GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo

JAK/STAT signaling is essential for a wide range of developmental processes in Drosophila melanogaster. The mechanism by which the JAK/STAT pathway contributes to these processes has been the subject of recent investigation. However, a reporter that reflects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stat92E target gene Socs36E, which contains at least two putative Stat92E binding sites, upstream of GFP, we generated three constructs that can be used to monitor JAK/STAT pathway activity in vivo. These constructs differ by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. We show that these reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reflect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP fluorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are specifically activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. In sum, we have generated in vivo GFP reporters that accurately reflect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila.     

JAK/STAT signaling promotes regional specification by negatively regulating wingless expression in Drosophila

During development, a small number of conserved signaling molecules regulate regional specification, in which uniform populations of cells acquire differences and ultimately give rise to distinct organs. In the Drosophila eye imaginal disc, Wingless (Wg) signaling defines the region that gives rise to head tissue. JAK/STAT signaling was thought to regulate growth of the eye disc but not pattern formation. However, we show that the JAK/STAT pathway plays an important role in patterning the eye disc: it promotes formation of the eye field through repression of the wg gene. Overexpression of the JAK/STAT activating ligand Unpaired in the eye leads to loss of wg expression and ectopic morphogenetic furrow initiation from the lateral margins. Conversely, tissue lacking stat92E, which cannot transduce JAK/STAT signals, is transformed from retinal tissue into head cuticle, a phenotype that is also observed with ectopic Wg signaling. Consistent with this, cells lacking stat92E exhibit ectopic wg expression. Conversely, wg is autonomously repressed in cells with hyperactivated Stat92E. Furthermore, we show that the JAK/STAT pathway regulates a small enhancer in the wg 3' cis genomic region. As this enhancer is devoid of Stat92E-binding elements, we conclude that Stat92E represses wg through another, as yet unidentified factor that is probably a direct target of Stat92E. Taken together, our study is the first to demonstrate a role for the JAK/STAT pathway in regional specification by acting antagonistically to wg.     

Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells

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.     

JAK/STAT signaling is required for hinge growth and patterning in the Drosophila wing disc

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.     

Differential requirement for STAT by gain-of-function and wild-type receptor tyrosine kinase Torso in Drosophila

Malignant transformation frequently involves aberrant signaling from receptor tyrosine kinases (RTKs). These receptors commonly activate Ras/Raf/MEK/MAPK signaling but when overactivated can also induce the JAK/STAT pathway, originally identified as the signaling cascade downstream of cytokine receptors. Inappropriate activation of STAT has been found in many human cancers. However, the contribution of the JAK/STAT pathway in RTK signaling remains unclear. We have investigated the requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) using a genetic approach in DROSOPHILA: Our results indicate that the JAK/STAT pathway plays little or no role in signaling by wild-type Tor. In contrast, we find that STAT, encoded by marelle (mrl; DStat92E), is essential for the gain-of-function mutant Tor (Tor(GOF)) to activate ectopic gene expression. Our findings indicate that the Ras/Raf/MEK/MAPK signaling pathway is sufficient to mediate the normal functions of wild-type RTK, whereas the effects of gain-of-function mutant RTK additionally require STAT activation.     

Cooperation of JAK/STAT and Notch signaling in the Drosophila foregut

Temporal and spatial regulation of morphogenesis is pivotal to the formation of organs from simple epithelial tubes. In a genetic screen for novel genes controlling cell movement during posterior foregut development, we have identified and molecularly characterized two alleles of the domeless gene which encodes the Drosophila Janus kinase (JAK)/STAT receptor. We demonstrate that mutants for domeless or any other known component of the canonical JAK/STAT signaling pathway display a failure of coordinated cell movement during the development of the proventriculus, a multiply folded organ which is formed by stereotyped cell rearrangements in the posterior foregut. Whereas the JAK/STAT receptor is expressed in all proventricular precursor cells, expression of upd encoding its ligand and of STAT92E, the signal transducer of the pathway, is locally restricted to cells that invaginate during proventriculus development. We demonstrate by analyzing gene expression mediated by a model Notch response element and by studying the expression of the Notch target gene short stop, which encodes a cytoskeletal crosslinker protein, that JAK/STAT signaling is required for the activation of Notch-dependent gene expression in the foregut. Our results provide strong evidence that JAK/STAT and Notch signaling cooperate in the regulation of target genes that control epithelial morphogenesis in the foregut.     

A novel functional activator of the Drosophila JAK/STAT pathway, unpaired2, is revealed by an in vivo reporter of pathway activation

Striking similarities continue to emerge between the mammalian and Drosophila JAK/STAT signaling pathway. However, until now there has not been the ability to monitor global pathway activity during development. We have generated a transgenic animal with a JAK/STAT responsive reporter gene that can be used to monitor pathway activation in whole Drosophila embryos. Expression of the lacZ reporter regulated by STAT92E binding sites can be detected throughout embryogenesis, and is responsive to the Janus Kinase hopscotch and the ligand upd. The system has enabled us to identify the effect of a predicted gene related to upd, designated upd2, whose expression initiates during germ band extension. The stimulatory effect of upd2 on the JAK/STAT reporter can also be demonstrated in Drosophila tissue culture cells. This reporter system will benefit future investigations of JAK/STAT signaling modulators both in whole animals and tissue culture.     

Patterns and functions of STAT activation during Drosophila embryogenesis

The JAK/STAT pathway mediates cytokine signaling in mammals and is involved in the function and development of the hematopoietic and immune systems. To investigate the biological functions of the JAK/STAT pathway during Drosophila development, we examined the tissue-specific localization of the tyrosine-phosphorylated, or activated form of Drosophila STAT, STAT92E. Here we show that during Drosophila embryonic development STAT92E activation is prominently detected in multiple tissues and in different developmental stages. These tissues include the tracheal pits, elongating intestinal tracks, and growing axons. We demonstrate that stat92E mutants are defective in tracheal formation, hindgut elongation, and nervous system development. Conversely, STAT92E overactivation caused premature development of the tracheal and nervous systems, and over-elongation of the hindgut. These results suggest that STAT activation is involved in proper differentiation and morphogenesis of multiple tissues during Drosophila embryogenesis.     

Cyclin D-Cdk4 and cyclin E-Cdk2 regulate the Jak/STAT signal transduction pathway in Drosophila

The JAK/STAT signal transduction pathway regulates many developmental processes in Drosophila. However, the functional mechanism of this pathway is poorly understood. In this report, we identify the Drosophila cyclin-dependent kinase 4 (Cdk4), which exhibits embryonic mutant phenotypes identical to those in the Hopscotch/JAK kinase and stat92E/STAT mutations. Specific genetic interactions between Cdk4 and hop mutations suggest that Cdk4 functions downstream of the HOP tyrosine kinase. We further show that Cyclin D-Cdk4 (as well as Cyclin E-Cdk2) binds and regulates STAT92E protein stability. STAT92E regulates gene expression for various biological processes, including the endocycle S phase. These data suggest that Cyclin D-Cdk4 and Cyclin E-Cdk2 play more versatile roles in Drosophila development.