• Components of the JAK/STAT pathway
  • domeless - a cytokine receptor
  • hopscotch - Drosophila's Janus kinase
  • outstretched (unpaired) - a cytokine
  • Signal-transducer and activator of transcription protein at 92E (marelle)
  • Suppressor of cytokine signaling at 36E

• Required for function of the JAK/STAT pathway
  • ken and barbie
  • Rab-protein 5
  • Sumo/smt3

• Targets of the JAK/STAT pathway
  • even-skipped
  • Sex lethal

Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, a genome-wide RNA interference (RNAi) screen was carried out in cultured Drosophila cells. One hundred and twenty-one genes were identified whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, it was found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, a key negative regulator of Drosophila JAK/STAT signaling was identified, protein tyrosine phosphatase PTP61F; it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. This study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway (Baeg, 2005).

To more clearly elucidate the roles of positive regulators, their requirement for the phosphorylation of STAT92E was assayed. Tyrosine phosphorylation is a key step in STAT activation upon cytokine/receptor stimulation. Thus, monitoring steady-state levels of phosphorylated STAT in dsRNA-treated cells would provide insight into the molecular functions of the candidate genes. As expected, Upd stimulation of S2-NP cells leads to a dramatic increase in tyrosine-phosphorylated STAT92E, as shown by Western blot analysis. The effect was measured of dsRNAs against the 29 positive regulators on Upd-induced STAT92E phosphorylation. Thirteen genes (besides STAT92E) were found to be required for Upd-induced STAT92E phosphorylation. As expected, these genes included the canonical components Dome and hop. In contrast to the initial assay in the primary screen, exogenous Upd was used to activate STAT92E phosphorylation, and thus it was not possible to identify genes that act upstream of the receptor, such as Upd2. Notably, two of the 13 genes (CG16790 and CG4329) that regulate STAT92E phosphorylation have no predicted function, yet clearly have human orthologs; further investigation of their molecular functions in JAK/STAT signaling in Drosophila may advance understanding of the mammalian pathway (Baeg, 2005).

Interestingly, this assay revealed that RNAi knockdown of the cyclin-dependent kinase 2 gene (cdc2) resulted in a decrease in STAT92E tyrosine phosphorylation, suggesting that cdc2 modulates JAK/STAT signaling by affecting tyrosine phosphorylation of STAT92E. Consistent with this observation, Warts/Lats, which has been shown both biochemically and genetically to interact with cdc2 and to negatively regulate its kinase activity, was identified in the screen as a potential negative regulator of JAK/STAT signaling. These results suggest that STAT92E plays an important role in Warts/Lats-mediated inhibition of cell proliferation (Baeg, 2005).

echinoid (ed) was identified as a positive regulator required for Upd-dependent STAT92E tyrosine phosphorylation. ed encodes a cell adhesion molecule and has been shown to be a negative regulator of the EGFR signaling pathway during Drosophila eye development. Previous experiments have shown both positive and negative interactions between the JAK/STAT pathway and the EGFR pathway. For example, STAT92E mutants phenocopy mutants in the EGFR pathway. Furthermore, studies using mammalian tissue culture systems have demonstrated that EGFR signaling activates both JAK1 and STAT1. In addition, EGFR-induced cell migration is mediated predominantly by the JAK/STAT pathway in primary esophageal keratinocytes. Similarly, ed has been shown to be responsible for defective cell migration in Caenorhabditis elegans. Therefore studying the role of ed in JAK/STAT signaling in different contexts may facilitate understanding of the genetic and biochemical mode of STAT activation by EGFR signaling, and provide insights into the mechanisms governing cancer cell metastasis in humans (Baeg, 2005).

Another step in the activation of the JAK/STAT signaling pathway is the translocation of STATs into the nucleus. In resting cells, STATs reside mainly in the cytoplasm. Upon cytokine stimulation, they are phosphorylated on key tyrosine residues and rapidly translocate to the nucleus, where they trans-activate target genes. Previous studies have shown that Importin alpha5 and Ran are required for the nuclear import of phosphorylated (activated) STATs. To reset the cells after stimulation, STATs are exported out of the nucleus into the cytoplasm in preparation for the next round of signaling using an Exportin-1/CRM-1-dependent mechanism. These observations suggest that defective nucleocytoplasmic shuttling of STATs can disrupt steady-state distribution of STATs and induce aberrant biological responses. Among all 121 candidates, seven genes were identified that are potentially involved in protein trafficking based on their predicted molecular functions and protein domains. These include Rab26, Ran, CG10225, which encodes the Drosophila homolog of Ran-binding protein 3 (RanBP3), CG11763, which encodes the Drosophila homolog of Ran-binding protein 10 (RanBP10), and the Drosophila homolog of Cellular Apoptosis Susceptibility gene product (CAS) that was initially identified as a Ran-binding protein. In addition, Drosophila homologs of Transportin 1 and Nucleoporin 196, which have been implicated in protein import and/or export in mammals, were identifed. The subcellular localization of phosphorylated STAT92E was examined under conditions where each of the seven candidates was depleted by RNAi except Rad26. As a control it was found that under resting conditions tyrosine phosphorylated STAT92E is detected predominantly in the cytoplasm. Moreover, a significant reduction was observed in phosphorylated STAT92E levels in the cytoplasm when cells were treated with dsRNA against the receptor dome. Upon stimulation with Upd, STAT92E accumulated in the nuclei of 27% of cells. These results illustrate the specificity and sensitivity of the assay. Interestingly, it was found that cells treated with dsRNAs against CG11763 or CG10225 displayed a significant increase in phospho-STAT92E nuclear accumulation upon Upd stimulation. This was not due to changes in the total phosphorylation levels of STAT92E. No significant effects of dsRNA-mediated knockdown of Cas or Trn on STAT92E translocation was detected. In contrast, the role of Ran and Nup98 in STAT92E translocation could not be assessed in this assay due to difficulties in introducing the Upd expression vector into cells upon RNAi knockdown of these two genes. Taken together, these results strongly suggest that the Drosophila homologs of RanBP3 and RanBP10 are novel regulators of JAK/STAT signaling that affect signal-dependent STAT92E nuclear transport (Baeg, 2005).

Another important step in the JAK/STAT signal transduction pathway is the dephosphorylation of the signaling molecules JAKs and STATs. In mammals, several PTPs have been implicated in the dephosphorylation of JAK and/or STAT proteins both in the cytoplasm and in the nucleus. In contrast, no PTPs have been identified that regulate JAK/STAT signaling in Drosophila. PTP61F was identified as a strong negative regulator in the screen. Knockdown of PTP61F by RNAi resulted in a more than fourfold increase in STAT92E-dependent reporter activity. PTP61F encodes the Drosophila homolog of mammalian PTP-1B, which has been shown to attenuate insulin, PDGF, EGF, and IGF-I signaling by dephosphorylating tyrosine residues of JAKs and/or STATs in mammalian tissue culture. Therefore the hypothesis was tested that PTP61F might serve as the tyrosine phosphatase for Hop. A dramatic increase was observed in tyrosine phosphorylation of Hop upon RNAi knockdown of PTP61F, suggesting that Hop may be a substrate of PTP61F. A significant increase was detected in STAT92E phosphorylation in cells treated with dsRNA against PTP61F. This is consistent with the notion that STAT92E is a downstream target of Hop, although the possibility that both Hop and STAT92E may be targets of PTP61F cannot be ruled out (Baeg, 2005).

In both mammals and Drosophila, SOCS, a negative regulator of the JAK/STAT pathway, has been shown to be transcriptionally activated by JAK/STAT signaling, thus generating a negative feedback loop. This prompted an examination of the expression pattern of PTP61F and whether its expression is responsive to JAK/STAT signaling in vivo. It was found PTP61F is expressed in a striped pattern, reminiscent of the STAT92E expression pattern. In addition, overexpression of Upd under the control of prd-Gal4 resulted in a dramatic increase in PTP61F transcript levels in the paired domain. Furthermore, levels of the PTP61F transcript were greatly reduced in embryos lacking Hop activity, suggesting that PTP61F transcription is dependent on active JAK/STAT signaling. Taken together, these results demonstrate that PTP61F expression responds to JAK/STAT signaling in vivo (Baeg, 2005).

These data suggested that loss of PTP61F would result in an increase in JAK/STAT signaling. Thus, the genetic interaction between PTP61F and canonical components of the JAK/STAT pathway was examined, using Df(3)ED4238, a deficiency uncovering the PTP61F gene. The interaction was tested in the Drosophila eye following overexpression of Upd using GMR-Gal4 driver, which causes a dramatic overgrowth and deformation of the adult eye. The severity of this phenotype is proportional to the strength of the JAK/STAT-mediated signal, because removing one copy of STAT92E significantly suppresses the GMR-Upd eye phenotype. Consistent with PTP61F being a negative regulator of the JAK/STAT signaling pathway, flies heterozygous for Df(3)ED4238 showed an enhanced deformed eye phenotype. A PTP61F transgene rescues this enhanced deformed eye phenotype in flies heterozygous for Df(3)ED4238. In addition, the PTP61F transgene also rescues lethality in flies carrying UAS-Upd GMR-Gal4/+; Df(3)ED4238/+, presumably caused by leaky expression of UAS-Upd in conjunction with PTP61F deficiency (Baeg, 2005).

The genetic interaction between PTP61F and Hop was examined. Flies carrying a dominant hyperactive Hop allele (Hop^Tum-l) display decreased viability and the formation of melanotic tumors. This tumor formation phenotype is sensitive to gene dosage. Previous studies have shown that reducing the levels of positive regulators, such as STAT92E, Cdk4, and CycE, increases the viability and/or decreases tumor formation. Therefore both viability and melanotic tumor formation were monitored in females heterozygous for Hop^Tum-l and these results were compared to females heterozygous for both Hop^Tum-l and Df(3)ED4238. Removing one copy of PTP61F in Hop^Tum-l heterozygous females leads to a significant decrease in survival rate and a dramatic enhancement in the formation of melanotic tumors. Altogether, these results demonstrate that PTP61F is a bona fide negative regulator of the JAK/STAT pathway in Drosophila (Baeg, 2005).

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

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

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

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

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

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

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

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

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

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

Nutrient-driven dedifferentiation of enteroendocrine cells promotes adaptive intestinal growth in Drosophila

Post-developmental organ resizing improves organismal fitness under constantly changing nutrient environments. Although stem cell abundance is a fundamental determinant of adaptive resizing, understanding of its underlying mechanisms remains primarily limited to the regulation of stem cell division. This study demonstrates that nutrient fluctuation induces dedifferentiation in the Drosophila adult midgut to drive adaptive intestinal growth. From lineage tracing and single-cell RNA sequencing, a subpopulation of enteroendocrine (EE) cells was identified that convert into functional intestinal stem cells (ISCs) in response to dietary glucose and amino acids by activating the JAK-STAT pathway. Genetic ablation of EE-derived ISCs severely impairs ISC expansion and midgut growth despite the retention of resident ISCs, and in silico modeling further indicates that EE dedifferentiation enables an efficient increase in the midgut cell number while maintaining epithelial cell composition. These findings identify a physiologically induced dedifferentiation that ensures ISC expansion during adaptive organ growth in concert with nutrient conditions (Nagai, 2023).

Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis

Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). This study shows a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. This work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche (Stine, 2014: PubMed).

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

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

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

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

Sox (SRY-related HMG-box) family transcription factors (TFs) are known to have diverse roles in cell-fate specification and differentiation in multicellular organisms. In mouse-small intestine, Sox9, a SoxE subfamily member, is expressed in ISCs to regulate ISC proliferation and differentiation, but whether it acts as an oncogene or a tumor suppressor is still in debate. In Drosophila midgut, Sox21a, a SoxB2 subfamily member, is specifically expressed in ISCs and transient progenitor cells, and is essential for progenitor cell differentiation into mature cells (Chen, 2016, Zhai, 2015, Zhai, 2017). This study identified Sox100B, the Drosophila ortholog of Sox9, as a common downstream gene target for both the JAK/STAT and the EGFR signaling in regulating ISC proliferation and differentiation. This study also revealed that an appropriate level of Sox100B is critical for its function in regulating ISC proliferation, in that it may allow it to serve as an important mediator for a balanced process of ISC proliferation and differentiation, thereby maintaining intestinal homeostasis (Jin, 2020).

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

Sox100B is a Sox family group-E transcription factor, homolog of mammalian Sox8/9/10. In mouse small intestine, Sox9 is expressed in stem cells and progenitor cells at the base of crypts, and loss of Sox9 in the intestinal epithelium causes ISC hyperplasia and failure of Paneth cell differentiation (Bastide, 2007, Mori-Akiyama, 2007). Interestingly, in the stem cell zone, Sox9 is expressed at low levels in ISCs and high levels in the quiescent or reserved stem cells that are also considered as the secretory progenitors. A possible explanation for these observations is that a low level of Sox9 sustains actively dividing ISCs, while an increase of SOX9 converts these proliferating ISCs into quiescent ISCs that will eventually differentiate into Paneth cells. Similarly, Sox9 is also implicated in regulating colorectal cancer cells, but there are conflicting data regarding whether Sox9 functions as an oncogene or a tumor suppressor. These seemingly contradictory results can be reconciled with a proposed model that Sox9 functions at an appropriate level, with a critical dose of Sox9 that exhibits proliferation-promoting activity, while increasing or decreasing this dose both result in proliferation-inhibitory activity. It is worthy to note that the differentiation-promoting function of Sox9 could potentially further complicate the interpretation of the mutant phenotype. It has been shown in Drosophila gut that defects in differentiation can induce a stressed microenvironment that promotes cell proliferation and propels tumor development (Jin, 2020).

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

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

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

Cell competition modifies adult stem cell and tissue population dynamics in a JAK-STAT-dependent manner

Throughout their lifetime, cells may suffer insults that reduce their fitness and disrupt their function, and it is unclear how these potentially harmful cells are managed in adult tissues. This question was addressed using the adult Drosophila posterior midgut as a model of homeostatic tissue and ribosomal Minute mutations to reduce fitness in groups of cells. A quantitative approach was taken, combining lineage tracing and biophysical modeling, and how cell competition affects stem cell and tissue population dynamics was addressed. Healthy cells were shown to induce clonal extinction in weak tissues, targeting both stem and differentiated cells for elimination. It was also found that competition induces stem cell proliferation and self-renewal in healthy tissue, promoting selective advantage and tissue colonization. Finally, winner cell proliferation was shown to be fueled by the JAK-STAT ligand Unpaired-3, produced by Minute^-/+ cells in response to chronic JNK stress signaling (Kolahgar, 2015).

Recent studies have shown that cell competition can also take place in adult tissues. This work has taken this notion forward and delineated quantitatively how adult stem cells and tissue population dynamics are affected by cell competition. In the subfit population, differentiated cells are killed by apoptosis followed by cell delamination; stem cells are also eliminated, possibly via induction of differentiation, as dying stem cells have not been detected. In parallel, as this study has shown, the healthy tissue expands due to an increase in stem cell proliferation and self-renewal. Indeed, biophysical modeling shows that changes in these parameters of a magnitude comparable to what observed experimentally is sufficient to recapitulate the stem cell dynamics of wild-type tissue undergoing Minute cell competition. Interestingly, accelerated proliferation of fitter stem cells has been seen in mouse embryonic stem cells using in vitro models of cell competition. However, in those studies, increased stem cell self-renewal has not been observed, probably because stemness in vitro is artificially maintained by exogenous factors in the culture medium (Kolahgar, 2015).

In many adult homeostatic tissues, stem cells stochastically differentiate or self-renew, and this leads to clonal extinction balanced by clonal expansion. This is known as neutral drift competition, because through this process, stem cell compartments stochastically tend toward monoclonality. It has also been shown that stem cell competition can be nonneutral (i.e., biased) when stem cells acquire a cell-autonomous advantage. In these cases, the bias derives from intrinsic differences (e.g., faster proliferation) and does not rely on cell interactions. This study shows instead that in adult homeostatically maintained tissues, competitive cell interactions can act as extrinsic cues that actively modify stem cell behavior, and that this confers on winners an advantage (e.g., as this study observed, increased proliferation rate and self-renewal) and on losers a disadvantage (e.g., as observed induced cell death), influencing tissue colonization. It is important to note that clones of wild-type cells that have lost proliferative capability because they are devoid of ISCs are equally able to induce death in neighboring M/+cells. This rules out the possibility that physical displacement due to a faster clonal expansion is the cause of cell competition in this case. This process instead, like the recent reports of cell competition in the mouse heart and fly nervous system, likely corresponds to the adult equivalent of the cellular competition observed in developing tissues (Kolahgar, 2015).

This work shows that M/+ midguts suffer from a chronic inflammatory response, which through JNK signaling activation and the ensuing production of the JAK-STAT ligand Upd-3 promotes wild-type tissue overgrowth. Thus, in this tissue, the overproliferation of winner cells stems from the increased availability of proliferative signals in the M/+ environment. The results suggest that wild-type cells respond more efficiently than M/+ cells to this proliferation stimulus, and that this difference results in their preferential overgrowth, contributing to cell competition. It has long been suggested that cell competition may result from the limiting availability of growth factors, which would compromise the viability of loser cells. Here it was instead found that excess production of a growth factor (Upd-3) can boost cell competition by promoting preferential proliferation of fitter cells. Given that JNK and JAK-STAT are frequently activated in response to stress or deleterious mutations, it would be interesting to test whether this is a general mechanism used by loser cells to promote the overgrowth of fitter neighbors. Notably, differences in JAK-STAT signaling are sufficient to trigger cell competition and, consistent with this, reducing JAK-STAT signaling in wild-type cells compromises their ability to eliminate scribble/losers. Thus, increased JAK-STAT signaling may in addition provide wild-type cells with a heightened fitness state and help promote the elimination of M/losers (Kolahgar, 2015).

Ribosomal mutations are linked with many adult disorders, not just in Drosophila but more importantly in humans, where they are associated with a number of severe pathologies, collectively known as ribosomopathies. Given that 79 proteins make up the eukaryotic ribosome (and several more are involved in ribosomal production) and that many Minute mutations are dominant, the sporadic insurgence of M/+ in adult tissues is likely to be one of the most common spontaneous generations of somatic mutant cells in our bodies. The elimination of these cells via cell competition is likely to play an unappreciated role in maintaining healthy adult tissues (Kolahgar, 2015).

A striking feature emerging from the results is that, in response to cell competition, normal cells can efficiently repopulate adult tissues, thus effectively replacing potentially diseased cells. This bears striking resemblance to the phenomenon of mosaic revertants, observed in a number of human skin and blood diseases. Spontaneous sporadic reversion of genetically inherited, disease-bearing mutations leads to the generation of revertant cells, which effectively repopulate tissues, at times ameliorating the condition. In some instances, the revertants' expansion is so efficient that selective advantage has been. Intriguingly, ichthyosis with confetti, a skin disease characterized by confetti-like appearance of revertant skin spots, is associated with a mutation in Keratin 10, which, due to its nucleolar mislocalization, could affect ribosome production similar to M-/+mutants. Thus, based on these findings, it is tentative to speculate that selective advantage in mosaic revertants could in some cases be driven by cell competition (Kolahgar, 2015).

The work shows that M/+ midguts suffer from a chronic inflammatory response, which through JNK signaling activation and the ensuing production of the JAK-STAT ligand Upd-3 promotes wild-type tissue overgrowth. Thus, in this tissue, the overproliferation of winner cells stems from the increased availability of proliferative signals in the M/+ environment. The results suggest that wild-type cells respond more efficiently than M/+ cells to this proliferation stimulus, and that this difference results in their preferential overgrowth, contributing to cell competition. It has long been suggested that cell competition may result from the limiting availability of growth factors, which would compromise the viability of loser cells. This study found instead that excess production of a growth factor (Upd-3) can boost cell competition by promoting preferential proliferation of fitter cells. Given that JNK and JAK-STAT are frequently activated in response to stress or deleterious mutations, it would be interesting to test whether this is a general mechanism used by 'loser' cells to promote the overgrowth of fitter neighbors. Notably, differences in JAK-STAT signaling are sufficient to trigger cell competition and, consistent with this, reducing JAK-STAT signaling in wild-type cells compromises their ability to eliminate scribble/ losers. Thus, increased JAK-STAT signaling may in addition provide wild-type cells with a heightened fitness state and help promote the elimination of M/+ losers (Kolahgar, 2015).

Ribosomal mutations are linked with many adult disorders, not just in but more importantly in humans, where they are associated with a number of severe pathologies, collectively known as ribosomopathies. Given that 79 proteins make up the eukaryotic ribosome (and several more are involved in ribosomal production) and that many Minute mutations are dominant, the sporadic insurgence of M/+ cells in adult tissues is likely to be one of the most common spontaneous generations of somatic mutant cells in our bodies. The elimination of these cells via cell competition is likely to play an unappreciated role in maintaining healthy adult tissues (Kolahgar, 2015).

A striking feature emerging from the current results is that, in response to cell competition, normal cells can efficiently repopulate adult tissues, thus effectively replacing potentially diseased cells. This bears striking resemblance to the phenomenon of mosaic revertants, observed in a number of human skin and blood diseases. Spontaneous sporadic reversion of genetically inherited, disease-bearing mutations leads to the generation of revertant cells, which effectively repopulate tissues, at times ameliorating the condition. In some instances, the revertants' expansion is so efficient that selective advantage has been proposed. Intriguingly, ichthyosis with confetti, a skin disease characterized by confetti-like appearance of revertant skin spots, is associated with a mutation in Keratin 10, which, due to its nucleolar mislocalization, could affect ribosome production similar to M/+ mutants. Thus, based on the current findings, it is tentative to speculate that selective advantage in mosaic revertants could in some cases be driven by cell competition (Kolahgar, 2015).

Methotrexate is a JAK/STAT pathway inhibitor

The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. There is a need for effective low cost treatments. This study used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. Methotrexate was shown to suppress human JAK/STAT signalling, without affecting other phosphorylation-dependent pathways. Furthermore, methotrexate significantly reduces STAT5 phosphorylation in cells expressing JAK2 V617F, a mutation associated with most human MPNs. Methotrexate acts independently of dihydrofolate reductase (DHFR) and is comparable to the JAK1/2 inhibitor ruxolitinib. However, cells treated with methotrexate still retain their ability to respond to physiological levels of the ligand erythropoietin. It is concluded that aminopterin and methotrexate act as competitive inhibitors of DHFR. Methotrexate is also widely used at low doses to treat inflammatory and immune-mediated conditions including rheumatoid arthritis. In this low-dose regime, folate supplements are given to mitigate side effects by bypassing the biochemical requirement for DHFR. Although independent of DHFR, the mechanism-of-action underlying the low-dose effects of methotrexate is unknown. Given that multiple pro-inflammatory cytokines signal through the pathway, it is suggested that suppression of the JAK/STAT pathway is likely to be the principal anti-inflammatory and immunosuppressive mechanism-of-action of low-dose methotrexate. In addition, it is suggested that patients with JAK/STAT-associated haematological malignancies may benefit from low-dose methotrexate treatments. These findings represent an important development with significant cost-saving future potential (Thomas, 2015).

Socs36E controls niche competition by repressing MAPK signaling in the Drosophila testis

Socs36E, which encodes a negative feedback inhibitor of the JAK/STAT pathway, is the first identified regulator of niche competition in the Drosophila testis. The competitive behavior of Socs36E mutant cyst stem cells (CySCs) has been attributed to increased JAK/STAT signaling. This study shows that competitive behavior of Socs36E mutant CySCs is due in large part to unbridled Mitogen-Activated Protein Kinase (MAPK) signaling. In Socs36E mutant clones, MAPK activity is elevated. Furthermore, it was found that clonal upregulation of MAPK in CySCs leads to their outcompetition of wild type CySCs and of germ line stem cells, recapitulating the Socs36E mutant phenotype. Indeed, when MAPK activity is removed from Socs36E mutant clones, they lose their competitiveness but maintain self-renewal, presumably due to increased JAK/STAT signaling in these cells. Consistently, loss of JAK/STAT activity in Socs36E mutant clones severely impairs their self-renewal. Thus, these results enable the genetic separation of two essential processes that occur in stem cells. While some niche signals specify the intrinsic property of self-renewal, which is absolutely required in all stem cells for niche residence, additional signals control the ability of stem cells to compete with their neighbors. Socs36E is the node through which these processes are linked, demonstrating that negative feedback inhibition integrates multiple aspects of stem cell behavior (Amoyel, 2016).

Stem cell niches are complex environments that provide support for stem cells through molecular signals. Several well-characterized niches provide not just one but multiple signals which stem cells must integrate and interpret in order to remain at the niche and self-renew. How this integration is achieved is not well understood at present. Furthermore, in order to maintain the appropriate number of stem cells and the homeostatic balance between self-renewal and differentiation, it is necessary that self-renewal cues be present in limiting amounts or that their activity be dampened to prevent excessive accumulation of stem cells. One general feature of many signal transduction pathways is the presence of feedback inhibitors. These are dampeners of signaling, transcriptionally induced by the signaling itself, that prevent signal levels from being aberrantly high. One such family of feedback inhibitors is the Suppressor of Cytokine Signaling (SOCS) proteins, which were identified as inhibitors of JAK/STAT signal transduction, and are SH2- and E3-ligase domain-containing proteins. The SH2 domain binds phosphorylated (i.e., activated) signal transduction components and the E3-ligase targets them for degradation by Ubiquitin-dependent proteolysis. In mammals, SOCS proteins can thus inhibit several tyrosine kinase-dependent signaling pathways, including JAK/STAT and Mitogen-Activated Protein Kinase (MAPK) (Amoyel, 2016).

The Drosophila testis is an ideal model system to study questions of signal regulation and integration in stem cells. The testis niche, called the hub, supports two stem cell populations. The first, germ line stem cells (GSCs), gives rise to sperm after several transit-amplifying divisions leading up to meiosis. The second, somatic cyst stem cells (CySCs), gives rise to cyst cells, the essential support cells for germ line development. Many ligands for signaling pathways are produced by the hub, including the JAK/STAT pathway agonist, Unpaired (Upd), the Hedgehog (Hh) pathway ligand Hh and the Bone Morphogenetic Protein (BMP) homologs Decapentaplegic (Dpp) and Glass Bottom Boat (Gbb). The latter two signals are also produced by CySCs and are required in GSCs for self-renewal, indicating that CySCs constitute part of the niche for GSCs along with the hub. CySCs require JAK/STAT and Hh activity for self-renewal (Amoyel, 2016).

CySCs and GSCs compete for space at the niche, a phenomenon that was revealed by the analysis of testes lacking the JAK/STAT feedback inhibitor Socs36E. In these animals, excessive JAK/STAT activity was detected in CySCs, and Socs36E mutant CySCs displaced the resident wild type GSCs. Additionally, it has been shown that CySCs with sustained Hh signaling or sustained Yorkie (Yki) activity also outcompeted neighboring wild type GSCs, indicating that several signaling pathways can control niche competition. Moreover, prior to out-competing GSCs, mutant CySCs displaced neighboring wild type CySCs, indicating that both intra- (CySC-CySC) and inter-lineage (CySC-GSC) competition take place in the testis. While the two types of competition appear related, in that one precedes the other, there are instances in which only intra-lineage competition takes place. While the competitive phenotype of Socs36E mutant CySCs was ascribed to increased JAK/STAT signaling, it was surprising to find that clonal gain-of-function in JAK/STAT signaling in CySCs did not induce competitive behavior, and it was concluded that loss of Socs36E did not mimic increased JAK/STAT signaling in CySC (Amoyel, 2016).

This study addressed whether other mechanisms could account for the competitive behavior of Socs36E mutant CySCs. Because SOCS proteins can inhibit MAPK signaling in cultured cells and in Drosophila epithelial tissues, this study examined if Socs36E repression of MAPK signaling underlied the Socs36E competitive phenotype. Indeed, it was found that Socs36E inhibits MAPK signaling in CySCs during self-renewal, and that gain of MAPK activity induces CySCs to outcompete wild type CySCs and GSCs at the niche. This study dissected the genetic relationship between Socs36E and the MAPK and JAK/STAT pathways and shows that loss of Socs36E can compensate for decreased self-renewal signaling within CySCs. Thus, CySCs integrate multiple self-renewal signals through the use of a feedback inhibitor that controls at least two signaling pathways regulating stem cell maintenance at the niche (Amoyel, 2016).

The data presented in this study implicate MAPK signaling as a major regulator of CySC competition for niche access and establish that the competitiveness of CySCs lacking Socs36E is derived primarily from their increased MAPK activity. The ability of a stem cell to self-renew reflects not only intrinsic properties but also extrinsic relationships with its neighbors. For instance, if a cell is unable to compete for space at the niche then it will be no longer able to receive short-range niche signals and will be more likely to differentiate. Conversely, if a cell is more competitive for niche space, this cell and its offspring will replace wild type neighbors and colonize the entire niche. (Amoyel, 2016).

These data show that CySCs with increased MAPK signaling out-compete neighboring stem cells in CySC-CySC as well as CySC-GSC competition and that CySCs with reduced MAPK activity are themselves out-competed. The interpretation is favored that MAPK regulates primarily competitiveness rather than self-renewal because while MAPK mutant clones are lost from the niche, lineage-wide inhibition of the pathway does not result in a complete loss of stem cells. This contrasts with the role of JAK/STAT signaling in CySCs. Stat92E mutant CySCs are lost and lineage-wide pathway inhibition results in pronounced and rapid stem cell loss. Based on these results, it is argued that JAK/STAT signaling in CySCs primarily controls their intrinsic self-renewal capability while MAPK signaling regulates their competitiveness. Interestingly, there are important similarities between Hh and MAPK function in CySCs in that CySCs lacking Hh signal transduction are out-competed and those with sustained Hh activity out-compete wild type neighbors. Lastly, it is noted that CySCs mutant for the tumor suppressor Hippo (Hpo) (which leads to sustained Yki activation) or Abelson kinase (Abl) also have increased competitiveness, suggesting the existence of multiple inputs controlling the ability of stem cells to stay in the niche at the expense of their neighbors. In the future, it would be interesting to determine if genetic hierarchies exist between competitive pathways or if they independently converge on similar targets. One outstanding question is how altering the competitiveness of CySCs affects the maintenance of the germ line. In the case of Socs36E, MAPK, Hh and Hpo, the competitive CySC displaces not only wild type CySCs but also wild type GSCs. While these observations suggest that out-competition of CySCs and GSCs is linked, the result that Abl mutant CySCs only compete with CySCs and not with GSCs indicates that these two competitive processes are separable genetically (Amoyel, 2016).

It is well established that Egfr/MAPK signaling is required in somatic cells for their proper differentiation and for their encystment of the developing germ line. In this study, an additional function for Egfr/MAPK was identified in the somatic stem cells, specifically that this pathway regulates competitiveness of CySCs, with each other and with GSCs. Regarding the latter, it is possible that the loss of GSCs when somatic cells have high MAPK signaling is linked to their possibly increased encystment by these cells. Indeed, recent work has shown that Egfr activity in CySCs regulates cytokinesis and maintenance stem cell fate in GSCs. It is tempting to speculate that increased somatic Egfr activity leads to increased encystment of GSCs and loss of stem cell fate in GSCs (Amoyel, 2016).

MAPK may play a conserved role in niche competitiveness as mouse intestinal stem cells that acquire activating mutations in Ras bias normal stem cell replacement dynamics and colonize the niche. Interestingly, the activating ligand Spi is produced by germ cells, suggesting that the germ line coordinates multiple behaviors in the somatic cell lineage. In addition to transducing signals from the germ line, CySCs also receive ligands from hub cells (including Hh and the JAK/STAT ligand Upd) and they have to integrate these various stimuli. If unmitigated, the combined effect of all of these signals could produce highly competitive CySCs, with overall negative effects on niche homeostasis. The data are consistent with a model in which the induction of Socs36E by the primary self-renewal pathway (JAK/STAT) results in the restraint of a competitive trigger (MAPK) in CySCs. In this way, Socs36E acts to integrate signals from different sources and maintain homeostatic balance between resident cell populations that share a common niche (Amoyel, 2016).

Socs36E limits STAT signaling via Cullin2 and a SOCS-box independent mechanism in the Drosophila egg chamber

The Suppressor of Cytokine Signaling (SOCS) proteins are critical, highly conserved feedback inhibitors of signal transduction cascades. The family of SOCS proteins is divided into two groups: ancestral and vertebrate-specific SOCS proteins. Vertebrate-specific SOCS proteins have been heavily studied as a result of their strong mutant phenotypes. However, the ancestral clade remains less studied, a potential result of genetic redundancies in mammals. Use of the genetically tractable organism Drosophila melanogaster enables in vivo assessment of signaling components and mechanisms with less concern about the functional redundancy observed in mammals. This study investigated how the SOCS family member Suppressor of Cytokine Signaling at 36E (Socs36E) attenuates Jak/STAT activation during specification of motile border cells in Drosophila oogenesis. Socs36E genetically interacts with the Cullin2 (Cul2) scaffolding protein. Like Socs36E, Cul2 is required to limit the number of motile cells in egg chambers. Loss of Cul2 in the follicle cells significantly increased nuclear STAT protein levels, which resulted in additional cells acquiring invasive properties. Further, reduction of Cul2 suppressed border cell migration defects that occur in a Stat92E-sensitized genetic background. These data incorporated Cul2 into a previously described Jak/STAT-directed genetic regulatory network that is required to generate a discrete boundary between cell fates. It was also found that Socs36E is able to attenuate STAT activity in the egg chamber when it does not have a functional SOCS box. Collectively, this work contributes mechanistic insight to a Jak/STAT regulatory genetic circuit, and suggests that Socs36E regulates Jak/STAT signaling via a Cul2-dependent mechanism, as well as by a Cullin-independent manner, in vivo (Monahan, 2015).

The Jak/STAT pathway is a highly conserved cytokine signal transduction cascade, which transmits information from an extracellular cue to an intracellular response through transcriptional regulation. Briefly, ligand binding to a catalytically inert cytokine receptor (Domeless/Dome in Drosophila) induces a conformational change in the receptor and the cytoplasmically associated, non-receptor tyrosine kinase, Jak. This change stimulates phosphorylation of the cognate Jaks and the cytoplasmic domain of the receptor. Monomeric STAT proteins bind phosphotyrosines along the receptor, are phosphorylated by Jak, dissociate, homo-dimerize, and translocate into the nucleus as an active transcriptional regulator. The Jak/STAT pathway is essential for several developmental and cellular processes, including stem cell maintenance, immune response and regulation, cell proliferation, cell migration, and hematopoiesis. Drosophila utilizes a minimal, yet fully functional, Jak/ STAT signaling cascade that is required in many of the same cellular processes as in vertebrates, including the process of cell migration (Monahan, 2015).

During Drosophila oogenesis, a STAT-mediated collective cell migration occurs. The ovary is comprised of 16-18 ovariole chains, each with multiple developing eggs (called egg chambers). Each egg chamber consists of a monolayer of somatic epithelial cells (called follicle cells) that encase the germline (the oocyte and 15 nurse cells). At approximately mid-oogenesis, a subset of anterior follicle cells acquires invasive properties. These cells cluster around two immotile polar cells to form the border cell cluster. This cell collective later detaches from the anterior end of the egg chamber, invades the nurse cells, and migrates as a group to the oocyte. This process is essential for female fertility and proper patterning of the developing egg and future embryo (Monahan, 2015).

Border cell motility requires a precise level of STAT activity, which is tightly regulated by a genetic circuit that includes attenuation mediated by Suppressor of Cytokine Signaling at 36E (Socs36E). The polar cells are the egg chamber's sole source of the Unpaired (Upd) family, which activates the Jak/STAT pathway in Drosophila. During stage 8, as Upd is released, surrounding follicle cells receive it and activate STAT in a spatial gradient. STAT promotes expression of the pro-migratory cue slow border cells (slbo) and the migratory inhibitor apontic (apt) in the anterior follicle cells. STAT activation is initially widespread, but must be dampened to produce an optimal number of invasive cells by stage 9. Apt feeds back to inhibit STAT activity, in part by regulating the expression of Socs36E and a Stat92E targeting microRNA (miR-279) to limit motility. The inability to shut off STAT signaling properly in anterior follicle cells enables an excessive number of cells to acquire invasive properties, which can impede border cell migration. However, while loss of Stat92E or slbo prevents border cell specification and migration, and significantly reduces female fertility, loss of apt, Socs36E, or miR-279 does not result in sterility, as not all egg chambers are equally affected. This reflects robust control of oogenesis, and underscores the complexity in the regulation of reproduction. While much is known about the genetic control of border cell migration, the molecular mechanisms that regulate signaling during border cell specification are less well understood (Monahan, 2015).

The SOCS family of proteins is a set of essential regulators of cytokine signaling that are conserved from humans to Drosophila. SOCS proteins possess two conserved domains: a Src Homology 2 (SH2) domain and a C-terminal SOCS box. However, the N-termini show no evidence of conserved domains, high sequence homology, or consistency in length between family members. Vertebrates contain eight SOCS proteins that are sub-divided into two groups: the vertebrate specific SOCS proteins (CIS and SOCS1-3) and the ancestral SOCS proteins (SOCS4-7). The vertebrate-specific SOCS proteins have strong phenotypes associated with their loss in vivo, which have led to extensive study both in vitro and in vivo. In contrast, ancestral SOCS members cause less severe loss of function phenotypes, likely due to genetic redundancies in mammals, and thereby there is a less clear understanding of how these proteins function. Since structural differences between the vertebrate-specific and ancestral SOCS proteins may mediate distinct mechanisms of action between the two groups, further characterization of the ancestral SOCS members is essential to elucidate their effects on signal transduction pathways. In contrast to mammals, Drosophila have only three SOCS proteins (Socs16D, Socs44A, and Socs36E). Socs36E, which is most similar to mammalian SOCS5, is the only one that appears to act in ovarian follicle cells (Monahan, 2015 and references therein).

The SOCS box interacts with Elongin B and C adaptor proteins and a Cullin scaffolding protein,which incorporates SOCS members into an E3 ubiquitin ligase complex to promote protein turnover. Specifically, the SOCS protein acts as the substrate recognition component of some RING finger E3 ligase complexes, as the SH2 domain may target the complex to specific substrate(s) for ubiquitination. In vitro studies have shown that SOCS proteins bind members of the Cullin family of scaffolding proteins, particularly Cullin2 (Cul2) and Cullin5 (Cul5). Most studies investigating SOCS proteins support a SOCS-Cul5 interaction, although some have shown an interaction with Cul2 (Monahan, 2015).

This study utilized border cell motility as an in vivo system to study the mechanism b ywhich Socs36E attenuates STAT signaling in the Drosophila egg chamber. Socs36E was determined to genetically interact with Cul2, and that loss of Cul2 was shown to result in mis-specification of additional invasive cells. This study found a significant increase in activated, nuclear STAT protein levels during border cell specification, an expanded border cell precursor population, and an excessive number of invasive cells at stage 10 when Cul2 was reduced in the anterior follicle cells. These phenotypes are similar to those of Socs36E deficient egg chambers. Importantly, it was determined that a reduction of Cul2 restored proper border cell migration when Stat92E was below endogenous levels, and that Cul2 genetically interacts with apt, another known STAT-regulator in the egg chamber. This study also discovered the SOCS box is not required for all functions of Socs36E in vivo. From this work, the STAT-regulatory genetic circuit in Drosophila egg chambers was refined by determining some modes of inhibition. It is proposed that Socs36E functions with Cul2 to restrict migratory fate to the border cell cluster through a ubiquitin-dependent mechanism, but that it can also regulate the Jak/STAT pathway in a SOCS-box independent manner (Monahan, 2015).

The involvement of SOCS proteins in RING Finger E3 ubiquitin ligases has been well-established, and is thought to be mediated by Cullin interaction. A previous report proposed that SOCS box proteins contain a Cullin5-box, while the highly related Von Hippel-Lindau (VHL) proteins have a Cullin2-box. However, the only SOCS family members assayed were SOCS1 and SOCS3, both of which have been shown to bind Cul5. Several reports have shown that proteins with a SOCS box (including SOCS1) are able to interact with and bind Cul2. Analysis of the predicted Cul5-box of SOCS1, SOCS3, SOCS5, and Socs36E revealed low sequence similarity across the four proteins. Furthermore, the proposed key sequence of the Cul5-box (LPLP) is only found in SOCS5 (Monahan, 2015).

This study found that Socs36E attenuates the Jak/STAT pathway in a Cul2- dependent manner. Reducing Cul2 function in the anterior follicle cells of stage 8 egg chambers significantly expanded the border cell precursor population (Slbo+ anterior follicle cells) and heightened nuclear STAT (nSTAT) protein levels. These data show Cul2 acts to attenuate STAT in the egg chamber. There did not appear to be a similar requirement for other Cullin family members that were tested, although in the absence of amorphic alleles, it remains a possibility. The expanded Slbo-positive population observed in egg chambers deficient for Cul2 is likely due to higher than normal STAT activity in follicle cells far from the polar cells, which can explain the additional invasive cell phenotype (similar to when STAT signaling is increased in apt or Socs36E mutants. In support of this, it was determined that lowering Cul2 rescues the delay in border cell migration that occurs when Stat92E is reduced. Collectively, these data strongly suggest that Cul2 limits STAT activity in the anterior follicle cells of the egg chamber (Monahan, 2015).

By finding that Cul2 genetically interacts with apt (a central component of STAT regulation in egg chambers), Cul2 can be incorporated into the previously described Jak/STAT regulatory circuit. It is postulated that Socs36E aids in the generation of a discrete boundary between migratory and non-migratory fates in the anterior follicle cells, by functioning as the substrate recognition component of a Cul2-E3 ubiquitin ligase complex. Given published biochemical data on SOCS family members and the current genetic and alignment results, it is suggested that SOCS proteins may have the potential to bind both Cul2 and Cul5; however the binding preference may be tissue- or context-dependent. Analyses of these interactions are well-suited for future in vivo study (Monahan, 2015).

Previous work established Socs36E as a regulator of the Epidermal Growth Factor Receptor (EGFR) pathway in some Drosophila tissues. After border cell specification, EGFR works redundantly with the PDGF- and VEGF receptor related (PVR) pathway to promote protrusive activity and to guide the migration of the cluster to the oocyte. EGFR is also required to direct the border cell cluster dorsally to the oocyte nuclei at stage 10B. No defects were observed in the directed migration of the border cell cluster when Cul2 was reduced, similar to previous results with Socs36E; thus it is not suspected that these genes are necessary for EGFR regulation in follicle cells. It is possible that the redundancy between EGFR and PVR in border cell chemotaxis masks Socs36E and Cul2 regulation of EGFR, therefore cannot be completely ruled out. However, the phenotypic results strongly suggest that the Jak/STAT pathway is the primary target of both Socs36E and Cul2 in the anterior follicle cells of the egg chamber (Monahan, 2015).

Previous work suggests that the Dome receptor is targeted for endocytosis after ligand binding, and that this event is required for proper migration of the border cells. While the mechanism has not been resolved in the egg chamber, a recent study using Drosophila Kc167 cells showed that Dome is degraded in the lysosome, in a ligand-dependent manner, and that loss of Socs36E delays receptor clearance. Knockout of Cul5 and Socs36E resulted in higher activation of Jak/STAT, relative to loss of Cul5 alone, although Cul2 was not assayed. These data may suggest a SOCS box-independent mechanism, but an additional interpretation could be that Socs36E also mediates receptor clearance in a Cul2-dependent manner. Stec also found that Socs36E can bind Dome, but only weakly interacts with Jak in a Dome-dependent manner. These data combined with the current work support a model in which Socs36E facilitates the degradation of an activated Dome-Jak complex in the anterior follicle cells of the egg chamber. This attenuates STAT signaling, which is essential to limit the acquisition of migratory fates (Monahan, 2015).

Other possible mechanisms of action were considered for Socs36E in the egg chamber. Several in vitro assays, including binding assays and crystallography, found the SOCS box directly interacts with Elongins B/C and Cullin. Many of these reports suggest that the SOCS box is essential for attenuation of cytokine signaling and SOCS protein stability. In contrast, several studies have found loss of the SOCS box impedes SOCS protein function, but does not eliminate it. These studies suggest that the SOCS box is necessary for complete SOCS-driven attenuation of cytokine signaling, but to a varying degree and possibly in a tissue specific manner. Because expression of UAS-Socs36EΔSB in a Socs36E deficient egg chamber restored approximately wild-type migration to Socs36E mutants, it is concluded that the SOCS box domain is largely dispensable for motile cell specification. However, it was not dispensable for normal cluster cohesion, as some invasive cells trailed behind the main cluster (Monahan, 2015).

Since SOCS proteins require the SOCS box to facilitate their incorporation into an E3 ligase complex, it is proposed that Socs36E can partially attenuate STAT activity independently of Cullin-E3 ligase activity in vivo. Consistent with vertebrate ancestral SOCS proteins, the current sequence analysis revealed the Socs36E N-terminus is intrinsically disordered. Intrinsically disordered proteins (IDPs) have a capacity to generate protein-protein interactions. Upon binding another protein, IDPs undergo an energetically favorable disordered to ordered transition. The intrinsic flexibility and ability to adopt several conformations and binding partners enables a single IDP to function in several signaling pathways and cellular processes (Monahan, 2015).

The long N-termini of SOCS5 and Socs36E have been proposed to play an essential role in the SOCS-receptor interaction and, in some cases, are critical for SOCS function. For example, the N-terminus of SOCS5 regulates T-cell differentiation by disrupting Jak1 association with IL4Rα, and a recent study proposed this region directly prevents Jak1/2 activity. Other studies have suggested that the N-termini of ancestral SOCS proteins play a critical role in SOCS-substrate interaction, including cell culture analysis of Socs36E. While this study did not locate a JIR consensus sequence in Socs36E, in vitro studies together with the current data suggest that the N-terminal region of Socs36E may be important for substrate binding. It is hypothesized that the N-terminus of Socs36E is intrinsically disordered and may play a role in limiting cytokine-activated signaling. It will be interesting to determine if and how the N-terminus functionally inhibits Jak/STAT signaling independently from a Cullin-E3-ligase complex. Many questions remain about IDPs, their interactions, structure, and other biochemical characteristics. New approaches will be required to fully study IDPs in a cellular context. It is suggested that the minimized pathway components, potent genetic tools, and high levels of genetic conservation make Drosophila an ideal system to study IDPs in an in vivo context. Thus, future work on Socs36E function in the ovary could provide further insight, not only into SOCS protein biology but also IDPs, in general (Monahan, 2015).

The SH2 domain of SOCS proteins is required for substrate binding, including the binding and turnover of Dome in Kc167 cells. The SH2 domain could also play an active role in cytokine attenuation. For instance, Socs36E may compete with STAT for the same or a proximal phosphotyrosine on the receptor, thereby preventing STAT phosphorylation, as has been proposed for CIS and SOCS2. These are not mutually exclusive ideas. Therefore, a model is favored in which Socs36E interacts with Cullin2 in an E3 ubiquitin ligase complex, but that can prevent Jak activity and/or block Dome/Jak access to STAT via its SH2 domain and/or N-terminus (Monahan, 2015).

The closest mammalian homolog of Socs36E, SOCS5, is a proposed tumor suppressor. Reduction of SOCS5 can result in loss of epithelial organization, increased tumor metastasis, and aggressive carcinomas. Thus, understanding its mechanism of action will enhance understanding of cancer progression, but analysis in mammals has proven challenging. This study shows that use of a genetic model organism enables in vivo assessment of SOCS proteins; this sheds light on how these proteins function (Monahan, 2015).

Developmental regulation of epithelial cell cuboidal-to-squamous transition in Drosophila follicle cells

Epithelial cells form continuous membranous structures for organ formation, and these cells are classified into three major morphological categories: cuboidal, columnar, and squamous. It is crucial that cells transition between these shapes during the morphogenetic events of organogenesis, yet this process remains poorly understood. All three epithelial cell shapes can be found in the follicular epithelium of Drosophila egg chamber during oogenesis. Squamous cells (SCs), are initially restricted to the anterior terminus in cuboidal shape. They then rapidly become flattened to assume squamous shape by stretching and expansion in twelve hours during midoogenesis. Previously, it was reported that Notch signaling activated a zinc-finger transcription factor Broad (Br) at the end of early oogenesis. This study reports that ecdysone and JAK/STAT pathways subsequently converge on Br to serve as an important spatiotemporal regulator of this dramatic morphological change of SCs. The early uniform pattern of Br in the follicular epithelium is directly established by Notch signaling at stage 5 of oogenesis. Later, ecdysone and JAK/STAT signaling activities synergize to suppress Br in SCs from stage 8 to 10a, contributing to proper SC squamous shape. During this process, ecdysone signaling is essential for the SC stretching, while JAK/STAT regulates SC clustering and cell fate determination. This study reveals an inhibitory role of ecdysone signaling in suppressing Br in epithelial cell remodeling. In this study single-cell RNA sequencing data was used to highlight the shift in gene expression which occurs as Br is suppressed and cells become flattened (Jia, 2022).

Germline protein, Cup, non-cell autonomously limits migratory cell fate in Drosophila oogenesis

Specification of migratory cell fate from a stationary population is complex and indispensable both for metazoan development as well for the progression of the pathological condition like tumor metastasis. Though this cell fate transformation is widely prevalent, the molecular understanding of this phenomenon remains largely elusive. This study employed the model of border cells (BC) in Drosophila oogenesis and identified germline activity of an RNA binding protein, Cup that limits acquisition of migratory cell fate from the neighbouring follicle epithelial cells. As activation of JAK-STAT in the follicle cells is critical for BC specification, these data suggest that Cup, non-cell autonomously restricts the domain of JAK-STAT by activating Notch in the follicle cells. Employing genetics and Delta endocytosis assay, Cup was demonstrated to regulate Delta recycling in the nurse cells through Rab11GTPase thus facilitating Notch activation in the adjacent follicle cells. Since Notch and JAK-STAT are antagonistic, it is proposed that germline Cup functions through Notch and JAK-STAT to modulate BC fate specification from their static epithelial progenitors (Saha, 2023).

JAK/STAT signalling mediates cell survival in response to tissue stress

Tissue homeostasis relies on the ability of tissues to respond to stress. Tissue regeneration and tumour models in Drosophila have shown that JNK is a prominent stress-response pathway promoting injury-induced apoptosis and compensatory proliferation. A central question remaining unanswered is how both responses are balanced by activation of a single pathway. JAK/STAT signalling, a potential JNK target, is implicated in promoting compensatory proliferation. While JAK/STAT activation in imaginal discs was observed upon damage, it was also found that JAK/STAT and its downstream effector Zfh2 promote survival of JNK-signalling cells instead. The JNK component fos and the pro-apoptotic gene hid are regulated in a JAK/STAT-dependent manner. This molecular pathway restrains JNK-induced apoptosis and spatial propagation of JNK-signalling, thereby limiting the extent of tissue damage, as well as facilitating systemic and proliferative responses to injury. It was found that the pro-survival function of JAK/STAT also drives tumour growth under conditions of chronic stress. Altogether, these results define JAK/STAT function in tissue stress and illustrate how crosstalk between conserved signalling pathways establish an intricate equilibrium between proliferation, apoptosis and survival to restore tissue homeostasis (La Fortezza, 2016).

JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling

A stable pool of morphogen-producing cells is critical for the development of any organ or tissue. This study presents evidence that JAK/STAT signalling in the Drosophila wing promotes the cycling and survival of Hedgehog-producing cells, thereby allowing the stable localization of the nearby BMP/Dpp-organizing centre in the developing wing appendage. The inhibitor of apoptosis dIAP1 and Cyclin A were identified as two critical genes regulated by JAK/STAT and contributing to the growth of the Hedgehog-expressing cell population. JAK/STAT was found to have an early role in guaranteeing Wingless-mediated appendage specification, and a later one in restricting the Dpp-organizing activity to the appendage itself. These results unveil a fundamental role of the conserved JAK/STAT pathway in limb specification and growth by regulating morphogen production and signalling, and a function of pro-survival cues and mitogenic signals in the regulation of the pool of morphogen-producing cells in a developing organ (Recasens-Alvarez, 2017).

Morphogens of the Wnt/Wg, Shh/Hh and BMP/Dpp families regulate tissue growth and pattern formation in vertebrate and invertebrate limbs. This study has unraveled a fundamental role of the secreted Upd ligand and the JAK/STAT pathway in facilitating the activities of these three morphogens in exerting their fate- and growth-promoting activities in the Drosophila wing primordium. Early in wing development, two distinct mechanisms ensure the spatial segregation of two alternative cell fates. First, the proximal-distal subdivision of the wing primordium into the wing and the body wall relies on the antagonistic activities of the Wg and Vn signalling molecules. While Wg inhibits the expression of Vn and induces the expression of the wing-determining genes, Vn, through the EGFR pathway, inhibits the cellular response to Wg and instructs cells to acquire body wall fate. Second, growth promoted by Notch pulls the sources of expression of these two morphogens apart, alleviates the repression of wing fate by Vn/EGFR, and contributes to Wg-mediated appendage specification. Expression of Vn is reinforced by a positive amplification feedback loop through the activation of the EGFR pathway. This existing loop predicts that, in the absence of additional repressors, the distal expansion of Vn/EGFR and its targets would potentially impair wing development. The current results indicate that Upd and JAK/STAT restrict the expression of EGFR target genes and Vn to the most proximal part of the wing primordium, thereby interfering with the loop and allowing Wg to correctly trigger wing development. Evidence is presented that JAK/STAT restricts the expression pattern and levels of its own ligand Upd and that ectopic expression of Upd is able to bypass EGFR-mediated repression and trigger wing development de novo. This negative feedback loop between JAK/STAT and its ligand is of biological relevance, since it prevents high levels of JAK/STAT signalling in proximal territories that would otherwise impair the development of the notum or cause the induction of supernumerary wings, as shown by the effects of ectopic activation of the JAK/STAT pathway in the proximal territories. Thus, while Wg plays an instructive role in wing fate specification, the Notch and JAK/STAT pathways play a permissive role in this process by restricting the activity range of the antagonizing signalling molecule Vn to the body wall region (Recasens-Alvarez, 2017).

Later in development, once the wing field is specified, restricted expression of Dpp at the AP compartment boundary organizes the growth and patterning of the whole developing appendage. Dpp expression is induced in A cells by the activity of Hh coming from P cells, which express the En transcriptional repressor. This study shows that JAK/STAT controls overall organ size by maintaining the pool of Hh-producing cells to ensure the stable and localized expression of the Dpp organizer. JAK/STAT does so by promoting the cycling and survival of P cells through the regulation of dIAP1 and CycA, counteracting the negative effects of En on these two genes. Since the initial demonstration of the role of the AP compartment boundary in organizing, through Hh and Dpp, tissue growth and patterning, it was noted that high levels of En interfered with wing development by inducing the loss of the P compartment. The capacity of En to negatively regulate its own expression was subsequently shown to be mediated by the Polycomb-group genes and proposed to be used to finely modulate physiological En expression levels. Consistent with this proposal, an increase was observed in the expression levels of the en-gal4 driver, which is inserted in the en locus and behaves as a transcriptional reporter, in enRNAi-expressing wing discs. The negative effects of En on cell cycling and survival reported in this work might also contribute to the observed loss of the P compartment caused by high levels of En. As is it often the case in development, a discrete number of genes is recurrently used to specify cell fate and regulate gene expression in a context-dependent manner. It is proposed that the capacity of En to block cell cycle and promote cell death might be required in another developmental context and that this capacity is specifically suppressed in the developing Drosophila limbs by JAK/STAT, and is modulated by the negative autoregulation of En, thus allowing En-dependent induction of Hh expression and promoting Dpp-mediated appendage growth. It is interesting to note in this context that En-expressing territories in the embryonic ectoderm are highly enriched in apoptotic cells. Whether this apoptosis plays a biological role and relies on En activity requires further study (Recasens-Alvarez, 2017).

Specific cell cycle checkpoints appear to be recurrently regulated by morphogens and signalling pathways, and this regulation has been unveiled to play a major role in development. Whereas Notch-mediated regulation of CycE in the Drosophila eye and wing primordia is critical to coordinate tissue growth and fate specification by pulling the sources of two antagonistic morphogens apart, the current results indicate that JAK/STAT-mediated regulation of CycA is critical to maintain the pool of Hh-producing cells in the developing wing and to induce stable Dpp expression. The development of the wing hinge region, which connects the developing appendage to the surrounding body wall and depends on JAK/STAT activity, has been previously shown to restrict the Wg organizer and thus delimit the size and position of the developing appendage. The current results support the notion that JAK/STAT and the hinge region are also essential to restrict the organizing activity of the Dpp morphogen to the developing appendage. Taken together, these results reveal a fundamental role of JAK/STAT in promoting appendage specification and growth through the regulation of morphogen production and activity, and a role of pro-survival cues and mitotic cyclins in regulating the pool of morphogen-producing cells in a developing organ. The striking parallelisms in the molecules and mechanisms underlying limb development in vertebrates and invertebrates have contributed to the proposal that an ancient patterning system is being recurrently used to generate body wall outgrowths. Whether the conserved JAK/STAT pathway plays a developmental role also in the specification or growth of vertebrate limbs by regulating morphogen production or activity is a tempting question that remains to be elucidated (Recasens-Alvarez, 2017).

JAK/STAT signaling is necessary for cell monosis prior to epithelial cell apoptotic extrusion

Epithelial cell extrusion is crucial for proper development and tissue homeostasis. Highly-sterotyped morphogenetic events are controlled by JAK/STAT signaling in a developmentally-programmed case of epithelial cell extrusion. Specialized somatic cells, Polar Cells (PCs), are produced in excess and then undergo apoptotic elimination from the follicular epithelium in the Drosophila ovary. This study shows that supernumerary PCs are systematically enveloped by PC neighbors on all sides in conjunction with highly-reinforced adherens junctions. The PC to be removed thus loses all contact with follicle cells, germline cells and the basement membrane in a process called cell 'monosis', for 'isolation' in Greek. PC monosis takes several hours, and always precedes, and is independent of, activation of apoptosis. JAK/STAT signaling is necessary within the surrounding follicular epithelium for PC monosis. Minutes after monosis is complete, PC apoptotic corpses are formed and extruded laterally within the epithelium. These apoptotic corpses are engulfed and eliminated by surrounding follicle cells, which are thus acting as non-professional phagocytes. This study therefore shows the non cell-autonomous impact of an epithelium, via JAK/STAT signaling activation, on cell morphogenesis events leading to apoptotic extrusion. It is likely that cell monosis and lateral extrusion within an epithelium are pertinent for other cases of epithelial cell extrusion as well (Torres, 2017).

Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment

Malignant tumors are caused by uncontrolled proliferation of transformed mutant cells that have lost the ability to maintain tissue integrity. Although a number of causative genetic backgrounds for tumor development have been discovered, the initial steps mutant cells take to escape tissue integrity and trigger tumorigenesis remain elusive. This study shows through analysis of conserved neoplastic tumor-suppressor genes (nTSGs) in Drosophila wing imaginal disc epithelia that tumor initiation depends on tissue-intrinsic local cytoarchitectures, causing tumors to consistently originate in a specific region of the tissue. In this "tumor hotspot" where cells constitute a network of robust structures on their basal side, nTSG-deficient cells delaminate from the apical side of the epithelium and begin tumorigenic overgrowth by exploiting endogenous JAK/STAT signaling activity. Conversely, in other regions, the "tumor coldspot" nTSG-deficient cells are extruded toward the basal side and undergo apoptosis. When the direction of delamination is reversed through suppression of RhoGEF2, an activator of the Rho family small GTPases, and JAK/STAT is activated ectopically in these coldspot nTSG-deficient cells, tumorigenesis is induced. These data indicate that two independent processes, apical delamination and JAK/STAT activation, are concurrently required for the initiation of nTSG-deficient-induced tumorigenesis. Given the conservation of the epithelial cytoarchitecture, tumorigenesis may be generally initiated from tumor hotspots by a similar mechanism (Tamori, 2016).

Tumor-induced disruption of the blood-brain barrier promotes host death

Cancer patients often die from symptoms that manifest at a distance from any tumor. Mechanisms underlying these systemic physiological perturbations, called paraneoplastic syndromes, may benefit from investigation in non-mammalian systems. Using a non-metastatic Drosophila adult model, this study found that malignant-tumor-produced cytokines drive widespread host activation of JAK-STAT signaling and cause premature lethality. STAT activity is particularly high in cells of the blood-brain barrier (BBB), where it induces aberrant BBB permeability. Remarkably, inhibiting STAT in the BBB not only rescues barrier function but also extends the lifespan of tumor-bearing hosts. This study identified BBB damage in other pathological conditions that cause elevated inflammatory signaling, including obesity and infection, where BBB permeability also regulates host survival. IL-6-dependent BBB dysfunction is further seen in a mouse tumor model, and it again promotes host morbidity. Therefore, BBB alterations constitute a conserved lethal tumor-host interaction that also underlies other physiological morbidities (Kim, 2021).

Insulin potentiates JAK/STAT signaling to broadly inhibit flavivirus replication in insect vectors

The World Health Organization estimates that more than half of the world's population is at risk for vector-borne diseases, including arboviruses. Because many arboviruses are mosquito borne, investigation of the insect immune response will help identify targets to reduce the spread of arboviruses. This study used a genetic screening approach to identify an insulin-like receptor as a component of the immune response to arboviral infection. Vertebrate insulin reduces West Nile virus (WNV) replication in Drosophila melanogaster as well as WNV, Zika, and dengue virus titers in mosquito cells. Mechanistically, insulin signaling was shown to activate the JAK/STAT, but not RNAi, pathway via ERK to control infection in Drosophila cells and Culex mosquitoes through an integrated immune response. Finally, insulin priming of adult female Culex mosquitoes through a blood meal reduces WNV infection, demonstrating an essential role for insulin signaling in insect antiviral responses to human pathogens (Ahlers, 2019).

Interferon functional analog activates antiviral Jak/Stat signaling through integrin in an arthropod

Drosophila Vago is a small antiviral peptide. Its ortholog in Culex mosquito was found to be an interferon-like cytokine that limits virus replication through activating Jak/Stat signaling. However, this activation is independent of Domeless, the sole homolog of vertebrate type I cytokine receptor. How Vago activates the Jak/Stat pathway remains unknown. Herein, this process is dependent on integrin in kuruma shrimp (Marsupenaeus japonicus). Shrimp Vago-like (MjVago-L) plays an antiviral role by activating the Jak/Stat pathway and inducing Stat-regulated Ficolin. Blocking integrin abrogates the role of MjVago-L. The interaction between MjVago-L and integrin β3 is confirmed. An Asp residue in MjVago-L is found critical for the interaction and MjVago-L's antiviral role. Moreover, Fak, a key adaptor of integrin signaling, mediates MjVago-L-induced Jak/Stat activation. Therefore, this study reveals that integrin, as the receptor of MjVago-L, mediates Jak/Stat activation. The establishment of the MjVago-L/integrin/Fak/Jak/Stat/Ficolin axis provides insights into antiviral cytokine signaling in invertebrates (Gao, 2021)

Maheshvara regulates JAK/STAT signaling by interacting and stabilizing hopscotch transcripts which leads to apoptosis in Drosophila melanogaster

Maheshvara (mahe), an RNA helicase that is widely conserved across taxa, regulates Notch signaling and neuronal development in Drosophila. In order to identify novel components regulated by mahe, transcriptome profiling of ectopic mahe was carried out and this revealed striking upregulation of JAK/STAT pathway components like upd1, upd2, upd3, and socs36E. Further, significant downregulation of the pathway components in mahe loss-of-function mutant as well as upon lowering the level of mahe by RNAi, supported and strengthened the transcriptome data. Parallelly, it was observed that mahe, induced caspase-dependent apoptosis in photoreceptor neurons, and this phenotype was significantly modulated by JAK/STAT pathway components. RNA immunoprecipitation unveiled the presence of JAK/STAT tyrosine kinase hopscotch (hop) transcripts in the complex immunoprecipitated with Mahe, which ultimately resulted in stabilization and elevation of hop transcripts. Additionally, the surge in activity of downstream transcription factor Stat92E, which is indicative of activation of the JAK/STAT signaling, was also observed, and this in turn led to apoptosis via upregulation of hid. Taken together, these data provide a novel regulation of JAK/STAT pathway by RNA helicase Maheshvara, which ultimately promotes apoptosis (Maurya, 2021).

Chromosomal instability-induced cell invasion through caspase-driven DNA damage

Chromosomal instability (CIN), an increased rate of changes in chromosome structure and number, is observed in most sporadic human carcinomas with high metastatic activity. This study used a Drosophila epithelial model to show that DNA damage, as a result of the production of lagging chromosomes during mitosis and aneuploidy-induced replicative stress, contributes to CIN-induced invasiveness. A sub-lethal role of effector caspases in invasiveness was unraveled by enhancing CIN-induced DNA damage and identify the JAK/STAT signaling pathway as an activator of apoptotic caspases through transcriptional induction of pro-apoptotic genes. Evidence is provided that an autocrine feedforward amplification loop mediated by Upd3-a cytokine with homology to interleukin-6 and a ligand of the JAK/STAT signaling pathway-contributes to amplifying the activation levels of the apoptotic pathway in migrating cells, thus promoting CIN-induced invasiveness. This work sheds new light on the chromosome-signature-independent effects of CIN in metastasis (Barrio, 2023).

Drosophila Larval Models of Invasive Tumorigenesis for In Vivo Studies on Tumour/Peripheral Host Tissue Interactions during Cancer Cachexia

Cancer cachexia is a common deleterious paraneoplastic syndrome that represents an area of unmet clinical need, partly due to its poorly understood aetiology and complex multifactorial nature. This study interrogated multiple genetically defined larval Drosophila models of tumourigenesis against key features of human cancer cachexia. The results indicate that cachectic tissue wasting is dependent on the genetic characteristics of the tumour and demonstrate that host malnutrition or tumour burden are not sufficient to drive wasting. This study shows that JAK/STAT and TNF-α/Egr signalling are elevated in cachectic muscle and promote tissue wasting. Furthermore, a dual driver system is introduced that allows independent genetic manipulation of tumour and host skeletal muscle. Overall, this study presents a novel Drosophila larval paradigm to study tumour/host tissue crosstalk in vivo, which may contribute to future research in cancer cachexia and impact the design of therapeutic approaches for this pathology (Hodgson, 2021).

A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells

The cellular mechanisms enabling temperature acclimation in ectotherms are still poorly understood. Cis-regulatory elements (CREs), which mediate increased transcription at cool temperature, and responsible transcription factors are largely unknown. The ectotherm Drosophila melanogaster with a presumed temperature optimum around 25°C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14-29°C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. This study analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D. Beyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. The identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range (Bai, 2021).

Proteasome α6 Subunit Negatively Regulates the JAK/STAT Pathway and Blood Cell Activation in Drosophila melanogaster

JAK/STAT signaling regulates central biological functions such as development, cell differentiation and immune responses. In Drosophila, misregulated JAK/STAT signaling in blood cells (hemocytes) induces their aberrant activation. This study identified several components of the proteasome complex as negative regulators of JAK/STAT signaling in Drosophila. A selected proteasome component, Prosα6, was studied further. In S2 cells, Prosα6 silencing decreased the amount of the known negative regulator of the pathway, ET, leading to enhanced expression of a JAK/STAT pathway reporter gene. Silencing of Prosα6 in vivo resulted in activation of the JAK/STAT pathway, leading to the formation of lamellocytes, a specific hemocyte type indicative of hemocyte activation. This hemocyte phenotype could be partially rescued by simultaneous knockdown of either the Drosophila STAT transcription factor, or MAPKK in the JNK-pathway. These results suggest a role for the proteasome complex components in the JAK/STAT pathway in Drosophila blood cells both in vitro and in vivo (Jarvela-Stolting, 2021).

A PtdIns(3,4,5)P(3) dispersal switch engages cell ratcheting at specific cell surfaces

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating contractile forces before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be ratcheting mechanisms that drive processive transformations in cell shape. Previous work has shown that force generation is coordinated with endocytic remodeling; however, how ratcheting becomes engaged at specific cell surfaces remains unclear. This study reports that PtdIns(3,4,5)P(3) is a critical lipid-based cue for ratcheting engagement. The Sbf RabGEF binds to PIP(3), and disruption of PIP(3) reveals a dramatic switching behavior in which medial ratcheting is activated and epithelial cells begin globally constricting apical surfaces. PIP(3) enrichments are developmentally regulated, with mesodermal cells having high apical PIP(3) while germband cells have higher interfacial PIP(3). Finally, this study shows that JAK/STAT signaling constitutes a second pathway that combinatorially regulates Sbf/Rab35 recruitment. Results elucidate a complex lipid-dependent regulatory machinery that directs ratcheting engagement in epithelial tissues (Miao, 2021).

Cell shaping processes use contractile force generation to drive the active contraction of specific cell surfaces that causes tissues to adopt new morphogenetic forms. This selective contraction of cell surfaces drives a diverse range of processes from tissue invagination to cell intercalation to epithelial cell extrusion and wound healing. A key discovery in the last decade of work on these processes is that they are often pulsatile in nature, with highly transient actomyosin populations that briefly apply a tensioning force to an area of the cell cortex. This has raised a central outstanding question-if actomyosin networks display such pulsed behaviors, how does processivity emerge from cyclic systems? To obtain lasting changes from such systems, there is a requirement for ratcheting mechanisms that gain irreversible changes out of periodic, contractile cycles (Miao, 2021).

Several potential molecular mechanisms of cell ratcheting have been discovered, ranging from processes that direct the turnover and reformation of elastic cortical networks to pathways that rely on direct remodeling of the plasma membrane. Previous work has shown that a membranous ratchet centered on SET-domain-binding factor (Sbf) RabGEF and Rab35 function is a critical part of the pathway used to remodel the cell surfaces during early morphogenesis in Drosophila. This membrane ratcheting module is deployable to specific cell surfaces-during cell intercalation, Sbf and Rab35 tubular invaginations of the plasma membrane are primarily found at planar polarized contracting interfaces, where they mediate the processive loss of AP (anterior-posterior) surfaces required for neighbor exchange through endocytic mechanisms. Alternatively, during the apical constrictions that drive invagination of the ventrally located mesoderm, Sbf and Rab35 are enriched at the apical surface, where they direct the ratcheted loss of apical surface areas. However, how this ratcheting activity is selectively recruited to specific cell surfaces is unknown. Although actomyosin function is required to terminate Sbf/Rab35 compartments, it is not required for Sbf and Rab35 recruitment. Thus, Sbf and Rab35 recruitment is not downstream of actomyosin function, suggesting that yet-to-be-identified mechanisms are in place to control Sbf/Rab35 compartmental formation (Miao, 2021).

One potential important cue for directing the localized activities of plasma-membrane-associated proteins are the lipid-signaling phosphoinositides (PIPs), which are often found in microdomains in the plasma membrane. PIPs have been shown to be potent regulators of both membrane trafficking and cytoskeletal networks. The various PIP phospho-species, especially PI(4,5)P2 and PI(3,4,5)P3, can regulate the activity of a number of endocytic-regulatory proteins, such as AP-2 and Dynamin. PIPs are also implicated in controlling actin assembly and plasma membrane-cytoskeletal linkage by binding directly and tightly to at least 30 regulatory proteins. PI(3,4,5)P3 in particular has been deeply implicated in the regulation of membrane trafficking processes such as regulated endocytosis and exocytosis. Upregulation of PIP3 levels induces recycling of the epidermal growth factor receptor to the cell surface, and PIP phospho-balance can regulate syncytial cleavage furrow lengths in the fly embryo. Further, recently published work suggests that PIPs may lay downstream of Toll receptor activity and Src activation in the germband epithelium. However, how PIPs regulate the dynamic cell shape changes that occur as epithelial sheets change dimensions is unclear, and the function of PIP phospho-species during Drosophila gastrulation remains to be closely examined. This study identified phosphatidylinositol phosphates as providing lipid-based membrane cues for morphogenesis and cellular gastrulation dynamics in the early Drosophila embryo. PI(3,4,5)P3 inhibition leads to a potent disruption of contractile cellular behaviors, and PIP3 function controls a 'constriction switch' that determines if epithelial cells enter into an apical constriction or cell interface contraction regime. This switch in contractile surfaces then determines which of two primary morphogenetic paradigms the developing tissue will follow-a primarily intercalatory process driven by neighbor exchange events, or the loss of apical areas that lead to furrow formation and tissue invagination (Miao, 2021).

The ability of epithelial cells to remodel specific cellular surfaces is central to determining cell dimensions as well as the neighbor community that they will interact with and adhere to. This ability to either grow or contract certain cell sides will determine overall cell shape, and the cumulative effects of these cell shape changes determines tissue behaviors and morphologies. By regulating the contraction of apical surfaces versus cell-cell interfaces, a tissue can drive events as diverse as furrow formation and cell ingression to cell intercalation and the intermixing of cells along the AP axis. However, it has been unclear whether direct cues reside within the plasma membrane that may guide and control the engagement of contractile forces. This study examined the function of plasma membrane phospholipids in recruiting Sbf-Rab35-driven ratcheting. PI(3,4,5)P3 was shown to regulates a switch in ratcheting engagement-a reduction in PIP3 levels causes a reorientation of Sbf-Rab35 compartment formation to apicomedial surfaces. This relocalization is sufficient to change the reversible oscillations in cell area that occur in the wild-type germband epithelium into a processive regime in which apical cell areas shrink and ectopic furrows are formed. Sbf, the guanine nucleotide exchange factor for Rab35, can directly bind PIP3, and PIP3 levels and sites of enrichment are differentially regulated between the germband and ingressing mesoderm to provide a differential lipid-based cue between these two tissues (Miao, 2021).

It is interesting to note that while ectopic furrows form after PIP3 disruption, these furrows are often disorganized and lack the regular appearance of the main ventral furrow that drives the ingression of mesoderm during gastrulation. In some respects, this is to be expected-whether the major portion of the embryo is transformed to attempt to contract apical surfaces, then cells will be engaged in a contractile tug-of-war against each other. This condition is likely shown by the juxtaposition of small and large cells, which is observed in both the germband as well as the ventral furrow after PIP3 disruption. The uneven contraction of apical surfaces may also reflect the different temporal dynamics of Sbf-Rab35 compartments after PIP3 disruption. The new medial compartments that form after PIP3 disruption are much more stable (indeed, lifetimes up to 10x longer in some measurements) and robust than those that form in either the germband or ventral furrow in control embryos. Previous work has shown the importance of contractile cycles to achieve a uniform overall contraction of the apical surfaces. For example, in circumstances where pulsatility, but not contractility, is compromised, contractile networks have been observed to tear and separate-this results in a similar loss of cell area uniformity as detected after PIP3 levels are downregulated. Thus, the change in Sbf-Rab35 compartment function to much more stable and longer cycles may enhance the tug-of-war element of the cell contractions previously referenced, producing 'winner' cells of much smaller apical areas and 'loser' cells that cannot shrink against the pulling forces of neighboring cells and thus possess larger apical areas. It is intriguing that the pulsatility of contraction appears to be such a fundamental element of contractile processes-pulsatility has been observed across a huge variety of contraction-driven processes ranging from wound healing to compaction of the mouse embryo to neuroblast ingression (Miao, 2021).

Another interesting aspect of this work is that both phosphatidyl inositol phosphate species and JAK/STAT-dependent signaling control where ratcheting engagement occurs. If either of these pathways is disrupted, then a medial signal dominates and Sbf-Rab35 compartment formation occurs in a central, apical location. The current model suggests that PIP3 and JAK/STAT signaling may provide a dispersal signal that guides the compartments from a single apico-central location to the cell periphery (in the case of germband epithelial cells) or to smaller, more dispersed apical locations (in cells of the ventral furrow). Based on the direct binding of PIP3 by the Sbf RabGEF in the PIP binding assay, this dispersal may be through a direct interaction. The experiments did not have the resolution to determine if small PIP3 microdomains exist in the plasma membrane, or if further systems direct the formation of smaller, compartmental assemblies. On the other hand, how does JAK/STAT direct ratcheting engagement? Previous work examining the apical constrictions driven by an absence of JAK/STAT signaling implicated a repression of WASP actin networks that, when activated, may cause the enhanced recruitment of apical myosin II populations. Interestingly, this fits with the current step detection measurements. In addition to the changes in Sbf-Rab35 localization, pulsed contractions are stronger and more sustained in JAK/STAT embryos than in PIP3-disrupted embryos. However, previous results have shown that the generation of Sbf-Rab35 compartments is independent of myosin II function. Thus, from these results, we suggest that, while PIP3 directly regulates ratcheting engagement, JAK/STAT may regulate both the underlying oscillatory machinery as well as the strength of the medial signal that directs ratcheting engagement. It is interesting to note that, through the use of automated step detection measurements based on mean squared displacements, it has been previously shown that the oscillatory machinery appears to be strengthened specifically in the ventral cells that will undergo processive apical constriction to form the ventral furrow, which suggests a commonality with the behaviors observed after JAK/STAT disruption (Miao, 2021).

Phosphatidylinositol phosphates and their function in morphogenetic processes PIP phospho-species are attractive candidates to provide important spatial information as they are directly embedded in target membranes. PIP3 has long been implicated in distinguishing the leading edge in migrating cells and is rapidly upregulated after cells are stimulated with chemoattractant where it promotes F-actin assembly necessary for cell crawling. However, how PIP3 regulates gastrulation events has been relatively unstudied. A recent work has demonstrated the Pi3K92E can bind to active Toll receptors and Src signaling complexes, and is planar polarized at AP interfaces, in keeping with our finding of developmental enrichment of PIP3 in the germband and suggesting an intriguing connection between planar positional information and PIP function (Tamada, 2021). PIP3 is necessary for cell migration events in the mesenchymal gastrulation movements that occur in zebrafish, while data from Drosophila have shown that PIP3 levels are upregulated after wounding and help cells recognize affected surfaces. Disrupting PIP3 levels disrupted dorsal closure in the late embryo, where, once again, PIP3 is found at higher levels specifically at those surfaces that are driving tissue remodeling. This work similarly finds the PIP3 levels are developmentally patterned, where they are enriched at contractile surfaces. Other work has shown that a PIP2/PIP3 balance affects actomyosin contractility during the cellularization process that creates the early embryonic epithelium through the recruitment of an actin stabilizer, bottleneck. Sbf-Rab35 compartments have represented an interesting convergence point between pathways that directly regulate cell membrane remodeling and those that control cortical force generation. Going forward, it will be interesting to examine if this convergence includes similar higher-level regulation of the protein networks that have been implicated in migrating systems (Miao, 2021).

This work demonstrates a fundamental switch in contractile behaviors depending on the activity and localization of the PI(3,4,5)P3 lipid cue. There were several limitations to these studies: first, many of the functional disruptions relied on pharmacological or shRNA knockdown lines which often produce only hypomorphic disruptions. Phenotypes were confirmed with secondary shRNA lines that targeted different regions of the selected mRNA and yet produced similar defects; however, deeper disruption of these genes may produce more severe defects at these stages or earlier in development. Second, a PIP3 biosensor (tGPH-GFP) was used to detect PIP3 localization and levels-this is the standard in the field but represents an indirect binder of PIP3, so future probe development may allow a better resolution of PIP3 behaviors. As was mentioned, it would be very interested to be able to better address if PIP3 microdomains exist in the plasma membrane, and if these may directly trigger site-specific Sbf/Rab35 compartmental formation, but these were not resolvable with the combination of probe and microscopy elements. Finally, gastrulation in the early Drosophila embryo shows a remarkable robustness to disruption, so additional phenotypes may be concealed by compensatory mechanisms that were not detected in our analysis (Miao, 2021).

Cao, X., Rojas, M. and Pastor-Pareja, J. C. (2022). Intrinsic and damage-induced JAK/STAT signaling regulate developmental timing by the Drosophila prothoracic gland. Dis Model Mech 15(1). PubMed ID: 34842272

Intrinsic and damage-induced JAK/STAT signaling regulate developmental timing by the Drosophila prothoracic gland

Development involves tightly paced, reproducible sequences of events, yet it must adjust to conditions external to it, such as resource availability and organismal damage. A major mediator of damage-induced immune responses in vertebrates and insects is JAK/STAT signaling. At the same time, JAK/STAT activation by the Drosophila Upd cytokines is pleiotropically involved in normal development of multiple organs. Whether inflammatory and developmental JAK/STAT roles intersect is unknown. This study shows that JAK/STAT is active during development of the prothoracic gland (PG), which controls metamorphosis onset through ecdysone production. Reducing JAK/STAT signaling decreased PG size and advanced metamorphosis. Conversely, JAK/STAT hyperactivation by overexpression of pathway components or SUMOylation loss caused PG hypertrophy and metamorphosis delay. Tissue damage and tumors, known to secrete Upd cytokines, also activated JAK/STAT in the PG and delayed metamorphosis, at least in part by inducing expression of the JAK/STAT target Apontic. JAK/STAT damage signaling, therefore, regulates metamorphosis onset by co-opting its developmental role in the PG. These findings in Drosophila provide insights on how systemic effects of damage and cancer can interfere with hormonally controlled development and developmental transitions (Cao, 2022).

PTP61F Mediates Cell Competition and Mitigates Tumorigenesis

Tissue homeostasis via the elimination of aberrant cells is fundamental for organism survival. Cell competition is a key homeostatic mechanism, contributing to the recognition and elimination of aberrant cells, preventing their malignant progression and the development of tumors. Using Drosophila as a model organism, this study have defined a role for protein tyrosine phosphatase 61F (PTP61F) (orthologue of mammalian PTP1B and TCPTP) in the initiation and progression of epithelial cancers. A Ptp61F null mutation confers cells with a competitive advantage relative to neighbouring wild-type cells, while elevating PTP61F levels has the opposite effect. Furthermore, it was shown that knockdown of Ptp61F affects the survival of clones with impaired cell polarity, and that this occurs through regulation of the JAK-STAT signalling pathway. Importantly, PTP61F plays a robust non-cell-autonomous role in influencing the elimination of adjacent polarity-impaired mutant cells. Moreover, in a neoplastic RAS-driven polarity-impaired tumor model, it was shown that PTP61F levels determine the aggressiveness of tumors, with Ptp61F knockdown or overexpression, respectively, increasing or reducing tumor size. These effects correlate with the regulation of the RAS-MAPK and JAK-STAT signalling by PTP61F. Thus, PTP61F acts as a tumor suppressor that can function in an autonomous and non-cell-autonomous manner to ensure cellular fitness and attenuate tumorigenesis (La Marca, 2021).

Ursolic Acid Protects Sodium Dodecyl Sulfate-Induced Drosophila Ulcerative Colitis Model by Inhibiting the JNK Signaling

Ursolic acid (UA) is a bioactive molecule widely distributed in various fruits and vegetables, which was reported to play a therapeutic role in ulcerative colitis (UC) induced by toxic chemicals. However, the underlying mechanism has not been well clarified in vivo. In this study, using a Drosophila UC model induced by sodium dodecyl sulfate (SDS), the defensive effect of UA on intestinal damage was investigated. The results showed that UA could significantly protect Drosophila from the damage caused by SDS exposure. Further, UA alleviated the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by SDS and upregulated the activities of total superoxide dismutase (T-SOD) and catalase (CAT). Moreover, the proliferation and differentiation of intestine stem cells (ISCs) as well as the excessive activation of the c-Jun N-terminal kinase (JNK)-dependent JAK/STAT signaling pathway induced by SDS were restored by UA. In conclusion, UA prevents intestine injury from toxic compounds by reducing the JNK/JAK/STAT signaling pathway. UA may provide a theoretical basis for functional food or natural medicine development (Wei, 2022).

Germline sex determination regulates sex-specific signaling between germline stem cells and their niche

Establishing germ cell sexual identity is critical for development of male and female germline stem cells (GSCs) and production of sperm or eggs. Germ cells depend on signals from the somatic gonad to determine sex, but in organisms such as flies, mice, and humans, the sex chromosome genotype of the germ cells is also important for germline sexual development. How somatic signals and germ-cell-intrinsic cues combine to regulate germline sex determination is thus a key question. This study found that JAK/STAT signaling in the GSC niche promotes male identity in germ cells, in part by activating the chromatin reader Phf7. Further, it was found that JAK/STAT signaling is blocked in XX (female) germ cells through the action of the sex determination gene Sex lethal to preserve female identity. Thus, an important function of germline sexual identity is to control how GSCs respond to signals in their niche environment (Bhaskar, 2022).

This study presents data that provides new insights into germline sex determination and the regulation of male versus female GSC identity. First, it was found that one key function of the JAK/STAT pathway in GSCs is to promote male identity and directly activate expression of the male germline chromatin regulator Phf7. Further, it was found that an important role for Sxl in female germ cells is to block the JAK/STAT pathway and prevent this signal from masculinizing the germline. Therefore, one key aspect of germline sexual identity is to regulate how GSCs respond to signals in their niche environment (Bhaskar, 2022).

Different findings have led to different conclusions about the role of the JAK/STAT pathway in male GSCs. When STAT activity is removed from individual GSCs, they are lost rapidly from the niche, indicating a role in GSC identity or maintenance. However, when STAT is removed from all GSCs, they exhibit defects in niche adhesion but can otherwise function as GSCs, although GSC loss is also observed. The JAK/STAT pathway has also been implicated in aging of GSCs and their niche. One interpretation of these diverse data would be that the JAK/STAT pathway is important for specific aspects of male GSC function, such as regulation of cell adhesion and the cell cycle, but it is not required for stem cell identity per se (Bhaskar, 2022).

A different role is proposed for the JAK/STAT pathway, which is to regulate GSC sexual identity. Previously it was reported that the JAK/STAT pathway is important for establishing male identity in the embryonic germline. This study shows that one defect observed in XX germ cells present in a male soma is that they exhibit reduced JAK/STAT signaling. Further, activation of the JAK/STAT pathway can partially rescue these XX germ cells, promoting a male identity and progression into spermatogenesis. Thus, it is proposed that the JAK/STAT pathway remains a key masculinizing signal for the germline throughout development and into adulthood. One possibility is that the JAK/STAT pathway regulates only GSC sex and that other roles, such as regulating a specific set of cell adhesion proteins, represent downstream consequences of altering sexual identity. Alternatively, the JAK/STAT pathway could regulate GSC sexual identity and other aspects of GSC behavior independently. One important way in which the JAK/STAT pathway promotes a male identity in the germline is by activating the male sex determination factor Phf7. Previously, it was shown that Phf7 is important for male identity in the germline and proper spermatogenesis. Phf7 likely promotes male germline identity by acting as a chromatin 'reader' and binding to histone H3 methylated at position K4. Phf7 is also toxic to female germ cells, making the sex-specific regulation of Phf7 extremely important. This study shows that the JAK/STAT pathway is a direct regulator of Phf7 expression in both embryos and adults. STAT protein can bind to the Phf7 locus, and consensus STAT binding sites near the male-biased promoter are essential for proper male expression of Phf7 and its ability to function in spermatogenesis. Expression from the male-biased promoter is important in part because the transcript from the downstream, 'female' promoter is subject to translational repression. Thus, Phf7 represents an important link between the JAK/STAT pathway and male identity in the germline (Bhaskar, 2022).

Sxl acts as a key regulator of sex determination in both the soma and the germline, and it is necessary and sufficient to confer female identity. However, the role of Sxl in the germline has remained mysterious. In the soma, Sxl regulates sexual identity through tra and dosage compensation through msl-2, but these genes do not play a role in the germline. Instead, this study has found that a key role of Sxl in the germline is to repress the JAK/STAT pathway in female germ cells (Bhaskar, 2022).

Initially, only the male somatic gonad expresses ligands for the JAK/STAT pathway and is capable of promoting JAK/STAT activation in the germ cells. However, ligands for the JAK/STAT pathway eventually become active in the germarium of the ovary, where they are important for the function or maintenance of the somatic escort cells. Sxl acts to repress JAK/STAT response in the female germ cells and thereby prevents activation of male-promoting factors such as Phf7. Somatic cells of the ovary such as the escort cells are still able to respond to these ligands and activate the JAK/STAT pathway, even though they also express Sxl. How Sxl is able to repress the JAK/STAT response in a germline-specific manner remains unknown, although the levels of Sxl appear higher in the GSCs than in the surrounding soma. However, the fact that an activated Hop (hop^Tum) can partially rescue the germline in XX males indicates that Sxl is repressing the pathway at the level of Hop or above. Interestingly, RNA for the JAK/STAT receptor domeless was identified in a pull-down experiment with Sxl, suggesting this could be a relevant target for regulation (Bhaskar, 2022).

These data support a model where the JAK/STAT pathway is important for activating male identity in the germline and expression of male genes such as Phf7, while this pathway is repressed in female germ cells by Sxl. Loss of Sxl from the female germline leads to both upregulation of JAK/STAT signaling and inappropriate expression of Phf7. In addition, suppression of the JAK/STAT pathway can partially rescue loss of Sxl in the female germline. Thus, regulation of the JAK/STAT pathway is one key aspect of how Sxl promotes female germline identity. However, no ability was observed for loss of Phf7 to rescue loss of Sxl from the female germline. This is in contrast to previously published results where loss of Phf7 was shown to rescue the female germline in sans fille mutants, which also primarily affects the germline by disrupting Sxl expression. This study has now reduced Phf7 function by both RNAi and using null Phf7 mutants, in both Sxl and sans fille loss-of-function backgrounds and observed no rescue or modification of the germline defects present. It is concluded that, while regulation of Phf7 by the JAK/STAT pathway and Sxl is clearly important for proper germline sexual development, ectopic expression of Phf7 is not the only defect present in Sxl mutant female germ cells; there must be additional targets for regulation by Sxl and JAK/STAT that are disrupted in Sxl mutants. In support of this view, loss of STAT from the male germline has a more severe phenotype than loss of Phf7. Previously, it has been shown that expression of another male-promoting factor in the germline, Tdrd5l, is regulated by Sxl. While this regulation appears to be, at least in part, via Sxl acting on the Tdrd5l mRNA to influence levels of Tdrd5l protein, it is possible that Tdrd5l is also regulated at the transcriptional level as an additional target of the JAK/STAT pathway (Bhaskar, 2022).

It is intriguing that Sxl acts as a negative regulator of the JAK/STAT pathway in both the soma and the germline but does so in different ways. In the soma, Sxl activates an alternative splicing cascade that leads to splicing of dsx in the female mode, creating the DSXF protein, while the DSXM protein is produced in males by default. An important sex-specific trait in the embryonic gonad is that male somatic cells produce ligands for the JAK/STAT pathway that activate JAK/STAT signaling specifically in male germ cells, and this is regulated in a manner dependent on dsx. Thus, in addition to being a negative regulator of JAK/STAT signal reception in the germline, Sxl acts as a negative regulator of JAK/STAT ligand production in the soma. Together, these independent aspects of regulation by Sxl combine to ensure that the masculinizing effects of the JAK/STAT pathway are restricted to male germ cells (Bhaskar, 2022).

An important conclusion from this work is that germline sex determination regulates how GSCs communicate with their surrounding stem cell niche. Germline sex determination is regulated by both germline autonomous cues, based on the germline sex chromosome constitution, and non-autonomous signals from the soma. The autonomous cues, acting through Sxl, regulate how signals from the niche are received and interpreted by the GSCs. In both the testis and ovary GSC cell niches, the JAK/STAT pathway is important for regulating somatic cells like the cyst stem cells in the testis and the escort cells in the ovary. However, this pathway is only required in the male GSCs and not female GSCs. It is proposed that it is essential to block JAK/STAT signaling in female GSCs to prevent their exposure to this masculinizing signal. Indeed, activation of the JAK/STAT signal is sufficient to promote male identity in XX germ cells, and removal of STAT is sufficient to partially rescue the defects observed in XX germ cells that have lost Sxl. Thus, a key aspect of how Sxl promotes female identity in the germline is to prevent female GSCs from being masculinized by activators of the JAK/STAT pathway present in the niche environment (Bhaskar, 2022).

It is important to note that, when the sex chromosome genotype affecting germline is referred to as 'sex determination' this could result from any contribution of sex chromosome genotype to successful spermatogenesis or oogenesis. For example, if dosage compensation is incomplete or non-existent in the germline, then the presence of two X chromosomes will lead to increased X chromosome gene expression, which may be incompatible with male germline differentiation. Similarly, a single X chromosome dose may be incompatible with oogenesis. It is also possible that the number of X chromosomes present in the germline has additional affects besides the presence or absence of Sxl expression. While XX germ cells present in a male soma exhibit severe atrophy and loss, the expression of Sxl in the male germline has a much weaker phenotype. Thus, there may be additional consequences of sex chromosome genotype on germline function beyond that which is controlled by Sxl. A better understanding of what germline sexual identity means in Drosophila, in particular at the level of whole-genome gene expression levels, is required before it will be possible to assess the true contribution of germline sex chromosome constitution to germline sex determination. Further, how the effects of X chromosome number on germline sexual development in Drosophila relate to infertility observed in patients with disorders of sexual development such as Klinefelter's and Turner's syndromes remains to be investigated (Bhaskar, 2022).

One limitation of this study is the relatively low frequency with which it was possible to rescue the XX germline in tra mutants by downregulating Sxl or upregulating the JAK/STAT pathway. This could be due to technical limitations of the timing or level of expression of the reagents used. Alternatively, this could mean that there is another interesting defect in XX germ cells present in a male somatic environment besides that is caused by expression of Sxl and downregulation of the JAK/STAT pathway. Another limitation of the study is that the molecular target for Sxl in regulating the JAK/STAT pathway remains unknown. RNA-seq analysis of Bam mutant testes and ovaries suggested that hop RNA splicing is differentially regulated between males and females. However, extensive experimental analysis failed to reveal a role for Sxl in regulating hop in the germline. Thus, this remains an important area for future research (Bhaskar, 2022).

Xu, D. C., Wang, L., Yamada, K. M. and Baena-Lopez, L. A. (2022). Non-apoptotic activation of Drosophila caspase-2/9 modulates JNK signaling, the tumor microenvironment, and growth of wound-like tumors. Cell Rep 39(3): 110718. PubMed ID: 35443185

Non-apoptotic activation of Drosophila caspase-2/9 modulates JNK signaling, the tumor microenvironment, and growth of wound-like tumors

Resistance to apoptosis due to caspase deregulation is considered one of the main hallmarks of cancer. However, the discovery of novel non-apoptotic caspase functions has revealed unknown intricacies about the interplay between these enzymes and tumor progression. To investigate this biological problem, this study capitalized on a Drosophila tumor model with human relevance based on the simultaneous overactivation of the EGFR and the JAK/STAT signaling pathways. The data indicate that widespread non-apoptotic activation of initiator caspases limits JNK signaling and facilitates cell fate commitment in these tumors, thus preventing the overgrowth and exacerbation of malignant features of transformed cells. Intriguingly, caspase activity also reduces the presence of macrophage-like cells with tumor-promoting properties in the tumor microenvironment. These findings assign tumor-suppressing activities to caspases independent of apoptosis, while providing molecular details to better understand the contribution of these enzymes to tumor progression (Xu, 2022).

Inter-organ Wingless/Ror/Akt signaling regulates nutrient-dependent hyperarborization of somatosensory neurons

Nutrition in early life has profound effects on an organism, altering processes such as organogenesis. However, little is known about how specific nutrients affect neuronal development. Dendrites of class IV dendritic arborization neurons in Drosophila larvae become more complex when the larvae are reared on a low-yeast diet compared to a high-yeast diet. A systematic search for key nutrients revealed that the neurons increase their dendritic terminal densities in response to a combined deficiency in vitamins, metal ions, and cholesterol. The deficiency of these nutrients upregulates Wingless in a closely located tissue, body wall muscle. Muscle-derived Wingless activates Akt in the neurons through the receptor tyrosine kinase Ror, which promotes the dendrite branching. In larval muscles, the expression of wingless is regulated not only in this key nutrient-dependent manner, but also by the JAK/STAT signaling pathway. Additionally, the low-yeast diet blunts neuronal light responsiveness and light avoidance behavior, which may help larvae optimize their survival strategies under low-nutritional conditions. Together, these studies illustrate how the availability of specific nutrients affects neuronal development through inter-organ signaling (Kanaoka, 2023).

Drosophila Stat92E Signaling Following Pre-exposure to Ethanol

Repeated exposure to alcohol alters neuromolecular signaling that influences acute and long-lasting behaviors underlying Alcohol Use Disorder (AUD). Recent animal model research has implicated changes in the conserved JAK/STAT pathway, a signaling pathway classically associated with development and the innate immune system. How ethanol exposure impacts STAT signaling within neural cells is currently unclear. This study investigated the role of Drosophila Stat92E in ethanol-induced locomotion, signaling activity, and downstream transcriptional responses. Findings suggest that expressing Stat92E-RNAi causes enhanced ethanol-induced hyperactivity in flies previously exposed to ethanol. Furthermore, alternative splicing of Stat92E itself was detected after repeated ethanol exposure, although no changes were found in downstream transcriptional activity. This work adds to growing understanding of altered neuromolecular signaling following ethanol exposure and suggests that STAT signaling may be a relevant target to consider for AUD treatment (Wilson, 2023).

Distinct signaling signatures drive compensatory proliferation via S-phase acceleration

Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, this study investigated models of compensatory proliferation in Drosophila imaginal discs. Compensatory proliferation was found to be associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models (Crucianelli, 2023).

Duox and Jak/Stat signalling influence disease tolerance in Drosophila during Pseudomonas entomophila infection

Disease tolerance describes an infected host's ability to maintain health independently of the ability to clear microbe loads. The Jak/Stat pathway plays a pivotal role in humoral innate immunity by detecting tissue damage and triggering cellular renewal, making it a candidate tolerance mechanism. This study found that in Drosophila melanogaster infected with Pseudomonas entomophila disrupting ROS-producing dual oxidase (duox) or the negative regulator of Jak/Stat Socs36E, render male flies less tolerant. Another negative regulator of Jak/Stat, G9a - which has previously been associated with variable tolerance of viral infections - did not affect the rate of mortality with increasing microbe loads compared to flies with functional G9a, suggesting it does not affect tolerance of bacterial infection as in viral infection. These findings highlight that ROS production and Jak/Stat signalling influence the ability of flies to tolerate bacterial infection sex-specifically and may therefore contribute to sexually dimorphic infection outcomes in Drosophila (Prakash, 2023).

Acetobacter and lactobacillus alleviate the symptom of insulin resistance by blocking the JNK-JAK/STAT pathway in Drosophila melanogaster

The dysregulation of intestinal microbiota is well-known to be one of the main causes of insulin resistance in both vertebrates and invertebrates. Specially, the acetobacter and lactobacillus have been identified as potentially capable of alleviating insulin resistance. However, the molecular mechanism underlying this effect requires further elucidation. In this study, Drosophila was used to delineate how intestinal microbiota disrupts the host intestinal signaling pathway, contributing to insulin resistance. These findings demonstrate that a long-term high-sugar diet leads to a reduction in the general diversity of intestinal microbiota in flies, as well as a marked decrease in the abundances of acetobacter and lactobacillus. Furthermore, symptoms of insulin resistance were alleviated by feeding flies with acetobacter or lactobacillus, indicating that these microorganisms play an essential role in maintaining blood sugar homeostasis in flies. Conversely, when all intestinal microbiota was removed, flies show severe symptoms of insulin resistance, confirming that the critical role of intestinal microbiota in maintaining host blood sugar homeostasis. These studies suggested that the intestinal but not fat body JNK pathway mediates the communication of intestinal microbiota and host insulin pathway. In flies, downregulation of JNK activity alleviates symptoms of insulin resistance by decreasing the activity of the JAK/STAT pathway. However, this offsets the therapeutic effects of supplying flies with acetobacter or lactobacillus, suggesting that the therapeutic function of these microorganisms is based on their interaction with JNK-JAK/STAT axis. Taken together, this study reveals that acetobacter and lactobacillus alleviate insulin resistance symptoms in a JNK-JAK/STAT pathway-dependent manner, indicating the therapeutic potential of probiotic supplementation and regulation of the activities of JNK-JAK/STAT pathway for diabetes control (Meng, 2023).

Anti-Tumor Effect of Turandot Proteins Induced via the JAK/STAT Pathway in the mxc Hematopoietic Tumor Mutant in Drosophila

Several antimicrobial peptides supress the growth of lymph gland (LG) tumors in Drosophila multi sex comb (mxc) mutant larvae. The activity of another family of polypeptides, called Turandots, is also induced via the JAK/STAT pathway after bacterial infection; however, their influence on Drosophila tumors remains unclear. The JAK/STAT pathway was activated in LG tumors, fat body, and circulating hemocytes of mutant larvae. The mRNA levels of Turandot (Tot) genes increased markedly in the mutant fat body and declined upon silencing Stat92E in the fat body, indicating the involvement of the JAK/STAT pathway. Furthermore, significantly enhanced tumor growth upon a fat-body-specific silencing of the mRNAs demonstrated the antitumor effects of these proteins. The proteins were found to be incorporated into small vesicles in mutant circulating hemocytes (as previously reported for several antimicrobial peptides) but not normal cells. In addition, more hemocytes containing these proteins were found to be associated with tumors. The mutant LGs contained activated effector caspases, and a fat-body-specific silencing of Tots inhibited apoptosis and increased the number of mitotic cells in the LG, thereby suggesting that the proteins inhibited tumor cell proliferation. Thus, Tot proteins possibly exhibit antitumor effects via the induction of apoptosis and inhibition of cell proliferation (Kinoshita, 2023).

MicroRNA miR-274-5p supresses Found-in-Neurons associated with Melanotic mass Formation and Developmental Growth in Drosophila

The hematopoietic system plays a crucial role in immune defense response and normal development, and it is regulated by various factors from other tissues. The dysregulation of hematopoiesis is associated with melanotic mass formation; however, the molecular mechanisms underlying this process are poorly understood. This study observed that the overexpression of miR-274 in the fat body resulted in the formation of melanotic masses. Moreover, abnormal activation of the and JAK/STAT signaling pathways was linked to these consequences. In addition to this defect, miR-274 overexpression in the larval fat body decreased the total tissue size, leading to a reduction in body weight. miR-274-5p was found to directly supress the expression of found-in-neurons (fne), which encodes an RNA-binding protein. Similar to the effects of miR-274 overexpression, fne depletion led to melanotic mass formation and growth reduction. Collectively, miR-274 plays a regulatory role in the fne-JNK signaling axis in melanotic mass formation and growth control (Kim, 2023).

Expression of human HIPKs in Drosophila demonstrates their shared and unique functions in a developmental model

Homeodomain-interacting protein kinases (HIPKs) are a family of four conserved proteins essential for vertebrate development, as demonstrated by defects in the eye, brain, and skeleton that culminate in embryonic lethality when multiple HIPKs are lost in mice. While HIPKs are essential for development, functional redundancy between the four vertebrate HIPK paralogues has made it difficult to compare their respective functions. Because understanding the unique and shared functions of these essential proteins could directly benefit the fields of biology and medicine, this study addressed the gap in knowledge of the four vertebrate HIPK paralogues by studying them in the fruit fly Drosophila melanogaster, where reduced genetic redundancy simplifies functional assessment. The single hipk present in the fly allowed the performance of rescue experiments with human HIPK genes that provide new insight into their individual functions not easily assessed in vertebrate models. Furthermore, the abundance of genetic tools and established methods for monitoring specific developmental pathways and gross morphological changes in the fly allowed for functional comparisons in endogenous contexts. Rescue experiments were performed to demonstrate the extent to which each of the human HIPKs can functionally replace Drosophila Hipk for survival and morphological development. The ability of each human HIPK to modulate Armadillo/β-catenin levels, JAK/STAT activity, proliferation, growth, and death, each of which have previously been described for Hipks, but never all together in comparable tissue contexts. Finally, novel developmental phenotypes induced by human HIPKs were characterized to gain insight to their unique functions. Together, these experiments provide the first direct comparison of all four vertebrate HIPKs to determine their roles in a developmental context (Kinsey, 2021).

Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration

Epithelial repair relies on the activation of stress signaling pathways to coordinate tissue repair. Their deregulation is implicated in chronic wound and cancer pathologies. Using TNF-α/Eiger-mediated inflammatory damage to Drosophila imaginal discs, this study investigate how spatial patterns of signaling pathways and repair behaviors arise. Eiger expression, which drives JNK/AP-1 signaling, was found to transiently arrest proliferation of cells in the wound center and is associated with activation of a senescence program. This includes production of the mitogenic ligands of the Upd family, which allows JNK/AP-1-signaling cells to act as paracrine organizers of regeneration. Surprisingly, JNK/AP-1 cell-autonomously suppress activation of Upd signaling via Ptp61F and Socs36E, both negative regulators of JAK/STAT signaling. As mitogenic JAK/STAT signaling is suppressed in JNK/AP-1-signaling cells at the center of tissue damage, compensatory proliferation occurs by paracrine activation of JAK/STAT in the wound periphery. Mathematical modelling suggests that cell-autonomous mutual repression between JNK/AP-1 and JAK/STAT is at the core of a regulatory network essential to spatially separate JNK/AP-1 and JAK/STAT signaling into bistable spatial domains associated with distinct cellular tasks. Such spatial stratification is essential for proper tissue repair, as coactivation of JNK/AP-1 and JAK/STAT in the same cells creates conflicting signals for cell cycle progression, leading to excess apoptosis of senescently stalled JNK/AP-1-signaling cells that organize the spatial field. Finally, this study demonstrated that bistable separation of JNK/AP-1 and JAK/STAT drives bistable separation of senescent signaling and proliferative behaviors not only upon tissue damage, but also in RasV12, scrib tumors. Revealing this previously uncharacterized regulatory network between JNK/AP-1, JAK/STAT, and associated cell behaviors has important implications for conceptual understanding of tissue repair, chronic wound pathologies, and tumor microenvironments (Jaiswal, 2023).

The Drosophila AWP1 ortholog Doctor No regulates JAK/STAT signaling for left-right asymmetry in the gut by promoting receptor endocytosis

Many organs of Drosophila show stereotypical left-right (LR) asymmetry; however, the underlying mechanisms remain elusive. This study has identified an evolutionarily conserved ubiquitin-binding protein, AWP1/Doctor No (Drn), as a factor required for LR asymmetry in the embryonic anterior gut. drn is essential in the circular visceral muscle cells of the midgut for JAK/STAT signaling, which contributes to the first known cue for anterior gut lateralization via LR asymmetric nuclear rearrangement. Embryos homozygous for drn and lacking its maternal contribution showed phenotypes similar to those with depleted JAK/STAT signaling, suggesting that Drn is a general component of JAK/STAT signaling. Absence of Drn resulted in specific accumulation of Domeless (Dome), the receptor for ligands in the JAK/STAT signaling pathway, in intracellular compartments, including ubiquitylated cargos. Dome colocalized with Drn in wild-type Drosophila. These results suggest that Drn is required for the endocytic trafficking of Dome, which is a crucial step for activation of JAK/STAT signaling and the subsequent degradation of Dome. The roles of AWP1/Drn in activating JAK/STAT signaling and in LR asymmetric development may be conserved in various organisms (Lai, 2023).

Many animals show directional left-right (LR) asymmetry in their body structure and function. Several mechanisms, such as cilia-generated flow, contribute to LR axis formation in vertebrates. However, the mechanisms underlying LR asymmetric development in invertebrates are relatively obscure and remain an elemental question in biology (Lai, 2023).

Stereotypical LR asymmetry is present in several organs of Drosophila, including the gut, testis, male genitalia, and brain. Of these organs, the embryonic gut is the first to show LR asymmetry during development. Intriguingly, the anterior and posterior parts of the embryonic gut are controlled by two distinct gene groups. The LR asymmetry of the posterior part is regulated by Myo1D, which determines cell chirality. An entirely different mechanism governs the LR asymmetry of the anterior part. The anterior gut of the embryo consists of the foregut (FG) and midgut (MG), which are complex structures with directional and stereotypical LR asymmetry. Various genetic pathways, including the JNK and Wnt pathways, play important roles in LR asymmetric development. A genetic screen suggested that the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway is also involved in LR asymmetric development. The present study reveals the involvement of JAK/STAT signaling in LR asymmetric development of the anterior gut (Lai, 2023).

JAK/STAT signaling, which is evolutionarily conserved from Drosophila to humans, is essential for morphogenesis, cell proliferation, cell differentiation, cell death, immunity and other biological events. In JAK/STAT signaling in Drosophila, the ligands are encoded by unpaired (upd; upd1), upd2 and upd3 and the receptor is encoded by domeless (dome). JAK is encoded by hopscotch (hop) in Drosophila. Moreover, similar to the mammalian JAK/STAT system, Hop constitutively associates with the intracellular domain of Dome in Drosophila. Ligand binding induces conformational changes in Dome that lead to the phosphorylation of JAK, which phosphorylates Dome to create docking sites for STATs, particularly Stat92E in Drosophila. JAK then tyrosine phosphorylates STATs, which are subsequently dimerized and translocated to the nucleus to bind to enhancers of target genes and activate transcription (Lai, 2023).

In mammals, endocytosis regulates JAK/STAT signaling through various mechanisms. Endocytosis and the regulation of JAK/STAT signaling activity are also closely connected in Drosophila. In the classic working model of endocytosis, membrane receptors are internalized into endosomal compartments, where they are degraded and recycled. This reduces the number of receptors available to transduce signaling. Moreover, receptor activity requires endocytic trafficking in some signaling pathways. Studies on the relationship between endocytosis and JAK/STAT signaling in Drosophila have provided contradictory results regarding whether endocytosis upregulates or downregulates JAK/STAT activity. To clarify these conflicting results, it would be helpful to identify and examine a factor that specifically regulates the endocytosis of Dome (Lai, 2023).

This study found that the Drosophila ortholog of AWP1 (associated with PRK1, also known as ZFAND6), which is referred to as Doctor No (Drn), positively regulates JAK/STAT signaling by facilitating endocytic trafficking of the Dome receptor, which is required for normal LR asymmetric development of the embryonic gut. Drosophila Drn was named after Ian Fleming's fictional character, whose heart was located on the right side of his chest. Drn protein contains an A20-type zinc finger at the N terminus and an AN1-type zinc finger at the C terminus; this is similar to the mammalian ortholog, which was first identified in humans and mice. In vertebrates, AWP1 proteins bind to ubiquitin, regulate NF-κB activity, and stimulate the export of Pex5 from the peroxisome, among other roles. During Xenopus development, AWP1 modifies Wnt and FGF signaling to specify neural crest cells. However, the molecular mechanisms by which AWP1/Drn proteins influence these various cell signaling pathways are not well understood. This study found that AWP1/Drn plays a crucial role in internalizing the Dome receptor, and a mechanism is proposed by which AWP1/Drn positively modulates JAK/STAT signaling (Lai, 2023).

This study demonstrates that drn, which encodes a Drosophila ortholog of AWP1, plays crucial roles in the LR asymmetric development of the FG and AMG by positively regulating JAK/STAT signaling. In this process, wild-type drn is required for the normal endocytic trafficking of Dome, which is the Drosophila JAK/STAT signaling receptor. Whether the internalization as well as endocytic trafficking of Dome is required to activate JAK/STAT signaling in Drosophila has been a topic of debate. Analyses of mutations affecting Dome endocytosis have revealed that Dome must be internalized into early endosomes to activate JAK/STAT signaling. In contrast, the RNAi-mediated knockdown of genes required for Dome endocytosis was found to enhance JAK/STAT signaling activity, demonstrating that Dome endocytosis negatively regulates JAK/STAT signaling. This discrepancy can be explained by the multiple ramifications and parallelism of the endocytic pathway, as described in recent studies. In particular, blocking a particular step along the pathway can divert endocytic trafficking into various positive or negative regulatory cascades of cell signaling pathways. The relative contributions of endocytic regulators can also change the course of endocytic trafficking, as is well documented in Notch signaling. Moreover, a change in the balance of regulators may change how endocytosis regulates cell signaling pathways according to the context. Hence, to understand comprehensively how endocytosis contributes to the activation of Dome, it is necessary to examine the point at which endocytosis is disrupted and the altered course of endocytic trafficking when normal endocytosis fails (Lai, 2023).

A study using RNAi of cultured Drosophila cells identified drn as a negative regulator of JAK/STAT signaling; this finding appears to be opposite to the current model. However, as other genes required for Dome endocytosis were also identified as negative regulators of JAK/STAT signaling in that analysis, it is likely that this discordance regarding the role of Drn in JAK/STAT signaling results from a discrepancy in the contribution of Dome endocytosis to JAK/STAT signaling, as discussed above. Regarding this discrepancy, it has been proposed that long-term, loss-of-function analyses, including analyses of mutants in which Dome endocytosis is disrupted, may reflect unexpected cell fate changes induced by altered endocytic pathways and their influence on JAK/STAT signaling. However, in this study, three developmental contexts were examined in which JAK/STAT signaling is reduced in drn mutants. One of these involves the regulation of eve expression, which occurs before cell fate specification and during a short period in early embryogenesis; thus, it does not fit well with a mechanism involving unexpected cell fate changes. Therefore, a model is proposed in which Drn is required in some steps of the endocytic trafficking of Dome, which plays a role in the subsequent activation of JAK/STAT signaling. This model is consistent with that proposed by in another study in which the endocytic trafficking of Dome activates JAK/STAT signaling and also provides a mechanism to regulate it quantitatively. However, as where and how Drn can control the endocytic trafficking of Dome remain unclear, the coincidence between previous and current results should be interpreted with caution (Lai, 2023).

Drn is the Drosophila ortholog of AWP1, which binds to ubiquitin and modulates the functions of ubiquitylated proteins in mammals and Xenopus species. It is known that ubiquitylation of the receptors involved in JAK/STAT signaling is important for regulating signaling activities in mammals. The sorting of ubiquitylated membrane proteins into intraluminal vesicles relies on protein complexes in the ESCRT family. ESCRT-0, ESCRT-I, and ESCRT-II include multiple ubiquitin-binding proteins and interpret ubiquitin as a signal to sort membrane proteins. In Drosophila mutants of the ESCRT-0 complex components Hrs and Stam, ubiquitylated membrane proteins, such as Notch and Dome, aggregate at the cell cortex and in intracellular compartments. As such intracellular compartments, including aggregated Dome, were stained with an anti-ubiquitin antibody, it has been proposed that Drosophila Dome is ubiquitylated and its endosomal sorting is controlled by ubiquitylation; however, the ubiquitylation of Dome has not been confirmed biochemically. A loss of Drn caused Dome to accumulate in large clumps that frequently colocalized with ubiquitin but were not labeled by markers of typical intracellular compartments. In addition, Drn occasionally colocalized with markers of various endocytic compartments, demonstrating that Drn is an endocytic protein. Hence, it is speculated that Drn plays a role in the ubiquitin-dependent internalization or sorting of Dome through its potential ubiquitin-binding activity. This idea is consistent with the observation that Drn often colocalizes with Dome in some intracellular vesicles in wild-type Drosophila. Thus, in this model, Dome may be misrouted to atypical endocytic compartments, where it fails to be phosphorylated in the absence of Drn. Conversely, differences in Dome trafficking routes between wild-type and drn mutant embryos should help identify the endocytic compartment where Dome is activated by phosphorylation. However, it is difficult to delineate incorrect Dome trafficking routes in the drn mutant because Dome did not specifically colocalize with typical markers of endocytic compartments under this condition. The model also predicts that such misrouting can consequently prevent the degradation of Dome in lysosomes and leave it to accumulate, as observed in drn mutants (Lai, 2023).

These analyses revealed that drn mutations induce marked accumulation of Dome but not of Notch or Fz2. The intracellular distribution of Notch and Fz2 in the drn mutant appeared to be similar to that in the wild type. Thus, Drn is not a general component of endosomal protein sorting but is specific to Dome; however, how such specificity is achieved remains unclear (Lai, 2023).

This study found that JAK/STAT signaling activity must be maintained at proper levels for normal LR asymmetric development of the FG and AMG. A similar phenomenon was previously observed in Wnt or JNK signaling activity. However, LR asymmetry was bit detectuve in the activity or the distribution of molecules involved in the JAK/STAT, Wnt and JNK signaling pathways. As these three signaling pathways are required for LR asymmetric rearrangement of nuclei in the visceral muscles of the MG, they may play permissive roles in rearranging these nuclei upon a common cue, which is yet unknown, of LR polarity (Lai, 2023).

AWP1/Drn is highly conserved from Drosophila to humans. As receptors in the mammalian JAK/STAT pathway are also ubiquitylated, it is suggested that the role of AWP1/Drn in JAK/STAT signaling and LR asymmetric development is evolutionarily conserved in various organisms (Lai, 2023).

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Zygotically transcribed genes

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