc is not normally expressed, but yet not ubiquitously, as Tnc had no effect on the epidermis (Syed, 2012).

Tnc-driven lumen expansion causes an increase in inner and outer tube diameter, associated with epithelial flattening. It is known that luminal volume expansion upon a hydrostatic pressure causes similar effects, for example during inflation of the zebrafish brain ventricle, expansion of the mouse blastocyst and in vitro growth of renal cysts. The results would therefore comply with a mechanism whereby luminal accumulation of Tnc forces an increase in lumen volume and, thereby, expansion of the surrounding tube wall. Since luminal Tnc appears to be a major O-glycan with low mobility in the lumen, an attractive hypothesis is that Tnc forms supra-molecular complexes that cause volume expansion due to hydration of the attached O-glycans. Secretion of Tnc into a confined luminal space would then cause a pressure on the tube wall and lumen dilation. In an attempt to further evaluate if the effect of Tnc requires O-glycosylation of the PTS domains, hindgut morphology and the size of Tnc was examined in mutants that lack different glycosyl transferases. However, the results were inconclusive, showing effects on both Tnc levels and secretion (Syed, 2012).

The current study also show that Tnc can steer regional differences in tube diameter expansion along the tube axis, according to its pattern of expression. Such a regional effect of Tnc presumably occurs during normal hindgut development, where the amount of Tnc produced by the small intestine is larger than the amount produced by large intestine. As a likely consequence, the small intestine undergoes a higher degree of diameter expansion than large intestine, and it also shows a larger reduction in diameter upon loss of Tnc (Syed, 2012).

In summary, this study has shown that Tnc forms a lumen-spanning complex that drives expansion of the surrounding tube wall. The local and dose-dependent effect of Tnc on tube dilation illustrates that a single protein can model differential lumen diameter along a tube. A model is suggested were Tnc causes a luminal pressure upon secretion and promotes tube dilation according to its voluminous expansion. Since the lumen of different epithelial organs have been shown to exhibit dynamic patterns of glycan distribution during development, it is possible that glycan-rich luminal components have a broad importance in shaping developing epithelial organs (Syed, 2012).

Chiral cell sliding drives left-right asymmetric organ twisting

Polarized epithelial morphogenesis is an essential process in animal development. While this process is mostly attributed to directional cell intercalation, it can also be induced by other mechanisms. Using live-imaging analysis and a three-dimensional vertex model, 'cell sliding', a novel mechanism driving epithelial morphogenesis, was identified in which cells directionally change their position relative to their subjacent (posterior) neighbors by sliding in one direction. In Drosophila embryonic hindgut, an initial left-right (LR) asymmetry of the cell shape (cell chirality in three dimensions), which occurs intrinsically before tissue deformation, is converted through LR asymmetric cell sliding into a directional axial twisting of the epithelial tube. In a Drosophila inversion mutant showing inverted cell chirality and hindgut rotation, cell sliding occurs in the opposite direction to that in wild-type. Unlike directional cell intercalation, cell sliding does not require junctional remodeling. Cell sliding may also be involved in other cases of LR-polarized epithelial morphogenesis (Inaki, 2018).

The morphogenesis of epithelial cell sheets in the absence of cell number change has been explained as a consequence of cell-shape changes and/or cell rearrangements. Regarding cell rearrangements, the cell intercalation in convergent extension, in which cells intercalate in a medial direction to induce cell sheets to elongate in an anterior-posterior direction, is well described. Directional cell intercalation is also important in the LR asymmetric rotation of the male genitalia in Drosophila. However, the present study revealed that the LR asymmetric rotation of the embryonic Drosophila hindgut is achieved by chiral cell sliding, in which cells change their relative position against their subjacent neighbors in one direction. In contrast to directional cell intercalation, this cell sliding does not necessarily require cell rearrangements. Indeed, a computer simulation in which cell-boundary remodeling was prohibited still rotated as well as the standard 3D vertex model hindgut. Thus, in the hindgut case, it is speculated that the accumulation of small chiral cell sliding events between adjacent cells in the hindgut epithelial tube are sufficient to induce the 90° rotation of the hindgut epithelial tube, because of the large difference between the radius and the height of this organ (Inaki, 2018).

Considering that cell sliding and cell rearrangement may not be mutually exclusive, the definition of cell sliding may be extended. For example, theoretical models of Drosophila male genitalia rotation include the sliding motion of cells, although this mechanism also requires cell rearrangements, because a large amount of cell displacement takes place. In any event, for the dynamic cell events induced by cell chirality, cell sliding appears to drive the LR asymmetric rotation of tissues with or without cell intercalations. Thus, cell sliding may be a commonly used mechanism in LR asymmetric morphogenesis, although it has not been demonstrated to date (Inaki, 2018).

Directional cell intercalation often requires a biased subcellular distribution of Myosin II. For example, during the LR asymmetric rotation of Drosophila genitalia, LR asymmetric cell intercalation is induced by the LR asymmetric distribution of Myosin II. However, in the Drosophila hindgut, a biased Myosin II distribution was not detected in fixed or live tissues. Given that chiral cell sliding requires a very small amount of change in the relative positions of cells, a large bias in the amount of Myosin II may not be required. Indeed, the simulation revealed that the anisotropic contraction of edges before but not during the rotation was sufficient to induce whole-organ rotation, whereas the anisotropic contraction of edges needs to be maintained in the computer simulation to recapitulate the rotation of Drosophila male genitalia (see Computer simulation of the hindgut epithelial tube using a 3D cell-based vertex model for tissues and cell chirality, recapitulating the in vivo situation). Thus, if there is any bias in the subcellular distribution of Myosin II, it might be too subtle to detect by the methods used. Alternatively, force-generating mechanisms other than the biased expression of Myosin II might be used in the LR asymmetric rotation of the hindgut (Inaki, 2018).

The computer simulation recapitulated the LR directional axial rotation of the gut tube, which is driven by a mechanical force bias induced by cell chirality. Analyses of the model cells in the simulations suggested that the rotation of the model gut tube is achieved through two steps: the first is a deformation in the shape of each cell (loss of cell chirality) without a change in the relative positions of anterior-posterior neighboring cells, and the second is a change in the relative positions of anterior-posterior neighboring cells (called cell sliding in this study), which presumably drives the rotation of the model gut tube. Although the cell deformation and cell sliding temporally overlapped in part, the following observations suggested that these two events are mechanically distinct. First, the initiation of cell chirality loss mostly preceded the onset of cell sliding. The timing of the model gut tube rotation coincided with that of the cell sliding, but not of the cell chirality loss, suggesting that the rotation of the model gut tube involves two cellular dynamic steps. Second, in a simulation prohibiting the tube rotation, cell sliding did not occur, although the loss of cell chirality was still observed, indicating that the loss of cell chirality is not always coupled with cell sliding. Based on these results, it is speculated that the loss of cell chirality and the cell sliding are distinct mechanical processes (Inaki, 2018).

The differences between the cell chirality loss and cell sliding could be explained by their distinct mechanical properties. In a simulation, the equalization of the initial anisotropic contractile force was the only source driving subsequent events. During the balancing processes, individual cells were deformed quickly; most of the deformation was achieved by t = 1.0, although the force balance had not yet been neutralized given that the deformation continued to t = 80.0 (see the Computer simulation). In vertex models, a viscoelastic property is generally reflected in the speed of the response upon a force application. Thus, it is speculated that changes in the relative positions of cells (cell sliding) slowly occurs after the major dissolution of cell chirality, due to the difference in viscoelastic properties of the individual cell versus the cell aggregate, until the initial anisotropy of the force was neutralized. In turn, completion of the cell sliding led to global stabilization of the model tube (Inaki, 2018).

The conclusions about the mechanical properties of chiral cell sliding were based on the computer simulation. However, the ideas did not contradict the observations made in the embryonic hindgut in vivo. In this study, it was also found that the hindgut itself generates the active force for driving its rotation. Considering that cell chirality was observed before the hindgut rotation and was lost during the rotation in vivo, the cancelation of cell chirality is probably a primary driving force for the subsequent events including the cell sliding and hindgut rotation, in vivo. This result also excludes the possibility that the cell sliding is a consequence of hindgut rotation driven by an external force, further supporting the validity of the computer simulation for analyzing the relationship between cell chirality loss and cell sliding. In addition, the enantiomorphism of the initial cell chirality is correlated with the LR direction of the cell sliding, based on analyses of the LR inversion mutant, Myo31DF. Thus, the LR direction of the cell sliding appears to depend on the initial cell chirality. These results are consistent with the conclusions obtained from the computer simulations. In conclusion, it is proposed that chiral cell sliding is a cell dynamic mechanism that connects cell chirality with the LR asymmetric rotation of the hindgut tube (Inaki, 2018).

In vertebrates, organ laterality is thought to be determined by the LR body axis established by Nodal cassette gene expressions, which is distinct from the mechanisms reported for Drosophila. However, a recent study revealed that LR asymmetry formation in the zebrafish heart does not depend on nodal gene expression. Moreover, explanted linear hearts of various species develop dextral looping in culture, suggesting that the heart LR asymmetry formation is tissue-intrinsic. In Drosophila, which lacks the Nodal cassette gene functions, various organs use cell chirality as a common strategy to establish LR asymmetry in a tissue-intrinsic manner, in the absence of an LR body axis. To date, cell chirality in tissues has been observed only in Drosophila. However, cell chirality has been reported in cultured cells, including those of vertebrates. Importantly, many cell types from various organs show a chiral cell shape. Therefore, cell chirality may play specific roles in the LR asymmetric organogenesis in vertebrates as well (Inaki, 2018).

E and ID proteins regulate cell chirality and left-right asymmetric development in Drosophila

How left-right (LR) asymmetry forms in the animal body is a fundamental problem in Developmental Biology. While the mechanisms for LR asymmetry are well studied in some species, they are still poorly understood in invertebrates. It has been shown that the intrinsic LR asymmetry of cells (designated as cell chirality) drives LR asymmetric development in the Drosophila embryonic hindgut, although the machinery of the cell chirality formation remains elusive. This study found that the Drosophila homolog of the Id gene, extra macrochaetae (emc), is required for the normal LR asymmetric morphogenesis of this organ. Id proteins, including Emc, are known to interact with and inhibit E-box-binding proteins (E proteins), such as Drosophila Daughterless (Da). The suppression of da by wild-type emc was essential for cell chirality formation and for normal LR asymmetric development of the embryonic hindgut. MyosinID (MyoID), which encodes the Drosophila Myosin ID protein, is known to regulate cell chirality. It was further shown that Emc-Da regulates cell chirality formation, in which Emc functions upstream of or parallel to MyoID. Abnormal Id-E protein regulation is involved in various human diseases. These results suggest that defects in cell shape may contribute to the pathogenesis of such diseases (Ishibashi, 2019).

Maintenance of hindgut reabsorption during cold exposure is a key adaptation for Drosophila cold tolerance

Maintaining extracellular osmotic and ionic homeostasis is crucial for organismal function. In insects, hemolymph volume and ion content is regulated by the secretory Malpighian tubules and reabsorptive hindgut. When exposed to stressful cold, homeostasis is gradually disrupted, characterized by a debilitating increase in extracellular K(+) concentration (hyperkalemia). Accordingly, studies have found a strong link between the species-specific cold tolerance and their ability to maintain ion and water homeostasis at low temperature. This is also true for drosophilids where inter- and intra-specific differences in cold tolerance are linked to the secretory capacity of Malpighian tubules. There is, however, little information on the reabsorptive capacity of the hindgut in Drosophila To address this, a novel method was developed that permits continued measurements of hindgut ion and fluid reabsorption in Drosophila. This assay is temporally stable (approximately 2 hours) and responsive to cAMP stimulation and pharmacological intervention in accordance with the current insect hindgut reabsorption model. Then, how cold acclimation or cold adaptation affected hindgut reabsorption at benign (24 degrees C) and low temperature (3 degrees C) was investigated. Cold tolerant Drosophila species and cold-acclimated D. melanogaster maintain superior fluid and Na(+) reabsorption at low temperature. Furthermore, cold adaptation and acclimation caused a relative reduction in K(+) reabsorption at low temperature. These characteristic responses of cold adaptation/ acclimation will promote maintenance of ion and water homeostasis at low temperature. This study of hindgut function therefore provides evidence to suggest that adaptations in osmoregulatory capacity of insects are critical for their ability to tolerate cold (Andersen, 2020).

The homeodomain transcription factor Orthopedia is involved in development of the Drosophila hindgut

The Drosophila hindgut is commonly used model for studying various aspects of organogenesis like primordium establishment, further specification, patterning, and morphogenesis. During embryonic development of Drosophila, many transcriptional activators are involved in the formation of the hindgut. The transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, is expressed in the hindgut and nervous system of developing Drosophila embryos, but due to the lack of mutants no functional analysis has been conducted yet. This study shows that two different otp transcripts, a hindgut-specific and a nervous system-specific form, are present in the Drosophila embryo. Using an otp antibody, a detailed expression analysis during hindgut development was carried out. otp was not only expressed in the embryonic hindgut, but also in the larval and adult hindgut. To analyse the function of otp, the mutant otp allele otp^GT was generated by ends-out gene targeting. In addition, two EMS-induced otp alleles were isolated in a genetic screen for mutants of the 57B region. All three otp alleles showed embryonic lethality with a severe hindgut phenotype. Anal pads were reduced and the large intestine was completely missing. This phenotype is due to apoptosis in the hindgut primordium and the developing hindgut. These data suggest that otp is another important factor for hindgut development of Drosophila. As a downstream factor of byn otp is most likely present only in differentiated hindgut cells during all stages of development rather than in stem cells (Hildebrandt, 2020).

The Drosophila embryonic hindgut is a single-layered ectodermally derived epithelium surrounded by visceral musculature. It arises from a group of cells at the posterior part of the blastoderm stage embryo referred to as the hindgut primordium. The hindgut primordium is a ring of about 200 blastoderm cells that is internalised during gastrulation to form a short, wide sac. In a relatively short time this epithelium sac is transformed into a long tube containing approximately 700 cells. The growth of the hindgut starting at stage 12 is not due to cell divisions, but a twofold endoreplication that leads to an increase in cell size, and as a consequence total length of the hindgut. During this process, the developing hindgut becomes subdivided along the anterior posterior (AP) axis and the dorsoventral (DV) axis. Along the AP axis, the hindgut forms three morphologically distinct regions: the small intestine, large intestine, and rectum. The small intestine is the most anterior part of the hindgut and is connected to the posterior midgut, whereas the large intestine is the central part of the hindgut and forms three distinct regions along the DV axis. The dorsal and ventral regions constitute the outer and inner portions of the hindgut loop, respectively. Two rows of boundary cells are organised between these two regions and as two rings at the anterior and posterior borders of the large intestine. The most posterior-most portion of the hindgut is the rectum, which connects to the anal pads (Hildebrandt, 2020).

Several genes are required to establish the hindgut primordium and to pattern the hindgut along the AP axis. At the blastoderm stage a group of posterior cells, called the proctodeal primordium, will later on give rise to the hindgut. In these cells the transcription factor Tailless (Tll) is expressed and subsequently activates other transcription factors like Brachyenteron (Byn), Fork head (Fkh) and Bowel (Bowl) as well as the signalling protein Wingless (Wg), which are all necessary for hindgut development. The transcription factor Caudal (Cad) is also expressed in the proctodeal ring, but independently of Tailless. Tll and Wg are necessary to establish the primordium, whereas Cad is necessary for the internalisation of the hindgut primordium later on. Proper gene expression in and maintenance of the large intestine requires byn, Dichaete (D), raw, lines (lin) and mummy (mmy), while bowl and drumstick (drm) are required for gene expression in and maintenance of the small intestine (Hildebrandt, 2020).

The Drosophila T-box gene brachyenteron (byn) is expressed in the ring of cells that internalise to form the embryonic hindgut and expression is maintained in the hindgut throughout embryogenesis. In byn mutants the hindgut is shortened due to apoptosis and the large intestine is missing. The Drosophila homeobox gene orthopedia (otp) is also expressed in the hindgut, anal pads and along the central nervous system (Simeone, 1994). It is located in 57B region of the second chromosome in close vicinity to the other homeobox genes Drosophila retinal homeobox (Drx) and homeobrain (hbn). In the hindgut, otp is directly activated by byn in a dose-dependent manner via multiple binding sites present in a regulatory element of otp (Hildebrandt, 2020).

Otp is highly conserved through evolution and has been identified in most multicellular organisms. Among these are several invertebrates such as sea urchins, the mollusc Patella vulgata, the annelid Platynereis dumerilii and several vertebrates such as zebrafish, that have two genes namely otp1 and otp2, chicken, mouse and human. otp genes of vertebrates have a major function in the development of the hypothalamic neuroendocrine system (see Del Giaccom 2008 for review) (Hildebrandt, 2020).

The function of otp during Drosophila development has been unknown so far as no mutants have been described. The present study shows that otp is required for proper hindgut development in Drosophila. One otp allele was generated by ends-out gene targeting and two additional otp alleles were isolated in an EMS-mutagenesis screen. All three otp alleles are characterised by a dramatically reduced hindgut lacking the complete large intestine. This reduction in hindgut length is due to apoptosis in the hindgut primordium and the developing hindgut (Hildebrandt, 2020).

This paper analysed the function of the transcription factor Orthopedia during hindgut development. In the embryo, otp is expressed in the hindgut primordium, the developing hindgut, and the anal pads. This expression is dependent on several upstream regulators, such as lines and byn. Byn directly activates otp through modular binding sites upstream of hindgut specific promoter of otp in a cooperative fashion. otp is then expressed in the large intestine, rectum and anal pads, unlike Byn, which is also expressed in the small intestine. Byn alone is therefore not sufficient to activate otp; lines expression might also be needed. In the small intestine where byn is expressed, lines is repressed by drumstick preventing otp activation. If lines is overexpressed in the small intestine, the repressive effect of drumstick can be overruled and otp expression can take place, supporting the proposed model that lines and byn are necessary for otp expression in the hindgut. Once otp is activated in the embryonic hindgut, its expression continues until the adult stage. The only region where otp in contrast to byn is not expressed is the larval hindgut proliferation zone in the anteriorly located pylorus which supposedly generates hindgut stem cells capable of replacing the larval hindgut cells undergoing apoptosis and building the adult hindgut. The presence of adult hindgut stem cells has been questioned when it was shown that proliferation only takes place in response to tissue damage. The current view is that all parts of the Drosophila intestinal tract maintain stem cells that could migrate across organ boundaries. Since otp expression was never shown in areas where intestinal stem cells are present, otp is rather expressed in and a marker for differentiated hindgut cells (Hildebrandt, 2020).

To analyse the function of otp, a mutant allele was generated by gene targeting via homologous recombination and using this targeting strain, two additional mutant alleles were identified by complementation among an EMS-induced collection of mutants from the 57B region. In the gene targeting mutant, part of exon 4 and exon 5 were missing resulting in an N-terminal deletion of the otp-PC protein form including most of the homeodomain. In the otp¹⁰²⁴ mutant, the N-terminus and most of the homeodomain were present, but the C-terminal part of the protein was missing. In both cases no otp protein expression was detected since the anti-Otp antibody was directed against the C-terminal part of Otp. In the case of otp¹³⁰⁶⁴, no protein was detected with anti-Otp antibody nor was a sequence alteration in the coding region detected. The splice sites were intact, but it cannot be ruled out that a cryptic splice site might be generated. The generation of cryptic splice sites by mutations is often the case in human genetic disorders like Neurofibromatosis type I (NFI) or Cystic fibrosis where such mutations generate pseudo-exons. Another possibility might be a mutation in a regulatory region of the gene. All otp alleles showed embryonic lethality with a strong hindgut phenotype. The loss of the large intestine led to a dramatic reduction in hindgut length to about one third of the wildtype length. This phenotype is comparable to the byn phenotype, since byn is directly regulating otp. The three transcriptional regulators drm, bowl and lin are required for patterning and cell rearrangements during elongation in the hindgut, but when compared to otp, showed only a reduction to 40% (drm and bowl) or 50% (lin) in the mutant phenotype, suggesting that the loss of otp function is much more severe compared to these genes. A gut specific function of otp like seen in Drosophila is not known for otp genes in higher organisms, where an expression in the nervous system and a function in various aspects of nervous system development is known. In Drosophila, otp is also expressed in the ventral nerve cord and the brain. Expression in the nerve cord seems to be post-transcriptionally regulated, since, in contrast to the mRNA expression posterior to segment A2, the otp protein is not expressed there. This might be due to the regulation via a miRNA as it was seen for other developmental processes (Hildebrandt, 2020).

The nervous system function of otp in higher organisms has been mainly analysed in various model organisms like zebrafish and mice. It was shown that otp is expressed in the hypothalamus that exerts influence on physiological function in various processes like blood pressure, circadian rhythmicity, energy balance and homeostasis. In zebrafish otp in the hypothalamus is required in the preoptic area for the production of the neurohypophysial peptide arginine vasotocin, for dopaminergic and neuroendocrine cell specification, regulation of stress response and through neuropeptide switching that impacts social behavior. In mice, a loss of otp leads to a progressive impairment of neuroendocrine cells in the hypothalamus, and homozygous mutant animals die soon after birth with a failure of terminal differentiation of neurosecretory cells. Recently, it was shown that a mutation in otp is associated with obesity and anxiety in mice. The otp gene from humans was cloned some time ago, but only during the last few years, it could be shown that otp has a high diagnostic value concerning pulmonary neuroendocrine tumours. Even if these tumours accounted for only 1%-2% of all lung tumours, their occurrence increased over the last decades. People with poor survival rates showed a strong downregulation of otp. otp that is normally located in the nucleus (nOTP) could also be detected in the cytoplasm (cOTP) or not be present at all. Patients with nOTP have a favourable disease outcome, those with cOTP have an intermediate outcome and those with no otp expression have the worst disease outcome demonstrating the diagnostic value of OTP. Due to these very interesting aspects of otp function in the nervous system of higher organisms, it would be interesting to analyse the function of otp during embryonic brain development of Drosophila, as well as later functions of otp during larval development and in the adult, using the newly generated otp alleles in the future (Hildebrandt, 2020).

Using gene expression analysis and newly generated mutant otp alleles, this study has shown that the Drosophila homeodomain transcription factor Orthopedia is an important factor for hindgut development. These findings demonstrate a requirement of otp to build the large intestine of the hindgut and also in the correct formation of the anal pads. This phenotype is caused by apoptosis at the beginning of hindgut development. otp as a downstream factor of byn is most likely present only in differentiated hindgut cells during all stages of development rather than in stem cells (Hildebrandt, 2020).

Drosophila melanogaster as a Model System to Assess the Effect of Epstein-Barr Virus DNA on Inflammatory Gut Diseases

The Epstein-Barr virus (EBV) commonly infects humans and is highly associated with different types of cancers and autoimmune diseases. EBV has also been detected in inflamed gastrointestinal mucosa of patients suffering from prolonged inflammation of the digestive tract such as inflammatory bowel disease (IBD) with no clear role identified yet for EBV in the pathology of such diseases. Since immune-stimulating capabilities of EBV DNA has been reported in various models, this study investigated whether EBV DNA may play a role in exacerbating intestinal inflammation through innate immune and regeneration responses using the Drosophila melanogaster model. Inflamed gastrointestinal tracts were generated in adult fruit flies through the administration of dextran sodium sulfate (DSS), a sulfated polysaccharide that causes human ulcerative colitis- like pathologies due to its toxicity to intestinal cells. Intestinal damage induced by inflammation recruited plasmatocytes to the ileum in fly hindguts. EBV DNA aggravated inflammation by enhancing the immune deficiency (IMD) pathway as well as further increasing the cellular inflammatory responses manifested upon the administration of DSS. The study at hand proposes a possible immunostimulatory role of the viral DNA exerted specifically in the fly hindgut hence further developing understanding of immune responses mounted against EBV DNA in the latter intestinal segment of the D. melanogaster gut. These findings suggest that EBV DNA may perpetuate proinflammatory processes initiated in an inflamed digestive system. These findings indicate that D. melanogaster can serve as a model to further understand EBV-associated gastroinflammatory pathologies. Further studies employing mammalian models may validate the immunogenicity of EBV DNA in an IBD context and its role in exacerbating the disease through inflammatory mediators (Madi, 2021).

Enteric neurons increase maternal food intake during reproduction

Reproduction induces increased food intake across females of many animal species, providing a physiologically relevant paradigm for the exploration of appetite regulation. By examining the diversity of enteric neurons in Drosophila melanogaster, this study identified a key role for gut-innervating neurons with sex and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. These findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain (Hadjieconomou, 2020).

Internal state has profound effects on brain function. Despite increasingly recognized roles for the gut-brain axis in maintaining energy balance, links between internal state and gastrointestinal innervation remain poorly characterized. Progress has been hindered by neuroanatomical complexity, which is only beginning to be parsed in mammals. The simpler-yet physiologically complex-intestine of Drosophila provides an alternative entry point into the study of gastrointestinal innervation (Hadjieconomou, 2020).

Innervation of the main digestive portion of the adult fly intestine, which encompasses the anterior midgut and the crop and central neurons of the pars intercerebralis (PI) in the brain. PI neurons directly innervate the anterior midgut and the crop, and include insulin-producing neurons and other peptidergic subtypes. The crop is further populated by processes that emanate from cells of the corpora cardiaca, which produce the glucagon-like adipokinetic hormone and are adjacent to the hypocerebral ganglion (HCG). Also adjacent to both the HCG and the corpora cardiaca are the corpus allatum cells, which produce juvenile hormone and extend short local projections. The thoracico-abdominal ganglion of the central nervous system might not innervate these gut regions (Hadjieconomou, 2020).

The crop-an expandable structure found in the intestines of insects-might be disregarded as a passive food store, but several observations suggest active regulation of its physiology. Refeeding flies after starvation results in enlarged, food-filled crops, pointing to modulation of food ingression into and out of the crop. Live imaging or temporal dissections of flies revealed that food always enters the crop before proceeding to the midgut. Additionally, food transit through the crop is dependent on both its palatability and its nutritional value. Therefore, in adult flies, all food transits through the crop, which is nutrient-sensitive and shows chemically and anatomically diverse innervation (Hadjieconomou, 2020).

The crop and anterior midgut are innervated by myosuppressin (Ms)-positive neurons located in the PI and the HCG. PI Ms neurons are distinct from known neuronal subsets, with the exception of eight Ms neurons that co-express the Taotie-GAL4 marker. Two PI Ms neuron populations can be distinguished by cell size: one comprises 18 large cells and another comprises 12 smaller cells. Single-cell clones of large Ms neurons reveal a single process that bifurcates into a longer, probably axonal projection to the gut-which arborizes in the HCG and extends further to innervate the crop-and a shorter, probably dendritic process that reaches the suboesophageal zone, where the axons of peripheral gustatory sensory neurons terminate. A subset of HCG Ms-expressing neurons also innervates the crop, whereas another subset projects locally. This study confirmed the expression of Ms using an endogenously tagged Ms reporter (Ms-GFP) and in situ hybridization Ms innervation was also observed of the hindgut, the rectal ampulla and the heart, and a subset of peripheral Ms-positive neurons innervating the female reproductive tract (Hadjieconomou, 2020).

This study selectively activated or silenced Ms neurons in adult flies. Activation resulted in greatly enlarged crops in flies that were fed ad libitum, consistent with the relaxant properties of Ms on insect muscles ex vivo. By contrast, silencing of Ms neurons prevented crop enlargement in a starved-refed condition in which the crop normally expands. Genetic downregulation or mutation of Ms (using a new mutant) prevented crop enlargement, albeit to a lesser extent than Ms neuron silencing. This could be due to another Ms-neuron-derived neurotransmitter or neuropeptide contributing to crop enlargement, or to loss of the Ms peptide during development in these experiments, resulting in adaptations that render the crop more active than it would be in response to acute loss of the Ms peptide. A Gal4 insertion into the Ms locus was generated that disrupts Ms production (MsTGEM). In contrast to the crop enlargement resulting from TrpA1-mediated activation from Ms-Gal4, TrpA1 expression from this (Ms mutant) MsTGEM-Gal4 driver failed to induce crop enlargement, further confirming a requirement for Ms. Ms neuron subtype-specific downregulations and activations enabled establishing that the PI Ms neurons (in particular, the Taotie-Gal4-positive subset of large PI Ms neurons) induce, and are indispensable for, crop enlargement through their production of Ms neuropeptide (Hadjieconomou, 2020).

The contributions of myosuppressin receptors 1 and 2 (MsR1 and MsR2) were explored. MsR1 expression was observed in crop muscles, in subsets of neurons including the PI and HCG Ms-positive neurons and neurons innervating the ovary and heart; no MsR1 expression was detected in ovarian or heart muscles. Expression of MsR2 was also detected in crop muscles. To investigate the function of the Ms receptor, MsR1 was downregulated specifically in adult crop muscles using two independent driver lines (vm-Gal4 and MsR1crop-Gal4). Both genetic manipulations led to reduced crop enlargement in a starvation-refeeding assay, comparable to that observed for Ms neuron silencing or Ms mutation. Downregulation of MsR2 did not affect crop enlargement. A role for MsR1 in mediating crop enlargement was confirmed using a MsR1TGEM mutant. MsR1 is therefore identified as the crop muscle receptor through which Ms signals to modulate crop enlargement (Hadjieconomou, 2020).

The physiological regulation of crop enlargement was explored and found that it is dependent on sex and on reproductive status: the crops of mated females fed ad libitum (which were used for all the experiments described above) were consistently more expanded than those of virgin female or mated male flies fed ad libitum. Because post-mating changes were not seen in Ms neuron projections, it was asked whether post-mating crop enlargement might result from the release of Ms preferentially in mated females. Ms peptide levels were lower in the PI neuron cell bodies of females only after mating. In the absence of Ms transcriptional changes this observation is consistent with a post-mating increase in the secretion of Ms peptide in females. This effect of mating on Ms levels was specific to mating: nutrient availability did not affect intracellular Ms levels. It was also observed that the Ms neurons of mated females had higher cumulative calcium levels and a reduced amplitude of calcium oscillations compared to virgin females, as detected both by in vivo GCaMP6 calcium imaging and by the calcium-sensitive reporter CaLexA, in which GFP expression is proportional to cumulative neuronal activity. Physiologically, and in contrast to observations in mated females, a reduction of Ms signalling in males or in virgin female flies failed to impair crop enlargement. Consequently, when Ms signalling to crop muscles was prevented, the size of the crop of mated females no longer differed from that of virgin females. Collectively, these findings support the idea that, in female flies, the activity of PI Ms neurons changes after mating to promote Ms release (Hadjieconomou, 2020).

Levels of the steroid hormone ecdysone, which promotes egg production and intestinal stem-cell proliferation, increase after mating. The ecdysone receptor (EcR) is expressed by all PI Ms neurons, which suggests that they might be sensitive to circulating ecdysone. Expression of a dominant-negative EcR-which targets all EcR isoforms-confined to the Ms neurons of adult flies was found to increase intracellular Ms levels in the Ms PI neuron cell bodies of mated females to the levels observed in virgin females, whereas it had no effect on virgin females. Downregulation of EcR (using RNA interference lines that target all isoforms or the B1 isoform specifically) produced comparable results. In both experiments, the amplitude of in vivo calcium oscillations in Ms neurons was increased to levels seen in virgin females. Compromising EcR signalling in adult Ms neurons significantly reduced crop enlargement preferentially in mated females; this phenotype was also apparent when the PI Ms neurons were targeted using Taotie-Gal4. Ecdysone therefore communicates mating status to Ms neurons through its B1 receptor (Hadjieconomou, 2020).

Previous work showed that the adult intestine is resized and metabolically remodelled after mating (Reiff, 2016), but did not investigate possible effects on its hormone-producing enteroendocrine cells. This study now observe a post-mating increase in the number of enteroendocrine cells, including a subset that expresses the hormone bursicon α (Burs), which is known to signal to adipose tissue through an unidentified neuronal relay. An endogenous protein reporter for the Burs receptor Rickets (Rk, also known as Lgr2) revealed its expression in subsets of neurons including all PI Ms neurons (including the Taotie-Gal4-positive subset) and in projections terminating in the HCG. Expression in a subset of the HCG Ms neurons was observed only sporadically (Hadjieconomou, 2020).

Consistent with the regulation of Ms neurons by the increase in Burs derived from enteroendocrine cells after mating, adult-specific downregulation of the Burs receptor gene rk in Ms neurons reverted intracellular Ms levels in the PI Ms neurons of mated females to levels observed in virgin females; there was no effect in virgin females. Like EcR downregulation, rk downregulation in Ms neurons also increased the amplitude of in vivo calcium oscillations in the Ms neuron cell bodies of mated females to values similar to those observed in virgin females. Functionally, both the downregulation of Burs in intestinal enteroendocrine cells and the adult-specific rk downregulation in Ms neurons-either in all neurons or in the Taotie-Gal4-positive subset in the PI-preferentially reduced crop enlargement in mated females. Conversely, stimulating the intestinal release of enteroendocrine hormones-including Burs-from enteroendocrine cells resulted in reduced Ms levels in the Ms neuron cell bodies of virgin females, similar to those observed in mated females, and greatly enlarged crops (Hadjieconomou, 2020).

Thus, steroid and enteroendocrine hormones communicate mating status to the brain. Acting through their receptors in the PI Ms neurons, these hormones change Ms neuronal activity, promoting the release of Ms after mating (Hadjieconomou, 2020).

To investigate the importance of Ms neuron modulation after mating, post-mating crop enlargement was selectively prevented by downregulating MsR1 in adult crop muscles using two independent strategies. This had no discernible effects in males or virgin females, but specifically prevented the increase in food intake that is normally observed in female flies after mating. Comparable results were obtained by blocking the post-mating ecdysone and Burs inputs into the Ms neurons. Downregulation of MsR2 had no such effect. The post-mating change in crop expandability, mediated by Ms and MsR1 signalling, thus causes the increased food intake observed in females after mating (Hadjieconomou, 2020).

The negative pressures that have been reported in the crops of larger insects suggest that the crop may draw food in by generating suction. The increased crop expandability enabled by Ms release after mating could therefore increase food intake through changes in suction. It was observed that mated females ingest more food per sip than virgin females, which is consistent with mated females needing to generate a higher suction pressure to facilitate bigger sips. Crop enlargement was therefore modeled using the Poiseuille equation for incompressible fluid flow in a pipe and found that the crop would need a suction pressure of the order of -1 kPa to achieve the previously reported intake volume per sip. This is in reasonable agreement with previously reported values measured in cockroach crops of between -0.5 and -1 kPa. The model predicts that mated flies would require a modest increase in suction pressure to -1.3kPa in order to facilitate the increased sip size (Hadjieconomou, 2020).

In the model, the change in crop volume drives food intake through increased suction. A crop that cannot enlarge, or a persistently enlarged crop, should therefore result in a comparable reduction in food intake by preventing the generation of suction. This was tested by persistently preventing crop enlargement (using crop-muscle-specific MsR1 knockdown) or by persistently inducing it (using TrpA1-mediated Ms neuron activation from Ms-Gal4 or Taotie-Gal4), after which the diet of these flies was switched from an undyed to a dye-laced food source to assess food intake. As predicted, both genetic manipulations reduced food intake. Conversely, increasing the rate at which the crop expands should increase food intake. This was tested by activating the Ms neurons as in the previous experiment, but this time the dye-laced food source was provided, and its intake was monitored at the same time as the neurons were activated (that is, as inducing greater crop expansion was being induced) rather than after a persistent activation (when the crop is already maximally expanded). Increased food intake was observed under these conditions in the absence of changes in the number of meals. Although further work will be required to elucidate the full dynamics of crop enlargement, filling and emptying, these experiments support the idea that the Ms-induced enlargement of the crop after mating increases food intake at least partly by increasing the suction power of the crop (Hadjieconomou, 2020).

Finally, given the links between nutrient intake and fecundity, it is proposed that the Ms-driven crop enlargement after mating might be adaptive and support reproduction. Crop enlargement was prevented selectively after mating by downregulating MsR1 from crop muscles, as in previous experiments. This resulted in reduced egg production, and the eggs that were produced had reduced viability. It is therefore conclude that the crop and its Ms innervation sustain the increase in food intake after mating, maximising female fecundity (Hadjieconomou, 2020).

These findings lead to a proposal that the maternal increase in food intake during reproduction is adaptive, that the crop is a key reproductive organ, and that Ms is a major effector of post-mating responses. In support of these ideas, the crop is absent in larvae-the juvenile stage of insects-and other Diptera have co-opted it for reproductive behaviours such as the regurgitation of nuptial gifts or the secretion of male pheromones. Ms receptors are also closely related to the Sex peptide receptor (the 'mating sensor' of female flies), and both diverged after duplication of an ancestral receptor that might have responded to the Myoinhibitory peptide (Mip) in the last common ancestor of protostomes. It will be interesting to explore possible links between Ms and Sex peptide signalling, and whether and how these mating signals affect recently described crop mechanosensing mechanisms that restrain ingestion as the crop expands in order to terminate large meals (Hadjieconomou, 2020).

This study has provided evidence for a gut-to-brain axis in Drosophila by identifying central Ms neurons as targets of the gut-derived hormone Burs. These central neurons innervate the gut, 'closing' a gut-brain-gut loop that connects midgut enteroendocrine signals to the crop, a more anterior gut region. This might allow for the functional coordination of different gut portions, while enabling central modulation by sensory cues (for example, gustatory). This study also identified the Ms neurons as the neural targets of ecdysone, which has been shown to promote food intake. Reproduction has pronounced, and in some cases lasting, effects on the human female brain; Ms neurons provide a tractable and physiologically relevant neural substrate for the investigation of the mechanisms involved (Hadjieconomou, 2020).

The human digestive system might be similarly modulated by reproductive cues to affect food intake. In mammals, enteric neurons express sex and reproductive-hormone receptors, and enteroendocrine hormone levels change during reproduction. It is suggested that pregnancy and lactation represent an attractive and relatively unexplored physiological adaptation for the investigation of nutrient intake regulation, organ remodelling and metabolic plasticity-mechanisms that might eventually be leveraged to curb appetite and/or weight gain (Hadjieconomou, 2020).

A neural circuit integrates pharyngeal sensation to control feeding

Swallowing is an essential step of eating and drinking. However, how the quality of a food bolus is sensed by pharyngeal neurons is largely unknown. This study finds that mechanical receptors along the Drosophila pharynx are required for control of meal size, especially for food of high viscosity. The mechanical force exerted by the bolus passing across the pharynx is detected by neurons expressing the mechanotransduction channel NOMPC (no mechanoreceptor potential C) and is relayed, together with gustatory information, to IN1 neurons in the subesophageal zone (SEZ) of the brain. IN1 (ingestion neurons) neurons act directly upstream of a group of peptidergic neurons that encode satiety. Prolonged activation of IN1 neurons suppresses feeding. IN1 neurons receive inhibition from DSOG1 (descending subesophageal neurons) neurons, a group of GABAergic neurons that non-selectively suppress feeding. These results reveal the function of pharyngeal mechanoreceptors and their downstream neural circuits in the control of food ingestion (Yang, 2021).

Overconsumption is harmful for animals. Although the drive to ingest can be overwhelming for a hungry animal in the initial stage of a meal, inhibition becomes more dominant with the processes of food intake. This study found that food flowing across the pharynx accumulates the satiety state in the brain, demonstrating that multiple strategies are used by the nervous systems to avoid overeating. These pharyngeal sensory neurons are sensitive to sugar and mechanical force, serving as a flow meter that monitors food quality and amount so that the brain knows how much food is ingested even before the food reaches the intestine. This circuit may coordinate with other satiety signals, such as those conveyed by mechanical feedback from the intestine, to control feeding (Yang, 2021).

Gustatory and mechanosensory neurons are well separated on the fly labellum before their axons reach the SEZ, where they interact with each other to regulate the perception of food quality. In contrast, the sensory neurons in the pharynx seem to adapt a different coding mechanism. Some of the pharyngeal neurons are polymodal because they respond to chemical and mechanical stimuli, with PM neurons being an example. A 'generalist' versus 'specialist strategy has been found in other sensory organs too. Being able to evaluate multiple properties of a bolus in the pharynx allow the animals to effectively control the feeding amount. There are sensory neurons in the pharynx that may be tuned to gustatory or mechanosensory cues. For example, the R41E11-GAL4 and nompC-QF labeled approximately 10 pairs of neurons in LSOs along the pharynx, similar to the number observed for mechanosensory neurons. Most of those neurons are likely 'generalist' and are tuned to mechanical stimuli only. It would be valuable to determine the full repertoire of these sensory neurons to understand how the swallowing maneuver is initiated, sustained, and terminated (Yang, 2021).

It has been proposed that IN1 neurons may function as a key node of the feeding control circuits to govern rapid feeding decisions. Previous studies have revealed that IN1 neurons are directly downstream of pharyngeal GRNs and that activation of IN1 neurons to sugar stimulation is correlated with a fly's motivation to feed. Because activation of IN1 neurons triggers proboscis extension to food, they are likely upstream of the motor circuit that controls feeding. IN1 neurons thus appear to function as a hub that integrates sensory information to initiate food ingestion. This study found that IN1 neurons' activity is under control of the fly's feeding states. IN1 neurons are directly downstream of DSOG1 neurons that non-selectively suppress ingestion. In fed flies, DSOG1 neurons impart inhibition on IN1 neurons, resulting in a transient and moderate response to a sugar sip that triggers a robust and sustained calcium response in fasted flies (Yang, 2021).

It has been proposed that DSOG1 neurons impart constant inhibition on the neuronal circuits that initiate food ingestion. However, the upstream circuit of DSOG1 neurons has not been identified. A cohort of neuropeptide receptor genes has been screened, but none of them seemed to function on DSOG1 neurons in feeding control. This study found that interrupting signaling of the neuropeptide MIP phenocopied overfeeding in flies with silenced DSOG1 neurons. It is tantalizing to hypothesize that MIP neurons are upstream of the DSOG1 circuit, either directly or indirectly. Because the receptors of MIP have not yet been identified, further experiments are need to differentiate between the two possibilities (Yang, 2021).

Besides PM neurons, there are many NOMPC-expressing mechanosensory neurons along the fly pharynx. Because of the lack of specific driver lines and the technique to record a single neuron's activity during feeding, their roles in feeding regulation are interesting open questions and await in-depth investigation. Moreover, the receptors of MIP peptide have not been identified, especially the ones involved in feeding regulation, making it difficult to establish a connection between MIP neurons and DSOG1 neurons (Yang, 2021).

Drosophila TRPgamma is required in neuroendocrine cells for post-ingestive food selection

The mechanism through which the brain senses the metabolic state, enabling an animal to regulate food consumption, and discriminate between nutritional and non-nutritional foods is a fundamental question. Flies choose the sweeter non-nutritive sugar, L-glucose, over the nutritive D-glucose if they are not starved. However, under starvation conditions, they switch their preference to D-glucose, and this occurs independent of peripheral taste neurons. This study found that eliminating the TRPgamma channel impairs the ability of starved flies to choose D-glucose. This food selection depends on trpgamma expression in neurosecretory cells in the brain that express Diuretic hormone 44 (DH44). Loss of trpgamma increases feeding, alters the physiology of the crop, which is the fly stomach equivalent, and decreases intracellular sugars and glycogen levels. Moreover, survival of starved trpgamma flies is reduced. Expression of trpgamma in DH44 neurons reverses these deficits. These results highlight roles for TRPgamma in coordinating feeding with the metabolic state through expression in DH44 neuroendocrine cells (Dhakal, 2022).

Orthopedia expression during Drosophila melanogaster nervous system development and its regulation by microRNA-252

During brain development of Drosophila melanogaster many transcription factors are involved in regulating neural fate and morphogenesis. In this study it was shown that the transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, plays an important role in this process. Otp is expressed in a stable pattern in defined lineages from mid-embryonic stages into the adult brain and therefore a very stable marker for these lineages. The abundance of the two different otp transcripts in the brain and hindgut during development was determined using qPCR. CRISPR/Cas9 generated otp mutants of the longer protein form significantly affect the expression of Otp in specific areas. An otp enhancer trap strain was generated by gene targeting and reintegration of Gal4 that mimics the complete expression of otp during development except the embryonic hindgut expression. Since in the embryo, the expression of Otp is posttranscriptionally regulated, putative miRNAs interacting with the otp 3'UTR were sought; microRNA-252 was identified as a candidate. Further analyses with mutated and deleted forms of the microRNA-252 interacting sequence in the otp 3'UTR demonstrate an in vivo interaction of microRNA-252 with the otp 3'UTR. An effect of this interaction is seen in the adult brain, where Otp expression is partially abolished in a knockout strain of microRNA-252. These results show that Otp is another important factor for brain development in Drosophila melanogaster (Hildebrandt, 2022).

CYP311A1 in the anterior midgut is involved in lipid distribution and microvillus integrity in Drosophila melanogaster

Lipids are either taken up from food sources or produced internally in specialized tissues such as the liver. Among others, both routes of lipid metabolism involve cytochrome P450 monooxygenases (CYPs). This study sought to analyze the function of Cyp311a1 that has been shown to be expressed in the midgut of the fruit fly Drosophila melanogaster. Using a GFP-tagged version of CYP311A1 that is expressed under the control of its endogenous promoter, it was shown that Cyp311a1 localizes to the endoplasmic reticulum in epithelial cells of the anterior midgut. In larvae with reduced Cyp311a1 expression in the anterior midgut, compared to control larvae, the apical plasma membrane of the respective epithelial cells contains less and shorter microvilli. In addition, reduction of neutral lipids was observed in the fat body, the insect liver, and decreased phosphatidylethanolamine (PE) and triacylglycerols (TAG) amounts in the whole body of these larvae. Probably as a consequence, they cease to grow and eventually die. The microvillus defects in larvae with reduced Cyp311a1 expression are restored by supplying PE, a major phospholipid of plasma membranes, to the food. Moreover, the growth arrest phenotype of these larvae is partially rescued. Together, these results suggest that the anterior midgut is an import hub in lipid distribution and that the midgut-specific CYP311A1 contributes to this function by participating in shaping microvilli in a PE-dependent manner (Dong, 2022).

A timer gene network is spatially regulated by the terminal system in the Drosophila embryo

In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the 'timer' genes caudal, Dichaete and odd-paired, whose expression dynamics correlate with the mode of segmentation. In Drosophila, the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small 'tail' region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless, but beyond this the regulation of the timer genes is poorly understood. This study used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. It is proposed that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation (Clark, 2022).

Transmission of beneficial yeasts accompanies offspring production in Drosophila-An initial evolutionary stage of insect maternal care through manipulation of microbial load

Parent-to-offspring transmission of beneficial microorganisms is intimately interwoven with the evolution of social behaviors. Ancestral stages of complex sociality-microbe vectoring interrelationships may be characterized by high costs of intensive parental care and hence only a weak link between the transmission of microbial symbionts and offspring production. This study investigate the relationship between yeast symbiont transmission and egg-laying, as well as some general factors thought to drive the "farming" of microscopic fungi by the fruit fly Drosophila melanogaster, an insect with no obvious parental care but which is highly dependent on dietary microbes during offspring development. The process of transmitting microbes involves flies ingesting microbes from their previous environment, storing and vectoring them, and finally depositing them to a new environment. This study revealed that fecal materials of adult flies play a significant role in this process, as they contain viable yeast cells that support larval development. During single patch visits, egg-laying female flies transmitted more yeast cells than non-egg-laying females, suggesting that dietary symbiont transmission is not random, but linked to offspring production. The crop, an extension of the foregut, was identified as an organ capable of storing viable yeast cells during travel between egg-laying sites. However, the amount of yeast in the crop reduced rapidly during periods of starvation. Although females starved for 24 h deposited a smaller amount of yeast than those starved for 6 h, the yeast inoculum produced still promoted the development of larval offspring. The results of these experiments suggest that female Drosophila fruit flies have the ability to store and regulate the transfer of microorganisms beneficial to their offspring via the shedding of fecal material. It is argue that this observation may represent an initial evolutionary stage of maternal care through the manipulation of microbial load, from which more specialized feedbacks of sociality and microbe management may evolve (Cho, 2023).

Senescent cells and macrophages cooperate through a multi-kinase signaling network to promote intestinal transformation in Drosophila

Cellular senescence is a conserved biological process essential for embryonic development, tissue remodeling, repair, and a key regulator of aging. Senescence also plays a crucial role in cancer, though this role can be tumor-suppressive or tumor-promoting, depending on the genetic context and the microenvironment. The highly heterogeneous, dynamic, and context-dependent nature of senescence-associated features and the relatively small numbers of senescent cells in tissues makes in vivo mechanistic studies of senescence challenging. As a result, which senescence-associated features are observed in which disease contexts and how they contribute to disease phenotypes remain largely unknown. Similarly, the specific mechanisms by which various senescence-inducing signals are integrated in vivo to induce senescence and why some cells become senescent while their immediate neighbors do not are unclear. This study identified a small number of cells that exhibit multiple features of senescence in a genetically complex model of intestinal transformation established in the developing Drosophila larval hindgut epithelium. These cells emerge in response to concurrent activation of AKT, JNK, and DNA damage response pathways within transformed tissue. Eliminating senescent cells, genetically or by treatment with senolytic compounds, reduces overgrowth and improves survival. This tumor-promoting role is mediated by Drosophila macrophages recruited to the transformed tissue by senescent cells, which results in non-autonomous activation of JNK signaling within the transformed epithelium. These findings emphasize complex cell-cell interactions underlying epithelial transformation and identify senescent cell-macrophage interactions as a potential druggable node in cancer. It is concluded that interactions between transformed senescent cells and macrophages drive tumorigenesis (Datta, 2023).

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date revised: 12 April 2022 

Genes involved in organ development

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