The Interactive Fly

Zygotically transcribed genes

Cytokine signaling: the JAK/STAT pathway

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

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

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

Methotrexate is a JAK/STAT pathway inhibitor

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

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

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

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

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


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 (HopTum-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 HopTum-l and these results were compared to females heterozygous for both HopTum-l and Df(3)ED4238. Removing one copy of PTP61F in HopTum-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).

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).

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).

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).


References

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Zygotically transcribed genes

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