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Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. In this study, we identified a testis‐specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C2H2 type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin‐protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587‐Gal4 > CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, we found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92EF06346 mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, our results indicated that CG3526 is a downstream effector gene in the JAK‐STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila. [ABSTRACT FROM AUTHOR]

A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling that may represent additional clinical interventions . Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK [Hopscotch (Hop)] and one STAT (Stat92E). hopTumorous-lethal (Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, we identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in ex, wts, and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling.

The maintenance of germline stem cells (GSCs) is essential for tissue homeostasis. JAK/STAT signaling maintains GSC fate in Drosophila testis. However, how JAK/STAT signaling maintains male GSC fate through its downstream targets remains poorly understood. Here, we identify p115, a tER/cis-Golgi golgin protein, as a putative downstream target of JAK/STAT signaling. p115 maintains GSC fate independent of GM130 and GRASP65. p115 localizes in cytosol, the ER and Golgi apparatus in germline cells and is required for the morphology of the ER and Golgi apparatus. Furthermore, depletion of p115 in GSCs results in aberrant spindle orientation. Mechanistically, p115 associates with and stabilizes STAT. Finally, ectopic expression of STAT completely restores GSC loss caused by p115 depletion. Collectively, JAK/STAT signaling and p115 form a feedforward loop to maintain male GSC fate. Our work provides new insights into the regulatory mechanism of how stem cell maintenance is properly controlled by JAK/STAT signaling., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Models that incorporate specific chemical mechanisms have been successful in describing the activity of Drosophila developmental enhancers as a function of underlying transcription factor binding motifs. Despite this, the minimum set of mechanisms required to reconstruct an enhancer from its constituent parts is not known. Synthetic biology offers the potential to test the sufficiency of known mechanisms to describe the activity of enhancers, as well as to uncover constraints on the number, order, and spacing of motifs. Results: Using a functional model and in silico compensatory evolution, we generated putative synthetic even-skipped stripe 2 enhancers with varying degrees of similarity to the natural enhancer. These elements represent the evolutionary trajectories of the natural stripe 2 enhancer towards two synthetic enhancers designed ab initio. In the first trajectory, spatially regulated expression was maintained, even after more than a third of binding sites were lost. In the second, sequences with high similarity to the natural element did not drive expression, but a highly diverged sequence about half the length of the minimal stripe 2 enhancer drove ten times greater expression. Additionally, homotypic clusters of Zelda or Stat92E motifs, but not Bicoid, drove expression in developing embryos. Conclusions: Here, we present a functional model of gene regulation to test the degree to which the known transcription factors and their interactions explain the activity of the Drosophila even-skipped stripe 2 enhancer. Initial success in the first trajectory showed that the gene regulation model explains much of the function of the stripe 2 enhancer. Cases where expression deviated from prediction indicates that undescribed factors likely act to modulate expression. We also showed that activation driven Bicoid and Hunchback is highly sensitive to spatial organization of binding motifs. In contrast, Zelda and Stat92E drive expression from simple homotypic clusters, suggesting that activation driven by these factors is less constrained. Collectively, the 40 sequences generated in this work provides a powerful training set for building future models of gene regulation. [ABSTRACT FROM AUTHOR]

A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling that may represent additional clinical interventions. Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK [Hopscotch (Hop)] and one STAT (Stat92E). hopTumorous-lethal (Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, we identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in ex, wts, and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling. [ABSTRACT FROM AUTHOR]

Tissue homeostasis is maintained by balancing stem cell self-renewal and differentiation. How surrounding cells support this process has not been entirely resolved. Herewe showthat the chromatin and telomerebinding factor Without children (Woc) is required for maintaining the association of escort cells (ECs) with germ cells in adult ovaries. This tight association is essential for germline stem cell (GSC) differentiation into cysts. Woc is also required in larval ovaries for the association of intermingled cells (ICs) with primordial germ cells. Reduction in the levels of two other proteins, Stat92E and its target Zfh1, produce phenotypes similar to woc in both larval and adult ovaries, suggesting a molecular connection between these three proteins. Antibody staining andRT-qPCRdemonstrate that Zfh1 levels are increased in somatic cells that contact germ cells, and thatWoc is required for a Stat92E-mediated upregulation of zfh1 transcription. Our results further demonstrate that overexpression of Zfh1 in ECs can rescue GSC differentiation in woc-deficient ovaries. Thus, Zfh1 is a major Woc target in ECs. Stat signalling in niche cells has been previously shown to maintainGSCs non-autonomously.We now show that Stat92E also promotes GSC differentiation. Our results highlight the Woc-Stat-Zfh1 module as promoting somatic encapsulation of germ cells throughout their development. Each somatic cell type can then provide the germline with the support it requires at that particular stage. Stat is thus a permissive factor, which explains its apparently opposite roles in GSC maintenance and differentiation. [ABSTRACT FROM AUTHOR]

Highlights: [•] JAK/STAT signaling is central to epithelial and hematopoietic tumors in Drosophila. [•] Unpaired is ectopically induced by loss of distinct tumor suppressors. [•] Ras and JAK/STAT pathways cooperate to induce metastatic tumors. [•] Dominant-active JAK mutations cause melanotic tumors, a fly leukemia. [•] Genetic screens have identified new pathway effectors in blood cell tumors. [Copyright &y& Elsevier]

Abstract: JAK/STAT signaling is localized to the wing hinge, but its function there is not known. Here we show that the Drosophila STAT Stat92E is downstream of Homothorax and is required for hinge development by cell-autonomously regulating hinge-specific factors. Within the hinge, Stat92E activity becomes restricted to gap domain cells that lack Nubbin and Teashirt. While gap domain cells lacking Stat92E have significantly reduced proliferation, increased JAK/STAT signaling there does not expand this domain. Thus, this pathway is necessary but not sufficient for gap domain growth. We show that reduced Wingless (Wg) signaling dominantly inhibits Stat92E activity in the hinge. However, ectopic JAK/STAT signaling does not perturb Wg expression in the hinge. We report negative interactions between Stat92E and the notum factor Araucan, resulting in restriction of JAK/STAT signaling from the notum. In addition, we find that the distal factor Nub represses the ligand unpaired as well as Stat92E activity. These data suggest that distal expansion of JAK/STAT signaling is deleterious to wing blade development. Indeed, mis-expression of Unpaired within the presumptive wing blade causes small, stunted adult wings. We conclude that JAK/STAT signaling is critical for hinge fate specification and growth of the gap domain and that its restriction to the hinge is required for proper wing development. [Copyright &y& Elsevier]

Intercellular communication depends on the correct organization of the signal transduction complexes. In many signalling pathways, the mechanisms controlling the overall cell polarity also localize components of these pathways to different domains of the plasma membrane. In the Drosophila ectoderm, the JAK/STAT pathway components are highly polarized with apical localization of the receptor, the associated kinase and the STAT92E protein itself. The apical localization of STAT92E is independent of the receptor complex and is due to its direct association with the apical determining protein Bazooka (Baz). Here, we find that Baz-STAT92E interaction depends on the presence of the Drosophila Src kinases. In the absence of Src, STAT92E cannot bind to Baz in cells or in whole embryos, and this correlates with an impairment of JAK/STAT signalling function. We believe that the requirement of Src proteins for STAT92E apical localization is mediated through Baz, as we can co-precipitate Src with Baz but not with STAT92E. This is the first time that a functional link between cell polarity, the JAK/STAT signalling pathway and the Src kinases has been established in a whole organism. [ABSTRACT FROM AUTHOR]

JAK-STAT signaling has been proposed to act in numerous stem cells in a variety of organisms. Here we provide an overview of its roles in three well characterized stem cell populations in Drosophila, in the intestine, lymph gland and testis. In flies, there is a single JAK and a single STAT, which has made the genetic dissection of pathway function considerably easier and facilitated the analysis of communication between stem cells, their niches and offspring. Studies in flies have revealed roles for this pathway as diverse as regulating bona fide intrinsic self-renewal, integrating response to environmental cues that control quiescence and promoting mitogenic responses to stress. [ABSTRACT FROM AUTHOR]

The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-γ- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans. [ABSTRACT FROM AUTHOR]

Abstract: JAK/STAT signalling in vertebrates is activated by multiple cytokines and growth factors. By contrast, the Drosophila genome encodes for only three related JAK/STAT ligands, Upd, Upd2 and Upd3. Identifying the differences between these three ligands will ultimately lead to a greater understanding of this disease-related signalling pathway and its roles in development. Here, we describe the analysis of the least well characterised of the Upd-like ligands, Upd3. We show that in tissue culture-based assays Upd3–GFP is secreted from cells and appears to interact with the extracellular matrix (ECM) in a similar manner to Upd, while still non-autonomously activating JAK/STAT signalling. Quantification of each of the Upd-like ligands in conditioned media has allowed us to determine the activity of equal amounts of each ligand on JAK/STAT ex vivo and reveals that Upd is the most potent ligand in this system. Finally, investigations into the effects of ectopic expression of Upd3 in vivo have confirmed its ability to activate pathway signalling at long-distance. [Copyright &y& Elsevier]

Abstract: The embryonic development of the tracheal system of the fruit fly Drosophila provides a paradigm for genetic studies of branching morphogenesis. Efforts of many laboratories have identified Branchless (Bnl, a fibroblast growth factor homologue) and Breathless (Btl, the receptor homologue) as crucial factors at many stages of tracheal system development. The downstream targets of the Bnl/Btl signalling cascade, however, remain mostly unknown. Misexpression of the bnl gene results in specific tracheal phenotypes that lead to larval death. We characterised the transcriptional profiles of targeted over-expression of bnl in the embryonic trachea and of loss-of-function bnl P1 mutant embryos. Gene expression data was mapped to high-throughput in situ hybridisation based ImaGO-annotation. Thus, we identified and confirmed by quantitative PCR 13 Bnl-dependent genes that are expressed in cells within and outside of the tracheal system. [Copyright &y& Elsevier]

Abstract: Specification of the terminal regions of the Drosophila embryo depends on the Torso RTK pathway, which triggers expression of the zygotic genes tailless and huckebein at the embryonic poles. However, it has been shown that the Torso signalling pathway does not directly activate expression of these zygotic genes; rather, it induces their expression by inactivating, at the embryonic poles, a uniformly distributed repressor activity. In particular, it has been shown that Torso signalling regulates accumulation of the Capicua transcriptional repressor: as a consequence of Torso signalling Capicua is downregulated specifically at the poles of blastoderm stage embryos. Extending the current model, we show that activation of the Torso pathway can trigger tailless expression without eliminating Capicua. In addition, analysis of gene activation by the Torso pathway and downregulation of Capicua unveil differences between the terminal and the central embryonic regions that are independent of Torso signalling, hitherto thought to be the only system responsible for confering terminal specificities. These data provide new insights into the mode of action of the Torso signalling pathway and on the events patterning the early Drosophila embryo. [Copyright &y& Elsevier]

The JAK/STAT signalling pathway mediates both antiproliferative responses following interferon stimulation and cellular proliferation in response to cytokines such as interleukins and growth factors. Central to these responses are the seven vertebrate STAT molecules, misregulation of which is implicated in a variety of malignancies. We have investigated the proliferative role of the single Drosophila STAT92E, part of the evolutionarily conserved JAK/STAT cascade. During second instar larval wing disc development pathway activity is both necessary and sufficient to promote proliferation of this epithelial cell type. However by later stages, endogenous STAT92E is stimulated by a noncannonical mechanism to exert pronounced antiproliferative effects. Ectopic canonical activation is sufficient to further decrease proliferation and leads to the premature arrest of cells in the G2?phase of the cell cycle. The single STAT92E present in Drosophila therefore mediates both proproliferative functions analogous to vertebrate interleukin-stimulated STAT3 and antiproliferative functions analogous to interferon-stimulated STAT1. Pro- and antiproliferative roles therefore represent ancestral activities conserved through evolution and subsequently assigned to distinct molecules.Oncogene (2005) 24, 2503-2511. doi:10.1038/sj.onc.1208487 Published online 7 February 2005 [ABSTRACT FROM AUTHOR]

The suppressor of cytokine signalling (SOCS) gene family was originally identified as an immediate early response to cytokine signalling and function as negative regulators of the Janus kinase (JAK)/signal tranducers and activators of transcription (STAT) signal transduction pathway [Krebs and Hilton, J. Cell Sci. 113 (2000) 2813; Starr and Hilton, Int. J. Biochem. Cell Biol. 30 (1998) 1081]. Although key components of the Drosophila JAK/STAT pathway have been identified [Brown et al., Curr. Biol. 11 (2001) 1700, reviewed in Zeidler et al., Oncogene 19 (2000) 2598], regulators of the pathway, and SOCS genes in particular, have not yet been characterised. Here we report the cloning of Drosophila SOCS36E and show its expression pattern during embryonic and imaginal disc development. SOCS36E is expressed in an essentially identical pattern to the Drosophila JAK/STAT pathway ligand unpaired (Upd). It is not expressed in upd mutant embryos and is upregulated in response to ectopic activation of the pathway during both embryonic and imaginal development. [Copyright &y& Elsevier]

The use of adult Drosophila melanogaster as a model for hematopoiesis or organismal immunity has been debated. Addressing this question, we identify an extensive reservoir of blood cells (hemocytes) at the respiratory epithelia (tracheal air sacs) of the thorax and head. Lineage tracing and functional analyses demonstrate that the majority of adult hemocytes are phagocytic macrophages (plasmatocytes) from the embryonic lineage that parallels vertebrate tissue macrophages. Surprisingly, we find no sign of adult hemocyte expansion. Instead, hemocytes play a role in relaying an innate immune response to the blood cell reservoir: through Imd signaling and the Jak/Stat pathway ligand Upd3, hemocytes act as sentinels of bacterial infection, inducing expression of the antimicrobial peptide Drosocin in respiratory epithelia and colocalizing fat body domains. Drosocin expression in turn promotes animal survival after infection. Our work identifies a multi-signal relay of organismal humoral immunity, establishing adult Drosophila as model for inter-organ immunity. • Adult Drosophila have an extensive blood cell reservoir at the respiratory epithelia • No sign of hematopoiesis in adult Drosophila , even after bacterial infection • Blood cells act as sentinels of infection via PGRP-LC/Imd/Upd3 signaling • In tissues of the reservoir, this induces Drosocin expression to promote survival Sanchez Bosch et al. study hemocytes (blood cells) in adult Drosophila. They do not find hematopoiesis in the adult system, but they do identify a major hemocyte reservoir at the respiratory epithelia and colocalizing fat body. Following bacterial infection, hemocytes relay signals to the tissues of the reservoir to mediate a protective humoral immune response. [ABSTRACT FROM AUTHOR]