STAT/marelle


REGULATION

Promoter Structure

A presumptive transcription start site or TATA box is found in the genomic DNA about 180 bp 5' to the start of the cDNA (Hou, 1996).

Transcriptional Regulation

To explore the function of domeless/mom in activating the Hop/Stat92E signal transduction pathway, protein levels and distributions of Stat92E were compared in wild-type embryos and mutant embryos of Upd, mom, and hop genes. Embryos were stained using affinity-purified anti-Stat92E antibodies. Although strong Stat92E expression is detected as 15 clear stripes during stage 9 in the wild-type embryo, Stat92E expression is dramatically reduced in Upd, mom, and hop mutant embryos. As in wild-type embryos, the remaining Stat92E protein in mutant embryos is localized in both the nucleus and cytoplasm. These data suggest that Mom and the Upd/Mom/Hop signaling pathway regulate Stat92E protein expression (Chen, 2002).

Previously unrecognized mRNAs originating from a dual promoter at the stat92E locus are described. One of these encodes a truncated protein, DeltaNSTAT92E, that lacks the N-terminal 133 amino acids. Antibodies detect both the full-length and truncated molecules early in embryogenesis (1-5 h), and mRNA detection by specific RT-PCR reactions accords with the protein distribution. Given that the N termini of mammalian STATs are known to have positive functions in transcriptional activation, the role of DeltaNSTAT92E has been explored early in embryogenesis. The regulation of transcription from the two different promoters allows for controlling the effects of the full-length STAT protein. Use of only promoter 1 produces only full-length protein. Transcription from promoter 1a produces both full-length STAT92E and DeltaNSTAT92E protein. Furthermore, in processing primary transcripts from promoter 1a, a choice of splice sites to include or exclude Exon 2 can occur. By increasing the DeltaNSTAT92E-to-STAT92E ratio in overexpression and RNAi experiments, phenotypes compatible with suppression of wild-type STAT92E activity are reported. It was therefore concluded that the short form of STAT92E is a naturally occurring dominant-negative product that can be added to the growing list of negative regulators of STAT activity (Henriksen, 2002).

To determine whether the steady-state concentration of mRNAs transcribed from the two stat92E promoters changes throughout development, Northern blots were performed on RNA extracted from different stages. A probe (Exons 3 and 4) that reacted with all mRNAs showed a large amount of maternal stat92E mRNA and decreasing amounts of mRNA later in embryogenesis, with stronger expression returning in third-instar larvae and adults. This same picture was mirrored in the mRNA that arises from promoter 1a. The later embryonic increase was not as prominent in mRNA arising from promoter 1. Quantitative RT-PCR to distinguish promoter 1a mRNAs encoding full-length or shorter protein revealed that the DeltaNstat92E mRNA is largely responsible for the strong signal in larval and adult samples. These results are consistent with the protein expression, where DeltaNSTAT92E predominates in first-instar larvae. Furthermore, there exists a regulated alternative splicing mechanism for transcripts arising from promoter 1a, opening the possibility for different functions for the full-length STAT92E protein compared with the shorter DeltaNSTAT92E protein (Henriksen, 2002).

The developmental effects of changing the ratio of STAT92E to DeltaNSTAT92E in embryogenesis were tested by overexpressing DeltaNSTAT92E. The pair-rule gene even-skipped (eve) is expressed in seven stripes along the anterior/posterior axis of the 2-3-hour-old embryo. The expression pattern is under the control of modular enhancers that integrate spatial information of localized transcription factors to produce the seven-stripe pattern. For instance, in loss-of-function (LOF) mutants of stat92E and hopscotch (the Drosophila JAK), eve stripes, most commonly stripes 3, 5, and 7, are variably reduced, whereas stripes 1 and 2 are usually unaffected. Activation of stripe 3 and 7 is the direct consequence of STAT92E binding to two neighboring STAT DNA-binding sites contained in the eve stripe 3 + 7 enhancer (3.5 kb upstream of the transcription start site). This was shown by point mutagenesis and reporter gene assays in embryos. Therefore, if DeltaNSTAT92E had a dominant-negative function, overexpression would be expected to suppress production of Eve protein at STAT92E-dependent stripes but not at other eve stripes (Henriksen, 2002).

The binary UAS-Gal4 expression system was used for conditional overexpression of DeltaNSTAT92E early in embryonal development. It was found that EVE production of stripes 3, 5, and 7 is variably reduced in 20%-40% of the embryos overexpressing DeltaNSTAT92E. Control embryos with only the NGT-GAL4 or embryos overexpressing STAT92E display a wild-type seven-stripe pattern. In addition, stat92E397 embryos show reduction of stripe 3 and poor spreading so that stripe count was not possible. Taken together, these experiments suggest that DeltaNSTAT92E can suppress transcriptional activation by STAT92E in vivo (Henriksen, 2002).

To remove specific forms of STAT92E for comparison with the overexpression results, RNAi experiments involving dsSTAT-encoding sequences were performed. Prior to injecting dsRNAs (double-strand RNAs) corresponding to Exon 2 or Exons 3 and 4 into embryos, their efficacy in decreasing various stat92E mRNAs was characterized by RT-PCR in S2 cells in culture. dsRNA corresponding to Exons 3 and 4 reduced all stat92E mRNA and proteins, whereas dsRNA corresponding to Exon 2 specifically reduced the DeltaNstat92E mRNA and protein with equal efficacy. Unfortunately, there is no exon combination that would leave stat92E mRNA unaffected and remove only the DeltaNstat92E mRNA. The JAK-STAT pathway not only affects stripe formation but also embryonic segmentation, and defects in segmentation were used to score the effect of the injection of dsRNAs. Loss of maternal hopscotch, domeless (the pathway receptor), or stat92E activity often results in the deletion of the fourth and fifth abdominal segments, observed in cuticle preparations. Null embryos lacking both maternal and paternal contributions from these genes can have additional defects in the head and tail region. When embryos (0-1 h) were injected with ds-Exon3/4 RNA, which should lower the concentration of all possible stat92E mRNA, 7 of 103 surviving larvae had a loss and/or fusion of abdominal segments four and five. No defects in head or tail segments were seen. Other embryos were injected with ds-Exon2 RNA, which would not affect the amount of DeltaNstat92E mRNA but would decrease all the mRNA encoding full-length protein regardless of which promoter was used. In the surviving larvae, 16 of 153 had fused segments four and five, and defects in the head and tail were also observed in another 12 of the 153. Upon translation of the maternally deposited mRNA (plus any new mRNA synthesized very early in embryogenesis), these latter injected embryos would have a higher ratio of DeltaNSTAT92E protein compared with full-length protein. That the more severe defective phenotype is seen in embryos where the ratio of DeltaNSTAT92E-protein-to-full-length protein was increased shows that DeltaNSTAT92E acts negatively. In contrast, when all the stat92E mRNAs are depleted, only the less severe defect is seen (Henriksen, 2002).

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. A transgenic animal has been generated 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 identification of 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 (Gilbert, 2005).

A systematic analysis of Tinman function reveals Eya and JAK-STAT signaling as essential regulators of muscle development

Nk-2 proteins are essential developmental regulators from flies to humans. In Drosophila, the family member tinman is the major regulator of cell fate within the dorsal mesoderm, including heart, visceral, and dorsal somatic muscle. To decipher Tinman's direct regulatory role, a time course of ChIP-on-chip experiments was performed, revealing a more prominent role in somatic muscle specification than previously anticipated. Through the combination of transgenic enhancer-reporter assays, colocalization studies, and phenotypic analyses, two additional factors within this myogenic network were uncovered: by activating eyes absent, Tinman's regulatory network extends beyond developmental stages and tissues where it is expressed; by regulating stat92E expression, Tinman modulates the transcriptional readout of JAK/STAT signaling. This pathway is essential for somatic muscle development in Drosophila and for myotome morphogenesis in zebrafish. Taken together, these data uncover a conserved requirement for JAK/STAT signaling and an important component of the transcriptional network driving myogenesis (Liu, 2009).

This article presents a global map of the genomic regions bound by Tinman during multiple stages of mesoderm development, together with functional studies on the direct regulation and phenotypes of selected target genes. This analysis revealed several important features of Tinman's regulatory network. Although Tinman is primarily associated with its conserved role in heart development, many more target genes involved in somatic muscle development were identified. This implies that a major component of Tinman function is to orchestrate the transcriptional network driving early events in this tissue, including myoblast specification. In this context, Tinman's regulatory network is not only active in the dorsal mesoderm; the results pinpoint multiple nodes by which Tinman's regulatory influence extends to lateral and ventral regions of the embryo. First, Tinman directly regulates a number of identity genes essential for lateral and ventral muscle specification. For example, Tinman targets enhancers of slouch, which specifies two ventral muscle fibers (VT1 and VA3), apterous, which specifies three lateral and two ventral muscle fibers (LT1, LT2, LT3, and VA2, VA3), and ladybird (lbe, lbl), which specifies the lateral muscle fiber, SBM. Second, Tinman influences lateral and ventral muscle formation through the regulation of additional transcriptional cascades. A good example is eya, which is an integral component of the Tinman-regulatory network and is essential for the specification of somatic muscle in dorsal, lateral, and ventral regions of the embryo. Similarly, the data suggest that Tinman also directly regulates D-six4 and pox meso expression, two TFs essential for lateral and ventral somatic muscle development. In this manner, Tinman contributes to the general robustness of muscle specification, regardless of the muscle's position along the dorsal-ventral axis (Liu, 2009).

While examining the eya locus, an enhancer was identifed that fully recapitulates the spatiotemporal expression of eya within the mesoderm. This element is occupied by Tinman in vivo and requires tinman function for activity, indicating that Tinman provides direct input into eya expression via this eya-meso enhancer. Despite the strong dependence of the enhancer on tinman function, the expression of the endogenous eya gene is only marginally reduced in tinman mutant embryos. This result indicates a requirement for additional enhancers to regulate high-levels of eya expression within the mesoderm, one of which is most likely regulated by Twist. The expression of the stat92E gene in mesoderm is regulated in a similar manner. These types of regulatory connections (acting partially redundantly or for fine tuning), are often masked in genetics studies, yet serve as important inputs in generating robust regulatory networks (Liu, 2009).

The molecular nature of the ChIP-on-chip approach also uncovered a link between JAK/STAT signaling and muscle development, which was most likely masked in genetics studies due to the pleiotropic function of this pathway. Given the diverse cellular responses to this signaling cascade, including proliferation, apoptosis, and differentiation, the response of Stat activation in the context of myoblasts is currently not clear. A recent study on stat1 in C2C12 cells, a tissue culture model for myogenesis, suggests a potential role in proliferation. While this could partially explain the Drosophila muscle phenotype, it does not readily explain the defects in myotome boundary formation observed in zebrafish (Liu, 2009).

Although there are clear parallels between the role of eya-six genes and the JAK/stat pathway between flies and vertebrates, it is interesting to note that the positions of these genes within the overall myogenic network have diverged significantly. In vertebrates, Pax-Eya-Six act at the top of the transcriptional hierarchy, and are thus involved in the initiation of the myogenic network. In contrast, this regulatory module appears to function further down in the transcriptional hierarchy in flies. As a consequence, the upstream regulators of Eya-Six expression are not conserved. Similarly, as Nkx2 genes are not expressed in somites, stat5.1 cannot be regulated by these TFs in vertebrates, but is rather more likely to be regulated by members of the myogenic bHLH proteins, such as Myf5. Therefore, while the requirement of these key regulators is conserved, the wiring of these nodes within the overall network is highly diverged (Liu, 2009).

In summary, the systematic nature of this approach has revealed important regulators of myogenesis and partially redundant regulatory connections, both of which are often very difficult to uncover using standard genetic approaches (Liu, 2009).

Drosophila cytokine Unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion

In Drosophila, the fat body (FB), a functional analog of the vertebrate adipose tissue, is the nutrient sensor that conveys the nutrient status to the insulin-producing cells (IPCs) in the fly brain to release Drosophila insulin-like peptides (Dilps). Dilp secretion in turn regulates energy balance and promotes systemic growth. This study identified Unpaired 2 (Upd2), a protein with similarities to type I cytokines, as a secreted factor produced by the FB in the fed state. When upd2 function is perturbed specifically in the FB, it results in a systemic reduction in growth and alters energy metabolism. Upd2 activates JAK/STAT signaling in a population of GABAergic neurons that project onto the IPCs. This activation relieves the inhibitory tone of the GABAergic neurons on the IPCs, resulting in the secretion of Dilps. Strikingly, it was found that human Leptin can rescue the upd2 mutant phenotypes, suggesting that Upd2 is the functional homolog of Leptin (Rajan, 2012).

Previous studies have postulated the existence of secreted factors, produced by the FB, that stimulate systemic growth by stimulating cell proliferation and that the FB - the fly nutrient sensor - couples Dilp secretion from the brain IPCs depending on the nutritional status. This study shows that the JAK/STAT ligand Upd2, a type 1 cytokine signal, is involved in the inter-organ communication between the FB and the brain IPCs. Human Leptin can rescue the upd2 mutant phenotypes, implying that an invertebrate model system is suited to address questions pertaining to Leptin biology. (Rajan, 2012).

Upd proteins have secondary structures predicted to have α-helices similar to that of type I cytokines belonging to the IL-6 family, and sequence alignments suggest that they show some similarity to vertebrate Leptins. Among the three Upd ligands that activate the Dome receptor, only Upd2 plays a significant role in communicating the nutritional status from the FB. This is somewhat surprising as all three Upd proteins are secreted JAK/STAT pathway agonists and are able to activate the JAK/STAT pathway non-autonomously in-vivo. However, the signal sequences of the different Upds appear to confer them with different biophysical properties, as illustrated by tissue culture assays showing that, while Upd1 and Upd3 associate primarily with the extracellular matrix, Upd2 is easily detectable in the media. In addition, secretion assays showed that Upd2 is able to condition tissue culture media more potently than either Upd1 or Upd3. Altogether, these results suggest that Upd2 activates JAK/STAT signaling at greater distances than Upd1 or Upd3 (Rajan, 2012).

As evidenced by the growth and metabolic phenotypes of FB-specific knockdown, Upd2 seems to be required only in the FB but the reason for this tissue specificity is unclear. A previous study, which analyzed the Upd2 protein using a hidden Markov model, suggested that Upd2 is probably not secreted via the 'classical' Golgi-ER machinery because it lacks a signal peptide. In fact, other type I cytokines involved in inter-organ cross-talk also lack the signal peptide and are secreted by unconventional secretory pathways. Thus, one possible reason for the tissue specificity of Upd2 could be that the FB is the only tissue that can secrete this protein in an active form. Future work, contingent on the development of techniques and reagents to detect Upd2 in the fly hemolymph, will clarify this issue (Rajan, 2012).

The identification of Upd2 as a nutrient regulated signal from the FB that does not depend on AAs but is produced in response to dietary fats and sugars reveals that different nutrient-specific secreted factors exist in the fly. Interestingly, the upregulation of upd2 levels in FB knockdown of slif suggests the existence of a homeostatic feedback loop whereby Upd2, in the context of low protein, promotes utilization of fat and carbohydrate energy sources. High sugar diets in flies have been shown to trigger a lipogenesis program akin to high fat diets in mammals, suggesting that Upd2 is most likely downstream of signals that are produced by increased fat stores. This is a highly significant finding given that it questions a broadly prevailing view that one dominant secreted factor downstream of AAs governs nearly all aspects of systemic growth and metabolism in flies. The findings support the model that the fly FB secretes numerous factors that regulate systemic growth and metabolism downstream of various components of the fly diet (Rajan, 2012).

The results indicate that STAT activation in GABAergic neurons inhibits their firing. Previous work has implied that the GABA-B-receptors in Dilp neurons inhibit Dilp release. Given that these GABAergic neurons are pre-synaptic to the IPCs, it is proposed that activation of STAT in GABAergic neurons relieves the IPCs from repression, thus resulting in Dilp release. This is reminiscent to the observation that first order-neurons responding to adipose-derived Leptin are the inhibitory GABAergic neurons expressing LepRs. When LepRs are activated by Leptin they regulate Stat3 phosphorylation which, by an unknown mechanism, inhibits the firing of the GABAergic neurons. This in turn relieves the repression on a neuronal group called POMC (propiomelanocortin) neurons allowing them to fire. Thus, this circuit module is strikingly reminiscent to what is observe in the fly. (Rajan, 2012).

There are many outstanding questions yet to be resolved regarding the signaling mechanisms by which the JAK/STAT pathway regulates GABAergic neurons. The target(s) of the JAK/STAT pathway in regulating neuronal firing in mammalian GABAergic neurons remains to be identified. It has been suggested that Leptin activation of STAT signaling may be required for the long-term effects of Leptin’s action on energy homeostasis rather than for acute effects of Leptin, and that the acute effects of Leptin on the membrane potential of certain neuronal groups require activation of PI3-K signaling rather than STAT. However, the role of JAK/STAT versus PI3-K signaling in modulating the electrophysiology of the presynaptic GABAergic neurons is yet to be clarified , especially as previous studies were done on non-GABAergic neuronal groups. Altogether, further investigations into the role of JAK/STAT signaling in modulating neurotransmission in GABAergic neurons will be necessary to clarify how JAK/STAT signaling regulates their activities. Importantly, based on the similarity of the circuits and the conservation of the signaling pathways, studies in the fly are likely to provide insights relevant to mammalian neurophysiology. (Rajan, 2012).

The physiology of Leptin signaling in vertebrates is undoubtedly more complex and different from the physiology of flies. upd2- mutant flies are smaller and leaner whereas mutations in Leptin in mammals are associated with obesity. There is however some striking parallels. It was found that under starvation upd2 mRNA steady-state levels drop significantly, and there is a significant increase of upd2 mRNA expression under high fat diets. This is similar to the alteration in Leptin levels during starvation and high fat diets. Examination of the role of Leptin in the physiology of starvation, by providing mice with exogenous Leptin during periods of nutrient restriction, revealed that the primary physiological role of Leptin is to regulate the neuroendocrine system during starvation. Leptin reduced the reproductive capacity and increased stress hormone levels, which in turn increases the survival capacity of the organism under adverse nutrient conditions. Consistent with this, flies with ablated IPCs, which are unable to produce insulin, perform much better under starvation conditions and increased stress conditions. Given that the role of Upd2 is to promote insulin secretion, the reduction of Upd2 levels during starvation decreases Dilp secretion and increases the chances of survival under starvation (upd2- mutants are more starvation resistant than the wild-type controls). Hence, in this context, the primary physiological role of Upd2 and Leptin converge (Rajan, 2012).

The sterile 20-like kinase tao controls tissue homeostasis by regulating the hippo pathway in Drosophila adult midgut

The proliferation and differentiation of adult stem cells must be tightly controlled in order to maintain resident tissue homeostasis. Dysfunction of stem cells is implicated in many human diseases, including cancer. However, the regulation of stem cell proliferation and differentiation is not fully understood. This study shows that the sterile-like 20 kinase, Tao, controls tissue homeostasis by regulating the Hippo pathway in the Drosophila adult midgut. Depletion of Tao in the progenitors leads to rapid intestinal stem cell (ISC) proliferation and midgut homeostasis loss. Meanwhile, it was find that the STAT signaling activity and cytokine production are significantly increased, resulting in stimulated ISC proliferation. Furthermore, expression of the Hippo pathway downstream targets, Diap1 and bantam, is dramatically increased in Tao knockdown intestines. Consistently, it was shown that the Yorkie (Yki) acts downstream of Tao to regulate ISC proliferation. Together, these results provide insights into understanding of the mechanisms of stem cell proliferation and tissue homeostasis control (Huang, 2014).

Targets of Activity

The dyad symmetric sequence TTCnnnGAA affords maximum MRL binding to DNA (Yan, 1996a).

With even-skipped stripe 3, the anterior borders are defined through repression by Hunchback and Knirps. mrl mutants show abnormal expression of runt and eve. The fifth stripe of run is lost and the third and fifth stripes of eve are diminished. A reporter gene containing the eve stripe 3 promoter is not expressed in mrl mutants. Similar effects are also observed for hopscotch mutants (Hou, 1996). Two separate sequences within the eve stripe 3 promoter, each containing the TTCN3CAA motif, bind to activated MRL (Yan, 1996a).

An single enhancer sequence consisting of 500bp mapping 3.3kb upstream of the transciption start site is sufficient to direct eve expression in both stripes 3 and 7. There are 5 KNI binding sites in the 3 + 7 enhancer and 11 HB sites. HB and KNI act as repressors of stripe 3 expression, while the JAK kinase HOP, acting through Marelle, is involved in activation, with the KNI and HB sites closely linked to two STAT binding sites. A model is presented in which the repressors provide short term quenching of widespread STAT activation (Small, 1996)

The determination of sexual identity in Drosophila depends upon a system that measures the X chromosome to autosome ratio (X/A). This system relies upon the unequal expression of X-linked numerator genes in 1X and 2X nuclei. The numerators activate a special Sex lethal promoter, Sxl-Pe, in 2X/2A nuclei, but not 1X/2A nuclei. By multimerizing a conserved Sxl-Pe sequence block, a gain-of-function promoter, Sxl-PeGOF, is generated that is inappropriately active in 1X/2A nuclei. GOF activity requires the X-linked unpaired (upd) gene, which encodes a ligand for the Drosophila JAK/STAT signaling pathway. upd also functions as a numerator element in regulating wild-type Sxl-Pe reporters. The JAK kinase, Hopscotch, and the STAT DNA-binding protein, Marelle, are also required for Sxl-Pe activation (Jinks, 2000).

The numerators most important for turning on Sxl are sis-a and sis-b (scute). They are expressed throughout the embryo, and mutations in both can have quite pronounced effects on Sxl-Pe activity. However, neither of these numerators is critical for the gain-of-function activity of the Sxl-PeGOF promoter. Instead, the two numerators that contribute most to Sxl-PeGOF activity are the segmentation genes runt and upd. At the syncytial blastoderm stage, run is expressed in a broad central domain, and it is in this region that Sxl activation is defective in 2X/2A run mutants. Except for a dorsal crescent in the head, the upd expression domain closely coincides with that of run. It is in this same central run-upd domain that the highest levels of Sxl-PeGOF promoter activity are observed. Moreover, in both run and upd mutant males, Sxl- PeGOF promoter activity is severely impaired. From these findings, it can be inferred that the multimerized 72 bp fragment contains cis-acting targets for run and upd action (Jinks, 2000).

Since Upd is a secreted ligand, it is unlikely that it interacts directly with sequences in the 72 bp fragment. Instead, the data suggests that Upd acts by turning on a Drosophila JAK/STAT signaling cascade consisting of the Hop protein kinase and the Mrl transcription factor. In this model, the extracellular Upd ligand would activate the Drosophila JAK protein Hop. Hop would in turn phosphorylate the D-STAT homolog Mrl, which would then enter the nucleus and activate Sxl-Pe. That the Mrl protein is critical for the activity of Sxl-PeGOF is demonstrated by the dramatic reduction in beta-galactosidase expression seen in both 1X/2A and 2X/2A embryos derived from homozygous mrl- germline clones (Jinks, 2000).

The 72 bp fragment has a sequence that closely matches the consensus D-STAT-binding site. Hence, a plausible hypothesis is that Sxl-PeGOF is activated in 1X and 2X embryos by the binding of multiple Mrl proteins to the reiterated STAT sites in the multimerized fragment. Since there are also potential target sites for Runt in the 72 bp fragment, it is possible that Runt and Mrl collaborate in promoter activation. There are precedents in mammals for synergistic interactions between STAT and other transcription factors. Although a definitive answer will require further study, it is interesting that Sxl-PeGOF is not activated in male embryos in the dorsal crescent region of the head where upd but not run is expressed (Jinks, 2000).

Since Sxl-PeGOF has regulatory properties not seen in other Sxl-Pe promoter constructs, an obvious question is whether the JAK/STAT signaling pathway is a part of the normal X/A counting system. Several lines of evidence suggest that it is. (1) Genetic studies indicate that the upd gene is an X chromosome-counting element. Deletions that remove upd show female lethal interactions with mutations in the numerator genes sis-a and sis-b, and with Sxl. (2) As would be expected for an X chromosome-counting element, deletion of upd in females heterozygous for either sis-a or sis-b compromises the activity of wild-type Sxl-Pe reporter constructs. (3) The gain-of-function hopTum allele enhances the activity of the Sxl-Pe promoter in 2X/2A embryos. Moreover, consistent with the idea that a target site for the JAK/STAT signaling pathway is contained in the multimerized 72 bp fragment, the minimal Sxl-Pe0.4kb promoter (from which the 72 bp fragment is derived) is activated by the hopTum-1 mutation. (4) The Sxl autoregulatory feedback loop is not properly turned on in 2X/2A embryos when the maternally derived mrl gene product is absent. The observed defects in SXL protein expression are regional and for the most part overlap with the domain in which the JAK/STAT signaling pathway would be activated by upd expression. (5) The failure to properly activate the Sxl autoregulatory feedback loop in the absence of maternal mrl appears to be due to a marked reduction in Sxl-Pe activity. For the full-length promoter construct, Sxl-Pe3.0kb, beta-galactosidase expression is almost completely eliminated except in the very anterior of the embryo. In this context, it should be noted that Sxl-Pe contains two consensus STAT/Mrl-binding sites, in addition to the one found in the minimal 0.4 kb promoter. Conceivably these two upstream sites could provide additional targets for Mrl binding and promoter activation in vivo (Jinks, 2000).

The gene encoding the JAK/STAT ligand, upd, is required in the zygote to activate Sxl-Pe. Hence, like other numerators, it is the dose of the upd gene product produced in 1X and 2X embryos that is critical to the X chromosome-counting mechanism. The JAK kinase, hop, and the STAT transcription factor, mrl, have a different function in the counting process. These experiments show that the mrl gene is required in the mother's germline, not in the zygote. The available evidence suggests that this is also true for the X-linked hop gene. Since the products of these two genes would be deposited in constant amounts in the egg during oogenesis, they correspond to signal transduction elements like da. While the findings indicate that the JAK/STAT pathway plays an important role in the choice of sexual identity, the effects of mutations in the pathway do not seem to be as great as those observed for mutations in other components of the X/A counting system. For example, mutations that disrupt the maternal deposition of DA essentially eliminate both Sxl-Pe activity and SXL protein expression in female embryos. By contrast, when maternal mrl is removed, Sxl-Pe is not completely turned off, and SXL protein expression can still be detected, particularly in the termini. This suggests that the JAK/STAT pathway plays a secondary rather than a primary role in X chromosome counting (Jinks, 2000).

It is now clear that transcription factors involved in many different aspects of development, from segmentation to neurogenesis, have been coopted by the sex determination system in Drosophila. These genes generally have cell-autonomous activities and, consequently, are readily adaptable to a process that requires counting the number of chromosomes in each nucleus. Hence, it is somewhat surprising that a JAK/STAT signaling pathway, which depends upon the production and reception of an extracellular ligand, has also been incorporated into the counting system. Moreover, the apparent ligand, upd, corresponds to the X chromosome-counting element. Since Upd is secreted, it could potentially influence the counting process not only in the nucleus that produced the protein to begin with but also in adjacent nuclei. Supporting this possibility, it has been found that upd mutant cells can generate a normal pattern when adjacent to wild-type cells. Except under special circumstances (e.g., in gynandermorphs where 1X and 2X cells are in close proximity), counting elements that function nonautonomously need not have detrimental consequences and might even offer some advantages. For example, the signaling cascade may respond in a nonlinear fashion to variations in the dose of the ligand. In this case, the JAK/STAT pathway may provide a mechanism for magnifying the initial 2-fold difference in the amount of ligand produced in 1X/2A versus 2X/2A nuclei. In addition, signaling between adjacent nuclei might compensate for stochastic differences in numerator expression and might further amplify the signal by a relay mechanism (Jinks, 2000).

Metazoans use diverse and rapidly evolving mechanisms to determine sex. In Drosophila an X-chromosome-counting mechanism determines the sex of an individual by regulating the master switch gene, Sex-lethal (Sxl). The X-chromosome dose is communicated to Sxl by a set of X-linked signal elements (XSEs), which activate transcription of Sxl through its 'establishment' promoter, SxlPe. A new XSE called sisterlessC (sisC) is described whose mode of action differs from that of previously characterized XSEs, all of which encode transcription factors that activate Sxl Pe directly. In contrast, sisC encodes a secreted ligand for the Drosophila Janus kinase (JAK) and 'signal transducer and activator of transcription' (STAT) signal transduction pathway and is allelic to outstretched (os, also called unpaired). sisC works indirectly on Sxl through this signaling pathway because mutations in sisC or in the genes encoding Drosophila JAK or STAT reduce expression of SxlPe similarly. The involvement of os in sex determination confirms that secreted ligands can function in cell-autonomous processes. Unlike sex signals for other organisms, sisC has acquired its sex-specific function while maintaining non-sex-specific roles in development, a characteristic that it shares with all other Drosophila XSEs (Sefton, 2000).

If os acts on SxlPe indirectly through effects on Drosophila JAK (encoded by hopscotch [hop]) and on Drosophila STAT (encoded by Stat92E), then the effect on Sxl Pe of eliminating either hop or Stat92E should be the same as eliminating os. This prediction was confirmed. Because only maternal rather than zygotic hop and Stat92E are likely to be relevant at the very early embryonic stage when SxlPe is activated, the maternal contribution of these two genes was eliminated by inducing homozygous mutant germline clones in mothers heterozygous for null alleles. Expression of SxlPe:lacZ in these experimentals was compared with that for control embryos derived from hop-/+ and Stat92E-/+ germ cells. Loss of maternal hop+ does not eliminate Sxl Pe expression, but expression is substantially reduced: although 49% of the experimental embryos expressed SxlPe:lacZ , essentially identical to the 50% figure for the controls, 32% of the experimental embryos were in the intermediate staining class compared with only 6% for the controls. The reduction was generally more uniform across the embryos than in the os experiment. Similar results were seen for Stat92E. Sixteen per cent of controls stained in the intermediate range, compared with 45% for the experimentals; thus, SxlPe expression was clearly reduced. Curiously, the fraction of experimental embryos staining above background is greater than 50%, suggesting that although loss of maternal Stat92E decreases SxlPe expression in females, it might also increase SxlPe expression in males. Alternatively, this increase might be due to effects on the lacZ enhancer trap present in Stat92E6346. The observation that Drosophila STAT is a regulator of SxlPe is consistent with the finding of STAT binding sites (TTCNNNGAA) 253, 393 and 428 bp upstream of the SxlPe transcription start site. The tandem arrangement of these sites in Sxl would facilitate the kind of cooperative binding of STAT dimers shown to be important in some systems (Sefton, 2000).

With the discovery of sisC, the collection of fly XSEs may be nearly complete. The impression given by this collection is that Drosophila relies on biochemically diverse proteins to assess X-chromosome dose, but they all act on Sxl at the level of transcription. In contrast, the XSEs of Caenorhabditis elegans include both transcriptional and post-transcriptional regulators of their target, xol-1. Characterization of sisC reveals that both C. elegans and Drosophila XSEs seem to include proteins that work extracellularly (Sefton, 2000).

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. The requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) has been investigated using a genetic approach in Drosophila. The JAK/STAT pathway plays little or no role in signaling by wild-type Tor. STAT, encoded by marelle (mrl; Stat92E), is essential for the gain-of-function mutant Tor (TorGOF) to activate ectopic gene expression. These 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 (Li, 2002).

How does TorGOF RTK activate Mrl? There are at least three possible mechanisms through which STAT activation by RTK could occur. RTK could directly bind and activate STAT proteins. Alternatively, STAT could be indirectly activated by the RTK, either via JAK or MAPK. Genetic evidence allows ruling out the possibilities that TorGOF activates Mrl via JAK or MAPK. (1) Whether or not removal of Hop activity modifies the torGOF phenotype was examined. Surprisingly, a hop null mutation does not suppress torGOF, indicating that unlike Mrl, Hop is not required for ectopic tll expression. (2) Removal of mrl does not suppress rlSevenmaker (rlSem), which encodes a GOF mutant form of Drosophila MAPK, suggesting that Mrl is not essential for the effects of GOF mutation in MAPK. To test for a physical interaction between Mrl and TorGOF, Tor was immunoprecipitated from wild-type and torGOF embryos, respectively, with anti-Tor antibody, and the presence of Mrl was examined in the immune complexes. A specific band corresponding to Mrl was detected in the immunoprecipitates. The Tor-Mrl association, however, is only observed in the presence of vanadate (a general tyrosine phosphatase inhibitor), suggesting that this interaction takes place only when the cytoplasmic protein phosphorylation status is preserved, or when Tor and/or Mrl have been activated by the presence of vanadate. Altogether, these results are consistent with a direct activation of Mrl by TorGOF, possibly following recruitment of Mrl to phosphotyrosine residues on the Tor RTK via SH2-phosphotyrosine peptide interaction (Li, 2002).

Since Mrl activation is required for ectopic tll expression induced by TorGOF, whether Mrl-binding sites (TTCNNNGAA) are present in the regulatory region of the tll gene was examined. A search in the tll regulatory region revealed two putative Mrl-binding sites with the consensus TTCNNNGAA located at –2357 (site 1) and –2462 (site 2) upstream of the tll transcription start site. These two sites are able to bind Mrl, although site 2 shows a much lower affinity. Interestingly, the two Mrl sites are located 105 bp apart in the tll regulatory region. This configuration is reminiscent of that existing in the eve stripe 3 enhancer, where cooperative binding of two Mrl homodimers has been demonstrated. To assess the functional relevance of the two Mrl sites in tll expression, transgenes containing the 5.9 kb regulatory fragment upstream of the tll transcription start site fused to the lacZ gene were introduced into flies. This 5.9 kb fragment had been shown previously to drive lacZ expression in a pattern almost identical to that of the endogenous tll gene. Accordingly, lacZ expression is greatly expanded in a torGOF background. A 5.9 kb fragment with the two Mrl binding sites mutated, showed wild-type activity for lacZ expression in wild-type embryos, suggesting that these Mrl-binding sites are dispensable for tll expression under normal Tor signaling. However, in a torGOF background, the mutant 5.9 kb fragment shows greatly diminished ability to drive lacZ expression in an expanded domain compared to the situation when the Mrl binding sites are wild type. These results are consistent with the genetic results that Mrl is required for the full activity of gain-of-function, but not wild-type Tor (Li, 2002).

To account for the involvement of Mrl in tll regulation, it is proposed that a hyperactivated RTK requires a downstream pathway that is not essential for wild-type RTK under normal physiological situations. In wild-type embryos, Tor is activated only in the two terminal regions and defines the spatial limits of tll expression domains by relieving the transcriptional repressors bound to the tll promoter. Mrl is not an essential factor for tll activation in the terminal regions, although it remains to be determined whether Mrl contributes to the activation of tll expression redundantly with other yet unidentified factors. In torGOF mutant embryos, TorGOF is constitutively active in all regions of the embryo and causes ectopic tll expression. In this case, Mrl activation is indispensable for the ectopic tll expression in the central regions of the embryo. The differential requirement for Mrl in central and terminal regions might be due to the lack of other activators of tll and/or the presence of additional repressors in the central region of the embryos. Consistent with this idea, in the absence of Tor signaling (such as in tor mutant embryos), tll can be induced by uniformly expressing activated forms of downstream signaling components (such as RasV12 or 14-3-3). The resulting induction of tll expression happens preferentially in the terminal regions. Thus tll expression could be determined by the balance between repressors and activators that can bind to the tll promoter (Li, 2002).

These findings may explain some of the conflicting observations on the role of STAT in RTK signaling in mammals. For example, thanatophoric dysplasia type II (TD II) dwarfism in humans is caused by mutations that lead to constitutive activation of a human RTK FGF receptor 3 (FGFR3). Similar to TorGOF activating Mrl, it has been shown that an activated mutant FGFR3 specifically activates STAT1 in both human patient tissues and mouse models. The activated STAT1 in this case induces expression of the cell-cycle inhibitor p21WAF1/CIP1, resulting in growth inhibition of bone tissues. However, STAT1 is not known to be required for bone development. STAT1 knockout mice have perfect bones, although they exhibit defective immune systems. This might be explained by a redundancy among different STAT proteins. Alternatively, STAT1 may not be required for normal FGFR3 signaling in bone development. The presence of several STAT genes in mammals makes it technically difficult to distinguish between the above two possibilities using the mouse as a genetic model. In contrast, the presence of a single JAK and a single STAT gene in Drosophila allows the relationship between RTK and JAK/STAT signaling to be examined, without being limited by gene redundancy. These observations in Drosophila suggest that the TD II syndrome in humans could be explained if STAT1 is not normally required for FGFR3 signaling, but it becomes essential only for the activating mutant FGFR3 (Li, 2002).

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).

Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes

A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. A new negative regulator of JAK/STAT signaling is described and a potential mechanism identified by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, Ken & Barbie (Ken), which is an ortholog of the mammalian proto-oncogene BCL6, has been identified as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, endogenous Ken is shown specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo (Arbouzova, 2006).

Analysis of phenotypes associated with mutations in Drosophila JAK/STAT pathway components have identified a wide variety of requirements for the pathway during embryonic development and in adults. What is less clear is how the repeated stimulation of a single pathway is able to generate this pleiotropy of developmental functions. In order to identify modulators of JAK/STAT signaling that may be involved in this process, a genetic screen was undertaken for modifiers of the dominant phenotype caused by the ectopic expression of the pathway ligand Unpaired (Upd) in the developing eye imaginal disc. Such misexpression by GMR-updΔ3′ results in overgrowth of the adult eye, a phenotype sensitive to the strength of pathway signaling activity. With this assay, one genomic region, defined by Df(2R)Chig320, was found to enhance the GMR-updΔ3′-induced eye overgrowth phenotype. Of the genes deleted by Df(2R)Chig320, only mutations in ken showed consistent and reproducible enhancement of the phenotype. In addition, other dominant phenotypes induced by transgene expression from the GMR promoter are not modulated by ken mutations, indicating that Ken is unlikely to interact with the misexpression construct used (Arbouzova, 2006).

The enhancement of the GMR-updΔ3′ phenotype after removal of one copy of ken implies that Ken normally functions antagonistically to JAK/STAT signaling. Therefore phenotypes associated with mutations in other pathway components were tested to establish the reliability of this initial observation. Consistent with this, genetic interaction assays between ken mutations and the hypomorphic loss-of-function allele stat92EHJ show a reduction in the frequency of wing vein defects normally associated with this stat92E allele. Moreover, the degree of suppression is consistent with the strength of ken alleles tested. Similarly, the frequency of “strong” posterior spiracle phenotypes caused by the dome367 allele of the pathway receptor is also reduced when crossed to ken alleles or the Df(2R)Chig320 deficiency, with a concomitant increase in “weak” phenotypes (Arbouzova, 2006).

Thus, multiple independent ken alleles all modify diverse phenotypes caused by both gain- and loss-of-function mutations in multiple JAK/STAT pathway components. Each of these components acts at different levels of the signaling cascade and show interactions indicating that Ken consistently acts as an antagonist of the pathway (Arbouzova, 2006).

The ken locus contains three exons encoding a 601 aa protein. Ken possesses an N-terminal BTB/POZ domain between aa 17 and 131 and three C-terminal C2H2 zinc finger motifs from aa 502 to 590. Strikingly, a number of Zn finger-containing proteins that also contain BTB/POZ domains have also been shown to function as transcriptional repressors—often via the recruitment of corepressors such as SMRT, mSIN3A, N-CoR, and HDAC-1 (Arbouzova, 2006).

Searches for proteins similar to Ken identified homologs in Drosophila pseudoobscura and the mosquito Anopheles gambiae. In vertebrates, human B-Cell Lymphoma 6 (BCL6) was the closest full-length homolog. Drosophila Ken and human BCL6 share the same domain structure and show 20.3% overall identity. Proteins listed as potential vertebrate homologs of Ken in Flybase are more distantly related (Arbouzova, 2006).

Expression of ken was also examined during development, where it is detected in a dynamic pattern from newly laid eggs, throughout embryogenesis, and in imaginal discs. As such, endogenous Ken is present in all tissues and stages in which genetic interactions were observed (Arbouzova, 2006).

Given the presence of potentially DNA binding Zn finger domains and the nuclear localization of GFPKen, the DNA binding properties of Ken was determined by using an in vitro selection technique termed SELEX (systematic evolution of ligands by exponential enrichment). With a GST-tagged Ken Zn finger domain and a randomized oligonucleotide library, ten successive rounds of selection were undertaken. Sequencing of the resulting oligonucleotide pool and alignment of 43 independent clones showed that all recovered plasmids were unique and each contained one, or occasionally two, copies of the motif GNGAAAK (K = G/T) (Arbouzova, 2006).

To confirm the SELEX results, GFPKen was expressed in tissue culture cells and these were used for electromobility shift assays (EMSA). A radioactively labeled probe containing the wild-type (wt) consensus binding site GAGAAAG gives a specific band, which can be supershifted by an anti-GFP antibody and therefore represents a GFPKen/DNA complex. In order to identify positions essential for binding, a competition assay was used in which unlabeled oligonucleotides containing single substitutions in each position from 1 to 7 were added to binding reactions. 10-fold excess of unlabeled wild-type consensus oligonucleotide greatly diminished the intensity of the GFPKen band, while 50- and 100-fold excess totally blocked the original signal. By contrast, competition with unlabeled m3 oligonucleotides containing a G to A substitution at position 3 failed to significantly reduce the intensity of the band even at 100-fold excess. With this approach, the positions 1 and 7 are found dispensable for DNA binding, whereas the central GAAA core is absolutely required. Similar results were obtained with the converse experiment with labeled mutant probes, although in this case the wt probe produces a stronger signal than the m1 and m7 mutant oligonucleotides. Taken together, these experiments not only define the core sequence for Ken binding, but also demonstrate the specificity of Ken as a site-specific DNA binding molecule. Interestingly, the core consensus bound by Ken is very similar to that identified for human BCL6, with the Zn fingers of the latter binding to a DNA sequence containing a core GAAAG motif (Arbouzova, 2006).

One initial observation made is that the core GAAA essential for Ken binding overlaps the sequence recognized by STAT92E. Consistent with this overlap, a 100-fold excess of unlabeled oligonucleotide containing the STAT92E consensus is sufficient to fully compete for Ken in EMSA assays. Given this finding, it is hypothesized that the negative regulation of JAK/STAT signaling by Ken observed in genetic interaction assays may occur via a mechanism of competitive DNA binding site occupation. Due to the incomplete overlap between the STAT92E and Ken core sequences, this hypothesis also implies the existence of STAT92E DNA binding sites to which both STAT92E and Ken could bind (STAT+/Ken+) as well as sites with which Ken cannot associate (STAT+/Ken) (Arbouzova, 2006).

To test this hypothesis, a cell culture-based assay was set up by using a luciferase-expressing reporter containing four STAT92E binding sites originally identified in the promoter of the Draf locus. In addition to this STAT+/Ken+ wild-type reporter, STAT+/Ken and STAT/Ken variants identical but for the binding sequences were generated. When transfected into the hemocyte-like Kc167 Drosophila cell line, both STAT+/Ken+ and STAT+/Ken reporters showed strong stimulation upon coexpression with the pathway ligand Upd, an assay previously shown to require an intact JAK/STAT cascade. When cotransfected with KenGFP, the activity of the STAT+/Ken+ reporter was reduced, an effect reproduced in three independent experiments with both KenGFP and Ken. While the reduction in reporter activity for the STAT+/Ken+ assay shown is statistically significant, the STAT+/Ken reporter was unaffected by the coexpression of Ken. Reporters containing binding sites mutated to prevent binding of both STAT92E and Ken (STAT/Ken) showed no activation after pathway stimulation and did not respond to Ken (Arbouzova, 2006).

These results indicate that Ken functions as a transcriptional repressor in this cell-culture system and shows that this effect is specific to the DNA sequence determined by SELEX and EMSA. This result is also consistent with a recent whole-genome RNAi-based screen, which used a reporter containing STAT+/Ken+ binding sites and includes Ken among the list of JAK/STAT regulators identified. In addition, recent reports have also demonstrated BCL6 binding to STAT6 sites in vitro and have shown that BCL6 can act as a repressor of STAT6-dependent target gene expression in cell culture. Although this repression is mediated by the binding to corepressors to the BTB/POZ domain of BCL6, no link between BCL6 and STAT activity has been demonstrated in vivo (Arbouzova, 2006).

Finally, it should also be noted that both the STAT+/Ken+ and STAT+/Ken reporters contain additional GAAA sequences that are not part of the characterized STAT92E binding sequences. However, despite the presence of these potential Ken binding sites within 15 bp of the STAT92E site, Ken expression did not affect the STAT+/Ken reporter, suggesting that Ken may require STAT92E to influence gene expression. Although no direct association between Ken and STAT92E has been demonstrated, this possibility cannot be excluded, and further analysis remains to be undertaken (Arbouzova, 2006).

Having established that Ken functions at the level of DNA binding in cell culture, it was asked whether Ken also acts as a transcriptional repressor of JAK/STAT pathway target genes in vivo. For this, the effect of ectopically expressed Ken on the expression of putative JAK/STAT pathway target genes was examined and, given the high levels of maternally loaded STAT92E present at blastoderm stage, focus was placed on targets expressed later in embryogenesis. These include the hindgut-specific expression of vvl, the expression of trachealess (trh) and knirps (kni) in the tracheal placodes, and the dynamic expression of Socs36E throughout the embryo (Arbouzova, 2006).

First, the effect of Ken was addressed on trh, whose expression precedes the formation of the tracheal pits in the embryonic segments T2 to A8. Levels of trh are greatly reduced in embryos uniformly misexpressing Ken driven by the daughterless-GAL4 (da-GAL4) line. Many tracheal placodes express little or no trh, and tracheal pits fail to form even in the presence of residual trh. Similar effects are seen in updOS1A mutant embryos lacking all pathway activity. Likewise, downregulation of Kni expression is also observed in embryos misexpressing ken. These results show that both endogenous trh and kni are downregulated by ectopically expressed Ken (Arbouzova, 2006).

Whether Ken can modulate the expression of socs36E, a Drosophila homolog of mouse SOCS-5, was tested. socs36E expression closely mirrors that of upd, showing JAK/STAT pathway-dependent upregulation in segmentally repeated stripes, tracheal pits, and the hindgut. By contrast to trh and kni, ectopically expressed Ken does not affect any aspect of socs36E transcription. However, controls expressing a dominant-negative form of the pathway receptor DomeΔCyt, using the same Gal4 driver line, show a strong downregulation of socs36E, an effect reproduced by the complete removal of all JAK/STAT pathway activity by the updOS1A allele. Taken together, these results illustrate that ectopic expression of Ken during Drosophila development is sufficient to downregulate the expression of only a subset of putative JAK/STAT pathway target genes (Arbouzova, 2006).

As part of this analysis, modulation of vvl by Ken was tested. In wild-type embryos, vvl is expressed in the developing trachea and lateral ectoderm (in a JAK/STAT-independent manner) and in the hindgut of stage 12–14 embryos, where it requires JAK/STAT signaling. In updOS1A mutants, no vvl expression in the hindgut can be detected, indicating that this locus is a target of pathway activation. When Ken is uniformly misexpressed throughout the embryo, vvl expression is no longer detectable in the hindgut. Thus vvl, like trh and kni, can be a target of Ken-mediated repression (Arbouzova, 2006).

Having established that ectopic Ken is sufficient to downregulate vvl in the hindgut, whether endogenous Ken performs a similar role was determined. One overlap between ken expression and regions known to require JAK/STAT signaling are the developing posterior spiracles, structures in which both the pathway ligand upd and ken are simultaneously expressed. However, vvl is never detected in the posterior spiracle primordia in wild-type embryos, despite JAK/STAT pathway activity induced by upd expression in these tissues. Intriguingly, in a heteroallelic combination of the strongest kenk11035 allele and Df(2R)Chig320, vvl transcript was detected not only in its normal expression domain within the hindgut but also in the posterior spiracles. This ectopic expression is initially detected from late stage 13 and rapidly strengthens during stage 14–15. When kenk11035/Df(2R)Chig320 embryos simultaneously mutant for the amorphic updOS1A allele were analyzed, upregulation of vvl in the presumptive posterior spiracles was never observed at the stage by which ectopic vvl expression was first detected in the ken mutant embryos. At later stages, JAK/STAT pathway activity is required for posterior spiracle morphogenesis, posterior spiracles do not form, and upregulated vvl is not present (Arbouzova, 2006).

These results demonstrate that Ken is not only sufficient to downregulate the JAK/STAT pathway-dependent expression of vvl in the hindgut, but its endogenous expression is also necessary for vvl repression in the posterior spiracles. In ken mutants, ectopic vvl expression in the posterior spiracles results from a derepression of endogenous STAT92E activity (Arbouzova, 2006).

The overlap between the consensus sequences bound by STAT92E and Ken, together with the analysis of reporters containing STAT+/Ken+ and STAT+/Ken binding sites, indicate that Ken is likely to selectively regulate only a subset of JAK/STAT target genes. In this model, some target genes are regulated by binding sites compatible with both STAT92E and Ken, while others contain sequences to which only STAT92E can associate. While the DNA binding site is critical in cell-culture systems, similar proof is more difficult to establish in vivo. In particular, only a limited number of JAK/STAT pathway target genes have been rigorously demonstrated to require STAT92E binding in vivo (Arbouzova, 2006).

Although studied in some detail, the regulatory domains controlling vvl expression in the developing hindgut have not been identified. Therefore, although these results predict that such a domain would contain STAT+/Ken+ binding sequences, further analysis is required to confirm this hypothesis. By contrast, the regulatory domain of socs36E required to drive gene expression in the blastoderm, tracheal pits, and hindgut comprises a 350 bp region containing three STAT+/Ken+ and two STAT+/Ken binding sites. Although not conclusive, the presence of STAT92E-exclusive sites in this region may explain the inability of Ken to downregulate socs36E in vivo (Arbouzova, 2006).

The findings also draw a parallel between Drosophila Ken and BCL6. The data presented demonstrate that both proteins show similar abilities to bind DNA and to mediate transcriptional repression with some evidence also linking BCL6 to JAK/STAT signaling as described here. Taken together, these similarities suggest that Ken and BCL6 represent functional orthologs of one another. Given this evolutionary conservation, it is tempting to speculate that the selective regulation of JAK/STAT pathway target genes is also conserved and may represent a general mechanism by which the pathway is modulated to elicit diverse developmental roles in vivo. Although many STAT targets undoubtedly remain to be identified, it will be intriguing to see which may also be coregulated by Ken/BCL6-dependent mechanisms (Arbouzova, 2006).

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, this study shows 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, the JAK/STAT pathway regulates a small enhancer in the wg 3' cis genomic region. Since this enhancer is devoid of Stat92E-binding elements, it is concluded that Stat92E represses wg through another, as yet unidentified factor that is probably a direct target of Stat92E. Taken together, this study is the first to demonstrate a role for the JAK/STAT pathway in regional specification by acting antagonistically to wg (Ekas, 2006).

Although the majority of functions attributable to STATs involve transcriptional activation, at least one STAT protein, the Dictyostelium Dd-STATa, acts as a functional repressor. Therefore the ability of Stat92E to directly repress wg was tested. A reporter called wg2.11Z, in which ß-galactosidase is driven by a 263 base pair enhancer from the 3' cis wg genomic region, was sufficient to recapitulate wg expression in the dorsal margin of the disc proper and in the dorsal peripodial membrane (Pereira, 2006). This reporter was ectopically expressed in mosaic stat92E clones as well as in stat92E M+ clones in a manner similar to that observed for wgP. Moreover, wg2.11Z was repressed autonomously in hop-expressing clones. These data indicate that Stat92E can regulate dorsal wg expression through the wg2.11Z enhancer. wg2.11Z does not contain any Stat92E binding sites (TTC(N)3GAA), strongly suggesting that Stat92E does not repress dorsal wg directly, but rather regulates another factor which represses wg (Ekas, 2006).

Thus, this paper reports a new role for the Drosophila JAK/STAT pathway. The study demonstrates that JAK/STAT signaling promotes formation of the eye field through repression of wg gene transcription in both the dorsal and ventral halves of the eye disc epithelium. By monitoring Upd expression and activity, it was shown that the JAK/STAT pathway is normally activated early in eye development, during first and second instar. Ectopic activation of this pathway leads to abnormal patterning of the head capsule and a reduction in the inter-eye distance through an increase in dorsal ommatidia. By contrast, loss of activity of this pathway, using strong hypomorphic stat92E mutations, frequently resulted in the development of a rudimentary head. When the head capsule did form, stat92E mutants often had small or ablated adult eyes and excessive head cuticle. wg was ectopically expressed in stat92E clones and hop mutant eye discs, and was repressed by ectopic activation of the JAK/STAT pathway. Reduction in the dose of wg partially rescued stat92E mutants by increasing the rate of eclosion and by mitigating the phenotypes of stat92E mutant animals. Lastly, it was shown that wg regulation by the JAK/STAT pathway is independent of the known wg regulators Eyg, Dac, Hth and Pnr (Ekas, 2006).

These results conflict with those of a previous study, which reported that JAK/STAT signaling does not repress wg in the eye disc. This conclusion was reached on the basis of wild-type Wg protein expression in eye discs that contained ectopic upd-expressing clones (Zeidler, 1999). However, this study found that in the absence of stat92E, wg was ectopically expressed in both dorsal and ventral halves of the eye disc. It is likely that the current examination of the wg gene using the wgP enhancer trap is a more sensitive measure of wg expression than monitoring Wg protein. Zeidler also reported that the JAK/STAT pathway negatively regulates mirr expression. This conclusion was drawn after finding a preponderance of dorsal, mirr-positive ommatidia in adult eyes containing unmarked upd loss-of-function clones (Zeidler, 1999). However, using marked clones, the current study showed that Mirr is expressed normally in eye tissue that is largely homozygous mutant for stat92E. Moreover, stat92E M+ adult eyes are largely composed of Mirr-negative ommatidia, which indicates their ventral origin. Thus, the current data indicate that mirr is not regulated by JAK/STAT pathway activity (Ekas, 2006).

Previous work has shown that the 3' cis region of the wg gene regulates its expression in imaginal discs. Several wg mutations that specifically affect imaginal disc development, as well as discspecific enhancers, map to this region. In this study, it was shown that Stat92E negatively regulates dorsal wg through a small enhancer (wg2.11Z) in the 3' cis genomic region of the wg gene. This enhancer is ectopically expressed in stat92E and hop mutants and is autonomously repressed by ectopic activation of Stat92E. The DNA binding preferences of Stat92E and other STAT proteins have been well characterized. Because there are no Stat92E binding sites in the wg2.11Z enhancer, the interpretation is favoredthat Stat92E does not directly repress dorsal wg but rather acts through another factor. This repressor may be encoded by a direct Stat92E target gene, because wg is autonomously repressed by the JAK/STAT pathway. However, the possibility cannot be ruled out that Stat92E regulates wg through other transcription factors, such as Dorsal or vHNF-4, which have putative sites in wg2.11Z (Pereira, 2006). It is also possible that there are cryptic Stat92E binding sites in this wg enhancer, through which Stat92E may directly repress wg. Additional experiments will be needed to test these possibilities (Ekas, 2006).

This study also demonstrated that Stat92E represses ventral wg in the eye disc epithelium. This is presumably independent of the wg2.11Z enhancer, which recapitulates wg expression in the dorsal but not the ventral eye disc (Pereira, 2006). Moreover, Stat92E negatively regulates pnr in peripodial cells. In the absence of JAK/STAT signaling, pnr is dramatically expanded into the posterior peripodial membrane. However, it is stressed that because pnr is an intracellular protein, the ectopic pnr in the peripodial membrane cannot account for the ectopic wg observed in the disc proper of stat92E mutants. Currently, it is not know whether Stat92E regulates wg in the ventral eye disc epithelium and the peripodial membrane in the same manner as in the dorsal eye. All three wg expression domains may be regulated by the same as yet unidentified factor. Alternatively, Stat92E may regulate wg expression domains through different mechanisms. For example, dorsal wg may be regulated indirectly, whereas ventral and peripodial wg may be regulated directly by Stat92E. The wg gene 3' cis genomic region contains one putative Stat92E binding site, which resides downstream of the wg2.11Z enhancer. Therefore, it is possible that Stat92E regulates ventral and peripodial wg through this site. Future work will be needed to address these issues (Ekas, 2006).

Co-ordinated control of cell adhesión, cell polarity and cytoskeleton underlies Hox-induced organogenesis in Drosophila: regulation of crb by STAT

Hox genes control animal body plans by directing the morphogenesis of segment-specific structures. As transcription factors, HOX proteins achieve this through the activation of downstream target genes. Much research has been devoted to the search for these targets and the characterization of their roles in organogenesis. This has shown that the direct targets of Hox activation are often transcription factors or signaling molecules, which form hierarchical genetic networks directing the morphogenesis of particular organs. Importantly, very few of the direct Hox targets known are 'realizator' genes involved directly in the cellular processes of organogenesis. This study describes a complete network linking the Hox gene Abdominal-B to the realizator genes it controls during the organogenesis of the external respiratory organ of the larva. In this process, Abdominal-B induces the expression of four intermediate signaling molecules and transcription factors, and this expression results in the mosaic activation of several realizator genes. The ABD-B spiracle realizators include at least five cell-adhesion proteins, cell-polarity proteins, and GAP and GEF cytoskeleton regulators. Simultaneous ectopic expression of the Abd-B downstream targets can induce spiracle-like structure formation in the absence of ABD-B protein. It is concluded that Hox realizators include cytoskeletal regulators and molecules required for the apico-basal cell organization. HOX-coordinated activation of these realizators in mosaic patterns confers to the organ primordium its assembling properties. It is proposed that during animal development, Hox-controlled genetic cascades coordinate the local cell-specific behaviors that result in organogenesis of segment-specific structures (Lovegrove, 2006).

To initiate spiracle organogenesis, ABD-B, in combination with local signaling molecules, activates a set of targets within the dorsal area of A8. This study shows that there may be as few as four direct targets for the posterior spiracle. The expression of the primary targets, with their corresponding cofactors, subdivides the organ into specific regions. After this patterning stage, specific cell behaviors are controlled by another set of transcription factors that include the GATA transcription factor Grn to bring about cell rearrangements, and the JAK/STAT signaling pathway, which induces posterior spiracle-cell elongation. The partially overlapping expression of these transcription factors has the potential to activate in particular subsets of spiracle cells different sets of realizator genes. In the spiracles, these realizators include cell-adhesion molecules, apico-basal polarity proteins, and cytoskeletal regulators. Thus, in this way, ABD-B activates a genetic cascade coordinating the local cell-specific behaviors that result in organogenesis (Lovegrove, 2006).

Two main issues may explain why identification of the realizator genes has been so difficult. Primarily, by nature, many of these molecules are required for general functions in all cells. A screen for Hox realizators based on finding segment-specific defects would miss molecules like E-Cad or the Rho GTPases because of generalized embryonic malformations. Thus, their realizator nature can only be uncovered when, through intermediate regulators, a link to the HOX protein is found. This is demonstrated in the case of crb where a specific spiracle enhancer was found, that directs its increased transcription. In the case of the cytoskeleton, the link is made through the use of specific regulatory GEF and GAP proteins that modulate the activity of the GTPases. A second problem has been that some of the realizator molecules function redundantly and therefore a mutational approach yields no result. This is the case with the nonclassic cad88C and cad96C, which only show a mutant phenotype if E-cad is also mutated. Although cell-adhesion molecules had been originally proposed to be realizators, it is surprising to find that there are four nonclassical cadherins with restricted expression in the spiracle (Lovegrove, 2006).

Another unexpected finding has been the observation that the expression of apical- and basolateral-membrane proteins is modulated in the spiracle during the elongation stages. This study has established a link between ABD-B and the apical determinant crb through the JAK/STAT pathway. During invagination, spiracle cells are going through major membrane reorganization, including apical constriction and basal elongation. Thus, Crb, which is required in many epithelia for maintenance of a proper zonula adherens, may be playing an important role for the polarized remodeling along the apico-basal cell axis. Crb upregulation is functionally important for cell elongation, but it is not the only function controlled by STAT. In this respect, it is important to note that spiracle-cell elongation occurs mainly through the increase of basolateral membranes. It is thus likely that the spiracle-gene network will also be controlling basal polarity determinants (Lovegrove, 2006).

A role for ABD-B in regulation of the cytoskeleton in the posterior spiracles was expected because of the initial observations on cell elongation taking place in the spiracular chamber. The observed effects of the dominant-negative and constitutively active forms of Rho GTPases on spiracle development support this hypothesis. The finding of Gef64C regulation by ABD-B in the spiracle cascade and the finding of spiracle invagination defects in RhoGAP cv-c mutants confirms that specific control of the Rho GTPases is an important feature of spiracle development (Lovegrove, 2006).

Although all the realizators analyzed in this study are activated indirectly by ABD-B, the possibility cannot be excluded that ABD-B can also activate some others directly. Direct regulation of realizator genes by HOX may be important for differentiation of specific cell types (Lovegrove, 2006).

This study has linked the activity of a HOX protein, through the regulation of a small number of intermediate regulators, to a battery of realizator genes. The local-specific modulation of these genes that in other contexts control cell adhesion, polarity, and organization of the cytoskeleton, would be sufficient to confer unique morphogenetic properties to the cells leading to the formation of a segment specific organ. Other examples in Drosophila include salivary-gland organogenesis, where SCR initially activates a cascade of downstream genes, and head formation where DFD activates Dll but similar processes must be occurring in Hox-controlled organogenesis in vertebrates (Lovegrove, 2006).

Drosophila JAK/STAT pathway reveals distinct initiation and reinforcement steps in early transcription of Sxl

X-linked signal elements (XSEs) communicate the dose of X chromosomes to the regulatory-switch gene Sex-lethal (Sxl) during Drosophila sex determination. Unequal XSE expression in precellular XX and XY nuclei ensures that only XX embryos will activate the establishment promoter, SxlPe, to produce a pulse of the RNA-binding protein, SXL. Once XSE protein concentrations have been assessed, SxlPe is inactivated and the maintenance promoter, SxlPm, is turned on in both sexes; however, only in females is SXL present to direct the SxlPm-derived transcripts to be spliced into functional mRNA. Thereafter, Sxl is maintained in the on state by positive autoregulatory RNA splicing. Once set in the stable on (female) or off (male) state, Sxl controls somatic sexual development through control of downstream effectors of sexual differentiation and dosage compensation. Most XSEs encode transcription factors that bind SxlPe, but the XSE unpaired (upd) encodes a secreted ligand for the JAK/STAT pathway. Although STAT directly regulates SxlPe, it is dispensable for promoter activation. Instead, JAK/STAT is needed to maintain high-level SxlPe expression in order to ensure Sxl autoregulation in XX embryos. Thus, upd is a unique XSE that augments, rather than defines, the initial sex-determination signal (Avila, 2007).

The question of how embryos differentiate between precise 2-fold differences in X-linked signal element (XSE) doses is central to understanding how genetic constitution defines sexual fate. Current X-chromosome-counting models posit that the female fate is set when XSE proteins exceed a threshold concentration and activate SxlPe. The XSE threshold is set by interactions between the XSEs and other proteins in the embryo. Some XSEs interact with maternally supplied proteins to form dose-sensitive transcription factors, such as Scute/Daughterless, that bind SxlPe, but XSE doses are also assessed with reference to maternally and zygotically expressed repressors. Three XSE proteins, SisA, Scute, and Runt, are viewed as acting similarly by binding directly to and activating SxlPe. The fourth XSE, unpaired (upd, also called outstretched or sisC), encodes a secreted ligand that signals through the JAK kinase (hopscotch) to activate the Stat92E transcription factor. Although upd meets the criteria of an XSE, its effects on sex determination are weaker than those of sisA, scute, and runt, and changes in its gene dose have only moderate effects on Sxl. To understand how this comparatively dose-insensitive XSE regulates sex, when and where upd, JAK, and STAT act on the Sxl switch was examined (Avila, 2007).

Using in situ hybridization, the early embryonic expression pattern of upd was defined. No evidence was found for maternally supplied transcripts and it was observed that upd mRNA was first detectable in nuclear cycle 13. The fact that the first upd transcripts are present throughout the embryo, including at the poles, is consistent with the distribution of phosphorylated Stat92E. As cellularization progresses past early cycle 14, the upd pattern resolves into indistinct stripes that developed into a 14 stripe pattern during gastrulation. These results show that upd expression begins later than that of the other XSEs (sisA in cycle 8; scute in cycle 9) and also, paradoxically, that it begins after the onset of transcription of its target, Sxl, in cycle 12 (Avila, 2007).

To understand how upd functions in Sxl activation and how it differs from other XSEs, upd mutations were examined for their effects on SxlPe by using in situ hybridization and on Sxl protein levels by using immunostaining with SXL antibody. Significantly, the RNA probes detected nascent Sxl transcripts, allowing monitoring of both the spatial and temporal responses of SxlPe on a cell-cycle by cell-cycle basis (Avila, 2007).

updsisC1, a loss-of-function mutation that appears to specifically affect sex determination was examined, because it has no observable effect on later upd functions. Consistent with the fact that upd has a modest effect on SxlPe, it was found that two-thirds of homozygous updsisC embryos expressed SxlPe in a manner indistinguishable from that of the wild-type. A small proportion of embryos, 15%, had within their middle sections several clusters of 5-15 nuclei that did not express SxlPe, whereas the remaining 18% had severe defects, with SxlPe expression being absent from most of the central regions of the embryos. Despite early aberrations in SxlPe activity, immunostaining revealed no lasting defect in the expression of SXL, because updsisC1 embryos that reached germband extension stained in a 1:1 male:female ratio. To determine the effects of a complete loss of zygotic upd activity, updYC43, a probable null mutation, and the deficiency Df(1)ue69, which deletes upd and the upd-like gene, upd3, were examined. With respect to SxlPe, it was found that upd-null-mutant females were more severely affected than were updsisC1 embryos. At cellularization, the defects ranged from embryos containing large clusters of nuclei that did not express SxlPe in the central part of embryo to those in which the entire central region failed to express the promoter. The poles, however, expressed SxlPe normally. Immunostainings of updYC43 and Df(1)ue69 embryo collections revealed that these alleles had strong but incompletely penetrant effects on the later distribution of SXL. The fact that an estimated 40% of mutant female embryos stage 6 and older failed to express SXL in their central regions is consistent with the observed defects in SxlPe activity. The remainder eventually expressed normal levels of SXL in all their tissues, indicating that most upd mutant females were able to compensate for reduced SxlPe activity and ultimately engaged autoregulatory Sxl mRNA splicing. Two upd-like genes, upd2 and upd3, map adjacent to upd. Loss of zygotic upd2 had no effect on SxlPe, and the effects of Df(1)ue69 (upd3-,upd-) appeared identical to those of updYC43 when analyzed in a common genetic background. This shows that XSE activity in this region of the X is due to upd alone (Avila, 2007).

Except for the ligands, each component of the JAK/STAT pathway is maternally deposited into the embryo. To eliminate JAK/STAT activity completely, the dominant female-sterile technique was used to generate females lacking maternal hopscotch (hop) or Stat92E, which encode the only JAK kinase and STAT in Drosophila. It was expected that by removing maternal hop, STAT would remain unphosphoryated, allowing a determination of the effects of the loss of the entire pathway on SxlPe (Avila, 2007).

When Sxl expression was examined in cycle 14 embryos derived from hopC111 germline clones, it was found that SxlPe was active in the anterior and posterior regions of the embryos but almost completely inactive in the central region of the embryos. In contrast to the results with upd mutants and deficiencies, all of which exhibited considerable embryo-to-embryo variation, loss of maternal hop had nearly identical effects on SxlPe in every embryo. This more potent effect of maternal hopC111 as compared to upd mutants suggests that zygotic Upd might not be the only activator of JAK in the precellular embryo (Avila, 2007).

The findings with hopC111 were confirmed by using the Stat92E06346 mutation. Cycle 14 embryos derived from Stat92E06346 germline clones also lacked nearly all SxlPe expression in their central regions, but they were even more strongly affected than hopC111 females because SxlPe activity was also reduced in the termini. These findings are contrary to predictions of a linear JAK/STAT pathway going from zygotic Upd through receptor and kinase to activated STAT. Instead, the progressive weakening of SxlPe by removal of upd and Stat92E suggests that there is hop-independent control of Stat92E function in sex determination. The possibility of cross-talk between signaling systems is supported by the finding that the torso receptor-tyrosine-kinase pathway activates STAT92E in the embryo termini (Avila, 2007).

Although the hopC111 and Stat92E06346 mutations had large effects on SxlPe during cycle 14, the period of maximum SxlPe expression, it was found that these mutations had little effect on SxlPe at earlier stages. In wild-type females, SxlPe is first activated in cycle 12. Expression increases throughout cycle 13 and reaches a peak in the first minutes of cycle 14. In embryos from hopC111 mothers, SxlPe was expressed as in the wild-type during cycles 12 and 13. However, upon entry into cycle 14, SxlPe activity ceased in the middle sections of the embryos. A similar phenomenon was observed in embryos carrying strong upd mutants and in those derived from Stat92E06346 germline clones. These results show that JAK/STAT, and thus upd XSE function, is not needed for the initial activation of SxlPe. Instead, upd must function as a different kind of XSE: one dispensable for the initial assessment of X-chromosome dose, but needed to maintain SxlPe activity in the final stage of the X-counting process (Avila, 2007).

When the progeny of hopC111 mutant mothers were examined for Sxl protein, it was found that defects in SxlPe expression led to a permanent failure to express SXL in the central regions in 35% of female embryos. This suggests that the loss of SxlPe activity in cycle 14 can reduce the level of early Sxl to below the threshold normally required to activate autoregulatory mRNA splicing. Although 35% of female embryos were defective for later Sxl expression, most females that completed gastrulation expressed Sxl uniformly. This striking discordance between the effects of hop (and upd and Stat92E) mutants on SxlPe activity and ultimate Sxl levels suggests that stable Sxl autoregulation can be established even when SxlPe function has been seriously compromised. Although some rescuing Sxl mRNA or protein may have diffused from the poles, an alternative explanation is that expression of SxlPe during cycles 12 and 13 might often have provided sufficient Sxl to trigger autoregulation once the maintenance promoter, SxlPm, had been activated (Avila, 2007).

SxlPe is thought to have two main functional elements: a proximal 390 bp X-counting region responsible for sex-specific activation, and a more distal (to -1.4 kb) element that elevates Sxl transcription. Three predicted STAT-binding sites are located in these elements at positions -253, -393, and -428 bp. To test their roles, consensus TTC sequences were changed to TTT because such changes block binding by STAT92E and the mammalian homologs STATs 5 and 5a. In situ hybridizations revealed that the mutation in the proximal STAT site, S1, greatly reduced the number of nuclei expressing SxlPe-lacZ, creating a patchy staining pattern and lower overall mRNA levels. Mutations in S1 and S2, or in all three sites together, caused a strong but variable loss of SxlPe-lacZ expression in most nuclei, resulting in dramatically reduced accumulation of lacZ mRNA. Although the S1, S2, S3 mutant appeared to have a slightly stronger effect than the double mutant, both transgenes exhibited phenotypes reminiscent of those seen in embryos derived from Stat92E06346 germline clones. These results show that STAT92E acts through the consensus binding sites at SxlPe (Avila, 2007).

SxlPe is remarkable for both its rapid response and exquisite sensitivity to X-chromosome dose. In male embryos, it is always off. In female embryos, SxlPe is strongly expressed, but only during a 35-40 min period from mid cycle 12 until about 10-15 min into cycle 14. Given these time constraints, many have assumed that all XSEs would function to establish the initial on or off state of SxlPe. However, it was found that upd behaved very differently than sisA and scute, both of which are required for SxlPe activation and expression. Loss of upd or the JAK/STAT pathway had little or no effect on SxlPe during cycles 12 or 13. Instead, JAK/STAT mutations blocked SxlPe expression late in the process, during cycle 14. This observation is interpreted as revealing that SxlPe is regulated in two mechanistically distinct phases: the first controlling the initial response to X-chromosome dose, and the second acting to maintain or reinforce the initial decision (Avila, 2007).

The relatively late actions of upd and hop offer explanations for several puzzling aspects of upd's function in sex determination. First, upd is considered a weak XSE. This is both because Sxl is comparatively insensitive to upd dose and because loss of upd or JAK/STAT function doesn't eliminate Sxl expression. Both effects are consistent with expectations of a two-step, initiation and maintenance, model for SxlPe function. JAK/STAT mutations would not be expected to eliminate all Sxl function in a two-step model because the STAT-independent initiation step would produce Sxl mRNA and protein. The exact gene dose of upd would not be particularly important for sex because excess active STAT could not induce SxlPe without the prior actions of the initiating XSEs and because even a single dose of upd+ could provide enough active STAT to augment an earlier decision to become female. Thus, the proposed STAT maintenance function explains both the failure of the constitutively active hoptum-l allele to induce ectopic SxlPe expression in males and the ability of hoptum-l to further stimulate SxlPe activity in females. Likewise, the requirement for STAT site S2, located just distal to the 390 bp X-counting region of SxlPe, and the finding that upd is first expressed after Sxl can be explained if STAT's role is to bolster transcription from SxlPe in embryos that already have counted two Xs. Although neither essential for SxlPe expression nor highly dose sensitive, upd, hop, and Stat92E nonetheless play important roles at SxlPe. In their absence, the period of SxlPe activity is cut short, reducing the concentration of Sxl and preventing a large fraction of embryos from engaging the maintenance mode of Sxl expression (Avila, 2007).

How might STAT92E function in a two-step model? One possibility is that STAT might antagonize the late-acting repressor Dpn. Alternatively, the STAT transcription factor might augment, stabilize, or replace earlier-acting XSE activator complexes as their concentrations diminish in cycle 14. BAP60, a core component of the Brahma chromatin-remodeling complex, has been shown to interact with two components of the sex-determination signal. If STAT92E also interacts with the Brahma complex, it might maintain SxlPe chromatin in an active state, facilitating the restoration of transcription after the 13th mitosis (Avila, 2007).

Understanding the commonalities and unique mechanisms STATs employ in their multitude of roles is a fundamental goal of research on this ubiquitous signaling pathway. It is also essential for understanding why the pathway has so often been co-opted into new roles during evolution. STATS seem primarily permissive rather than instructive. They are rarely the primary signals defining cell fate. In these respects, comparison of the even-skipped (eve) stripe 3 enhancer and SxlPe reveals interesting parallels. Both SxlPe and eve stripe 3 are regulated by the balance between several activators and repressors. The responses of both elements to JAK/STAT signaling are extremely rapid, occurring within the dynamic environment of the precellular embryo. Stat92E is important for each, but its roles augment the actions of other factors, rather than being responsible for defining the initiating signals (Avila, 2007).

With respect to the evolution of the sex signal, it has been proposed that a diffusible JAK/STAT signal might have been recruited to allow non linear signal amplification or, alternatively, that a diffusible ligand might render SxlPe less sensitive to random fluctuations in cell-autonomous XSE protein concentrations. Although the weak dose dependence of upd argues against signal amplification, a buffering function is consistent with existing data. These findings suggest another possibility. STAT proteins respond rapidly to a range of regulatory signals; it may be this ability to act within a matter of minutes that brought JAK/STAT into the temporally dynamic X-chromosome-counting process (Avila, 2007).

Two distinct but convergent groups of cells trigger Torso receptor tyrosine kinase activation by independently expressing torso-like

Cell fate determination is often the outcome of specific interactions between adjacent cells. However, cells frequently change positions during development, and thus signaling molecules might be synthesized far from their final site of action. This study analyzed the regulation of the torso-like gene, which is required to trigger Torso receptor tyrosine kinase activation in the Drosophila embryo. Whereas torso is present in the oocyte, torso-like is expressed in the egg chamber, at the posterior follicle cells and in two separated groups of anterior cells, the border cells and the centripetal cells. JAK/STAT signaling regulates torso-like expression in the posterior follicle cells and border cells but not in the centripetal cells, where torso-like is regulated by a different enhancer. The border and centripetal cells, which are originally apart, converge at the anterior end of the oocyte, and both groups contribute to trigger Torso activation. These results illustrate how independently acquired expression of a signaling molecule can constitute a mechanism by which distinct groups of cells act together in the activation of a signaling pathway (Furriols, 2007; full text of article).

Although tsl is expressed in three different groups of follicle cells, these cells are not completely unrelated. Thus, for example, both the BCs and the CCs are derived from a common pool of anterior follicle cells and express and require some of the same genes for their development. Likewise, many similarities have also been recognized between the BCs and the PFCs. This raises the possibility that a common mechanism could single out these cells for tsl expression. Alternatively, each of these groups of follicle cells could be independently targeted to express tsl. As a first attempt to address how these distinct groups of follicle cells acquire the ability to express a common signaling factor, an analysis of the tsl promoter was undertaked (Furriols, 2007).

As a first indication of what constitutes the tsl regulatory region, the P-element insertion carrying the lacZ gene upstream of the 5'-UTR exon in the tsl0617 mutant, thereafter tsl0617-lacZ, was know to reproduced all of the features of tsl expression in the follicle cells, as judged by comparison with the tsl in situ hybridization pattern. By transformation of lacZ reporter constructs using different regions upstream of the coding sequences of tsl, it was found that a single fragment of ~1,500 bp upstream of the 5'-UTR exon (see Distinct Enhancers Regulate tsl Expression in Specific Groups of Follicle Cells) reproduces the tsl wild-type pattern. Further dissection allowed splitomg the tsl promoter into two nonoverlapping regions responsible for a different subset of the tsl expression pattern. In particular, it was found that a 604-bp sequence drives expression only in the CCs, hereafter referred to as the CC enhancer, whereas an adjacent 954-bp sequence drives expression in both the BCs and the PFCs. Comparison between the different constructs suggested that the enhancer for BCs and PFCs could be further refined to a region of 298 bp (fragment K). This assumption was confirmed by establishing that two copies of fragment K are sufficient to drive expression in BCs and PFCs, hereafter referred to as the BC/PFC enhancer. Thus, in summary, two different regions of the tsl promoter are responsible for distinct subsets of tsl expression. It is remarkable that a single promoter fragment (fragment K) drives tsl expression in two independent group of follicle cells (the BCs and PFCs), whereas separate enhancers (fragments K and F) are responsible for tsl expression in the BCs and CCs, which are derived from a common pool of anterior follicle cells (Furriols, 2007).

This study found that tsl expression is controlled by different cis-regulatory regions and different transactivating factors independently in different cell populations: a single promoter fragment responds to JAK/STAT signaling and activates tsl expression in both the BCs and PFCs, whereas another enhancer drives tsl expression in the CCs. Moreover, putative STAT binding sites (consensus TTCNNNGAA) were found in the identified BC/PFC enhancer. Mutations in those sites greatly reduce tsl-lacZ expression in the BCs and PFCs, pointing to a direct regulation by the JAK/STAT pathway. The fact that some reporter expression can occasionally be detected in those cells even when these sites are mutated could be attributed to regulation by other factors, which could also contribute to tsl expression in BCs and PFCs. In this regard, microarray analysis has shown that activity of the slbo transcription factor, which has been shown to function as a simple transcriptional activator and whose expression is also dependent on the JAK/STAT pathway, induces a 2-fold increase of tsl expression. In summary, these results show that the JAK/STAT pathway acts as a primary regulator of tsl expression in the BCs and PFCs (Furriols, 2007).

The JAK/STAT pathway is triggered in the Drosophila egg chamber by localized expression of its ligand, Upd, in two polar cells at each end of the chamber. Signaling from this pathway is responsible for the patterning of the follicle cells at both ends of the egg chamber, and the results show now that it is also responsible for tsl expression in the BCs and the PFCs. Thus, these results indicate that a common mechanism is responsible for initially patterning the egg chamber terminal epithelium and later triggering the mechanism that specifies the embryonic terminal regions (Furriols, 2007).

At the anterior end of the egg chamber, three populations of follicle cells can be distinguished: the BCs, the CCs, and the stretched cells in between. Among those, BCs and CCs, but not stretched cells, express tsl. Although the role of the JAK/STAT pathway in patterning the follicle cells at both ends of the egg chamber is well established, there are conflicting data about whether a gradient of its ligand, Upd, could indeed be responsible for patterning all of the anterior follicle cells. If that was the case, it might be expected that the JAK/STAT pathway could play a role in tsl expression in both the BCs and the CCs. In this scenario, absence of tsl expression in the stretched cells could be due to specific mechanisms of tsl gene repression in those cells. Conversely, the current results show that the JAK/STAT pathway does not have a specific role in the activation of tsl in the CCs. These results do not necessarily argue against a gradient of Upd. It could be argued, for example, that lower levels of JAK/STAT signaling in the CCs might not be sufficient to trigger activation of the BC/PFC enhancer. Alternatively, it could also be the case that a specific repressor element in this enhancer might inhibit its expression in the CCs. However, irrespective of a role of the Upd gradient in patterning the follicle cells, the results show that tsl expression in the CCs is independent of JAK/STAT. This result indicates that there are JAK/STAT-independent differences within the anterior epithelial cells of the egg chamber, as has been hypothesized (Furriols, 2007).

Feedback inhibition of JAK/STAT signaling by Apontic is required to limit an invasive cell population

In both normal development and in a variety of pathological conditions, epithelial cells can acquire migratory and invasive properties. Border cells in the Drosophila ovary provide a genetically tractable model for elucidating the mechanisms controlling such behaviors. An apontic (apt) mutant has been identified in which the migratory population expands and separation from the epithelium is impeded. This phenotype resembles gain-of-function of JAK/STAT activity. Gain-of-function of APT also mimics loss of function of STAT and its key downstream target, SLBO. APT expression is induced by STAT, which binds directly to sites in the apt gene. The data suggest that a regulatory circuit between STAT, APT, and SLBO functions to convert an initially graded signal into an all-or-nothing activation of JAK/STAT and thus to proper cell specification and migration. These findings are supported by a mathematical model, which accurately simulates wild-type and mutant phenotypes (Starz-Gaiano, 2008).

In many migratory cell types, including metastatic carcinomas, motile cells must detach from an epithelium to move to their final location. However the precise mechanisms by which cells disengage from their neighbors remain poorly understood, and in most cases it is not possible to view the process directly in vivo. Border cells in the Drosophila ovary represent a model for studying epithelial cell migration in vivo that is amenable both to genetic approaches and live imaging. This study reports the identification of a mutant, apt, in which the distinction between invasive and noninvasive cells was compromised. In apt mutants, as the border cell cluster moved away from the epithelium, additional migratory cells -- the stretched border cells -- ingressed in between the nurse cells. These stretched border cells maintained connections with both the main cluster of border cells, and the outer epithelial cell layer, resulting in a defect in detachment (Starz-Gaiano, 2008).

Recent technological advances have enabled analysis of border cells throughout their six hour migration by live imaging. Time-lapse movies of wild-type egg chambers revealed that the process of border cell detachment is surprisingly slow. This indicates that the ability to extend and retract protrusions is not sufficient for the border cells to exit the epithelium, and that there is sufficient time for transcriptional events to contribute to the process. In apt mutants, the border cells rounded up and advanced in between the nurse cells normally, but cells with an apparently intermediate identity were frequently trapped in between the border cell cluster and the follicle cell epithelium, unable to detach from either one. Thus, the two cell types must be clearly distinguished in order for them to be able to disconnect from one another (Starz-Gaiano, 2008).

In a variety of contexts throughout development, a graded distribution of a signaling molecule in a field of cells can elicit discrete cellular responses. Such threshold-like behavior can be achieved by positive autoregulation. Therefore, prior to the current work, it would have been reasonable to propose that STAT autoregulation could convert initially graded activity in the follicular epithelium to 'on' and 'off' states. In wild-type, the migrating border cell cluster takes the source of JAK/STAT activation (UPD expressed by the polar cells) with it, reinforcing SLBO expression in the migratory cells and removing the source of JAK/STAT activation from the anterior follicle cells. So, one could have postulated that the physical separation of the JAK/STAT signaling center from the anterior follicle cells was sufficient to create a significant difference in levels of STAT activity between the migrating cells and those left behind, and thus to distinguish the two cell types and behaviors. However, unexpectedly this study showed that neither STAT autoregulation nor the movement of the signaling center is sufficient to convert the gradient into a step function in the absence of APT (Starz-Gaiano, 2008).

It is proposed instead that feedback inhibition of JAK/STAT combined with the mutual repression of APT and SLBO is responsible for generating the stepwise activation pattern. When two genes mutually repress each other, a slight increase in the activation of one leads to a stronger repression of the second, which, in turn, leads to a further increase of the first. Thus, together these two genes behave as an autocatalytic system. Since apt and slbo are both targets of STAT activity, a three-component regulatory circuit is proposed. The mathematical model demonstrates that this circuit is sufficient to explain what is observed in vivo. In the absence of APT, JAK/STAT activation takes place in an enlarged region and, remarkably, the 'on-off' character of the JAK/STAT activation is lost. This suggests that the threshold behavior of the system does not result from JAK/STAT autoregulation but from the mutual repression of APT and SLBO (Starz-Gaiano, 2008).

The model that most accurately simulates the wild-type and mutant phenotypes is one in which SLBO antagonizes APT activity more strongly than its expression. This is consistent with experimental observation that overexpression of SLBO completely mimics the apt loss-of-function phenotype, but only reduces and does not eliminate APT expression (Starz-Gaiano, 2008).

It is striking that different patterns of SLBO and APT are induced by the same gradient of JAK/STAT activity. An important consequence is that, at high concentrations of active STAT, more SLBO is produced than APT. One way this could be explained is through the observation that STAT binds four different sites in the slbo enhancer with differing affinities. In cells with high concentrations of STAT, more sites, including low affinity sites, should be occupied and thus a higher level of slbo expression generated than in cells with lower STAT levels. In contrast, the apt gene contains only two detectable STAT binding sites, to which STAT can bind nearly as well as it binds the optimal STAT consensus sequence. Thus, apt expression should turn on in response to lower levels of active STAT than slbo and also should saturate at relatively low concentrations of active STAT, yielding a broad and shallow expression gradient across the anterior field of follicle cells. These are precisely the expression patterns observed. Therefore, in cells adjacent to the polar cells, SLBO wins the competition whereas further away from the source of UPD, APT wins the APT/SLBO competition. Higher levels of SLBO block the repression of JAK/STAT by APT in the cells next to the polar cells, causing an even stronger JAK/STAT activation and so on (Starz-Gaiano, 2008).

In addition, evidence was found for a low level of JAK/STAT-independent APT expression, which was also incorporated into the model. This baseline APT expression depended on the transcription factor known as Eyes absent, and based on the model it is proposed that its function could be to prevent any possibility of a renewed trigger of the JAK/STAT pathway in the cells that remain in the anterior epithelium (Starz-Gaiano, 2008).

The JAK/STAT pathway is highly conserved from insects to mammals and is critically important in development, immunity, and inflammation. Intriguingly, Drosophila APT is expressed in many domains where JAK/STAT signaling occurs, including embryonic trachea and the hub of the testes. In addition, apt has been uncovered as a downstream target of STAT in microarray analysis of testis and border cells. Therefore, apt may be a downstream target of STAT signaling in a variety of cell types (Starz-Gaiano, 2008).

It is also possible that this relationship is conserved in other animals, since genes highly related to apt are found in all sequenced insect genomes. In humans, the closest gene to apt is fibrinogen silencer-binding protein (FSBP). Interestingly, two strong loss-of-function alleles of apt contain missense mutations in well-conserved residues, demonstrating the functional significance of this region. Although FSBP has not been extensively characterized, it has been reported to be a negative regulator of the gamma chain of fibrinogen transcription. Fibrinogen is highly expressed in hepatocytes in response to inflammatory cytokine-mediated activation of the JAK/STAT pathway, and there are at least three STAT3 binding sites on the human gamma-fibrinogen promoter. This suggests that APT and FSBP could fulfill similar functions as negative regulators of STAT-responsive genes (Starz-Gaiano, 2008).

All of the major growth factor and cytokine signaling pathways are subject to extensive positive and negative feedback regulation, which is crucial to generate appropriate physiological responses. The work presented here establishes APT as a feedback inhibitor of JAK/STAT signaling and cell invasion (Starz-Gaiano, 2008).

Zfh-1 controls somatic stem cell self-renewal in the Drosophila testis and nonautonomously influences germline stem cell self-renewal

The ability of adult stem cells to maintain their undifferentiated state depends upon residence in their niche. While simple models of a single self-renewal signal are attractive, niche-stem cell interactions are likely to be more complex. Many niches have multiple cell types, and the Drosophila testis is one such complex niche with two stem cell types, germline stem cells (GSCs) and somatic cyst progenitor cells (CPCs). These stem cells require chemokine activation of Jak/STAT signaling for self-renewal. The transcriptional repressor Zfh-1 has been identified as a presumptive somatic target of Jak/STAT signaling, demonstrating that it is necessary and sufficient to maintain CPCs. Surprisingly, sustained zfh-1 expression or intrinsic STAT activation in somatic cells caused neighboring germ cells to self-renew outside their niche. In contrast, germline-intrinsic STAT activation was insufficient for GSC renewal. This data reveals unexpected complexity in cell interactions in the niche, implicating CPCs in GSC self-renewal (Letherman, 2008).

Thus Zfh-1 function is required to block differentiation of CPCs. zfh-1 may have conserved function as a stem cell factor, since it is expressed in somatic stem cell types in the Drosophila ovary, and the mammalian homologue zfhx1b is expressed in human ES cells (Boyer, 2005). As zfh-1 is also required to specify the CPC lineage, it joins a growing list of genes that act within a cell lineage to both to specify the lineage and later to maintain that cell type in the stem cell state (Letherman, 2008).

In the testis, the enrichment of Zfh-1 in CPCs and the fact that zfh-1 can be somatically induced in response to STAT activation suggested that zfh-1 is a target of the STAT self-renewal signal in CPCs. Consistent with this, Zfh-1 accumulation is reduced when STAT function is compromised. While it is not suspected that there will be other STAT targets in CPCs -- two candidates are the CPC-enriched genes SOCS36e and CG2264. zfh-1 is likely a key target, since sustained expression of zfh-1 elicits the same phenotype as somatic activation of STAT (the maintenance of stem cell fate in somatic daughter cells). However, it is also noted that Zfh-1 did not disappear upon stat inactivation, suggesting that another input(s) to CPCs remains to be identified (Letherman, 2008).

The data suggest strongly that the key activity of Zfh-1 is transcriptional repression. This is definitively the case from a gain-of-function test, and given the requirement found for the co-repressor CtBP in the somatic lineage, likely also so for Zfh-1 in CPCs in the niche. This is significant as recent embryonic stem cell studies suggest that a primary function for the self-renewal factors Nanog, Oct4, and Sox2 is to repress master regulators of differentiation pathways. Zfh-1 and its mammalian homologue zfhx1b repress various differentiation genes, including mef2 for muscle differentiation, brachyury for mesoderm formation, and E-cadherin for epithelial differentiation. In the testis niche, eya may be a target of Zfh-1 repression, since it is rapidly activated when zfh-1 is removed. Similarly, the Eya transcription factor is an essential differentiation factor in the somatic lineage. Thus, Zfh-1 shares functional similarity with the ES cell self-renewal factors in blocking differentiation (Letherman, 2008).

Among other potential targets for Zfh-1 in the CPCs is the repression of genes regulated by EGFR/raf signaling. Prior work showed that EGFR signaling is required in somatic cells, and the transcriptional branch of the EGFR pathway culminates in the activation of ETS transcription factors, regulating gene expression in the somatic cells. Interestingly, transcriptional repression by members of the Zfh-1 family can be overcome by ETS proteins acting in synergy with the transcription factor c-Myb. Perhaps in the testis niche Zfh-1 prevents differentiation by inhibition of ETS targets until an essential ETS synergizing factor is produced, or until Zfh-1 levels simply decay in CPC daughters (Letherman, 2008).

The ability of sustained zfh-1 expression to non-autonomously cause continual GSC self-renewal is a surprising result that questions the idea that germline Jak/STAT activation is instructive for GSC self-renewal. The data shows that Jak/STAT activation in the GSCs is not sufficient for GSC renewal outside the niche; rather, Jak/STAT activation in CPCs (with Zfh-1 activation) is necessary for niche independence. The gain-of-function experiment suggests several possibilities for normal GSC renewal. One is that Zfh-1 normally inhibits a differentiation signal sent from somatic to germ cells, and as a consequence of sustaining Zfh-1, GSCs stay in a 'default' stem cell state. This option is supported by the ability of GSCs to proliferate in the absence of EGFR activation in somatic cells. A second possibility is that both STAT activation and a zfh-1-dependent signal delivered by somatic cells are required for GSC renewal. For instance, the zfh-1-dependent signal could activate a transcription factor in germ cells that assisted phosphorylated STAT in GSC gene regulation. In this scenario, a requirement for STAT phosphorylation might be bypassed if enough of the second factor is produced (as when zfh-1 expression is sustained in CPC daughters). Interestingly, mouse ES cells require two signals for self-renewal: LIF activation of STAT3 and BMP activation of SMADs. A requirement for BMP signaling in testis GSCs has already been demonstrated, and it has been suggested that Jak/STAT signaling leads to production of the BMP ligand Dpp. However, a BMP ligand could only constitute part of the signal, as overexpression of BMP does not cause the same phenotype as does sustaining zfh1 expression (Letherman, 2008).

A requirement for a second signal assisting in GSC self-renewal has precedent in the Drosophila ovarian niche. There, a BMP was the first signaling pathway discovered to act in GSC self renewal. However, newly identified somatic escort stem cells (ESCs) surround GSCs, just as CPCs do in the testis. Like CPCs, ESCs intrinsically require STAT, as STAT loss non-autonomously causes GSC loss, demonstrating the requirement for an ESC-dependent signal in GSC renewal. In both the female and male niches, if somatic stem cell types are indeed required for GSC renewal, this would provide a means of balancing the two stem cell populations, since GSCs would not be able to survive without CPCs/ESCs, and an overabundance of CPCs/ESCs might increase the available GSC 'renewal' signal, causing additional germ cells to become GSCs via dedifferentiation or symmetric stem cell division. Proof of such a requirement for CPCs in the testis will require removing zfh-1 from many or most CPCs; since zfh-1 mutant CPCs are lost so rapidly, it has not been possible to follow the fate of neighboring GSCs. Attempts to knockdown zfh-1 in all CPCs by dsRNA transgenesis have thus far been unsuccessful in achieving significant reduction of Zfh-1 protein (Letherman, 2008).

This work suggests the existence of a hub to CPC to GSC self-renewal relay signal: Upd, secreted by hub cells, activates Zfh-1 in CPCs, which in turn causes a signal to be sent to the GSCs resulting in their self-renewal. Whether this relay is a required component of the GSC renewal, or simply an amplification of a hub renewal signal, it demonstrates a higher degree of complexity in cell-cell interactions than has been previously found in a stem cell niche. Recent work in other niches increasingly points toward the existence of (and a need for) such complex interactions. For example, in the Drosophila ovary, a feedback loop between stem cells and niche cells has recently been discovered. Similar to the Drosophila testis, the mammalian hair follicle niche supports two stem cell populations -- melanocyte and hair follicle stem cells. The potential for coordination between these two populations has not been explored, and could be relevant to the prevention of melanocyte stem cell loss, which results in hair graying. Neural stem cells were recently found to be much more diverse than expected, and their identity is dependent on their location, implying that niche signals for these stem cells must do more than just keep them in an undifferentiated state. Finally, hematopoietic stem cells reside in two distinct niches, associated with either osteoblasts or endothelial cells. Both niches require a second cell type, CXCL12-abundant reticular cells, for stem cell maintenance. How signals from these different cell types interact to coordinate self-renewal is completely unknown. Models based on the self-renewal relay described here will be a starting point for beginning to explore the complex cell interactions in these niches (Letherman, 2008).

chinmo is a functional effector of the JAK/STAT pathway that regulates eye development, tumor formation, and stem cell self-renewal in Drosophila

The Drosophila STAT transcription factor Stat92E regulates diverse functions, including organ development and stem cell self-renewal. However, the Stat92E functional effectors that mediate these processes are largely unknown. This study shows that chinmo is a cell-autonomous, downstream mediator of Stat92E that shares numerous functions with this protein. Loss of either gene results in malformed eyes and head capsules due to defects in eye progenitor cells. Hyperactivation of Stat92E or misexpression of Chinmo results in blood cell tumors. Both proteins are expressed in germline (GSCs) and cyst stem cells (CySCs) in the testis. While Stat92E is required for the self-renewal of both populations, chinmo is only required in CySCs, indicating that Stat92E regulates self-renewal in different stem cells through independent effectors. Like hyperactivated Stat92E, Chinmo misexpression in CySCs is sufficient to maintain GSCs nonautonomously. Chinmo is therefore a key effector of JAK/STAT signaling in a variety of developmental and pathological contexts (Flaherty, 2010).

This study has revealed important information about chinmo and its role in Stat92E-dependent biological processes, including eye development, hematopoeisis, and stem cell self-renewal. chinmo was identified as a cell-autonomously induced downstream mediator of JAK/STAT activity that shares loss- and gain-of-function phenotypes with Stat92E in several tissues. Although chinmo was originally identified in a screen for genes required for temporal identity of mushroom body neurons, no factors that regulate its expression had been identified. The fact that chinmo and Stat92E exhibit a high degree of functional overlap suggests that chinmo performs multiple Stat92E-dependent functions, including growth of the eye disc, formation of melanotic tumors, proliferation of mature hemocytes, self-renewal of adult stem cells, and repression of Ser. Furthermore, the results raise the interesting hypothesis that the JAK/STAT pathway is also required for the temporal identity of neurons in the mushroom body. It was also shown that Chinmo, like stabilized Stat92E, is expressed in GSCs and in CySCs in the testis. However, unlike Stat92E, Chinmo is required intrinsically only for the self-renewal of CySCs and not of GSCs. These data clearly indicate that Stat92E acts through distinct effector genes in these stem cells to promote cell-autonomous self-renewal. Finally misexpression of chinmo in CySCs results in the expansion of GSCs and CySCs, a phenotype also observed with misexpression of hopTum-l or of zfh1 in somatic cells. This provides additional evidence for the coordination of self-renewal and differentiation between adjacent GSCs and CySCs (Flaherty, 2010).

The BTB domain mediates protein-protein interactions, including dimerization, recruitment of transcriptional repressors to DNA, and protein degradation by acting as adaptors for Cul-3 E3 ubiquitin ligases. In the antenna, Ser was found to be cell-autonomously repressed by both Stat92E and Chinmo (this study; Flaherty, 2009). These data suggest that Chinmo might act as a transcriptional repressor, at least in the antennal disc, and that Ser might be one of its transcriptional targets. It should be stressed that these results do not rule out the possibility that Chinmo, through its BTB domain, can also act as an adaptor for Cul-3 and promote protein degradation. In fact, recent work has revealed that even BTB-ZF transcription factors like promyelocytic leukemia zinc finger (PLZF) can interact with Cul-3. Whereas the role of the BTB domain in PLZF-dependent transcriptional repression has been well documented, the physiological role of the PLZF-Cul-3 interaction and the proteins it modifies are as yet unknown. Future experiments will be needed to determine if Chinmo can act both as a transcriptional repressor per se and as an adaptor for E3 ligases. In either scenario, factors modified by Chinmo, in addition to Ser, will need to be identified and characterized (Flaherty, 2010).

This study shows that Chinmo, like Zfh1, is essential for the self-renewal of CySCs in the Drosophila testis. Furthermore, sustained expression of Chinmo in somatic cells, like that of Zfh1, is sufficient to induce the expansion of GSCs and CySCs. In addition, chinmo and zfh1 do not regulate each other's expression. chinmo and zfh1 both appear to act downstream of Stat92E to maintain CySCs, which raises the possibility that these factors function either in an epistatic or parallel manner in the somatic lineage (this study; Leatherman, 2008). chinmo does not act through zfh1, but it was not possible to determine if the reciprocal was true. However, it is hypothesized that if zfh1 is upstream of chinmo then CySCs lacking either of these factors should differentiate at the same time point. In fact, CySCs lacking zfh1 differentiate faster than those lacking chinmo, suggesting that zfh1 may not reside upstream of chinmo. Despite the unresolved genetic relationship between zfh1 and chinmo, the data are consistent with a model in which they function in a parallel pathway in the self-renewal of CySCs and the expansion of GSCs and CySCs (Flaherty, 2010).

Zfh1 has been shown to act as a transcriptional repressor (see Leatherman, 2008). The current data suggest that Chinmo may inhibit transcription directly or by post-translational modification of factors that silence genes. Zfh1 is expressed highly in CySCs and at low levels in early cyst cells. It is not expressed in late cyst cells. In contrast, Chinmo is expressed at high and comparable levels in CySCs and early cyst cells, but not in late cyst cells. Taking into account all of these results, two models are proposed to explain the function of Chinmo in the somatic lineage of the testis. The first hypothesizes that Zfh1 and Chinmo regulate distinct downstream effectors, all of which are required for the maintenance of CySCs. In this model, early cyst cells, which express high levels of Chinmo but low levels of Zfh1, can become late cyst cells only when Chinmo expression is sufficiently decreased there, allowing for full cyst cell differentiation and the complete development of mature germ cells. In the second model, Chinmo and Zfh1 regulate different genes critical for CySC self-renewal, but in contrast to the first, Chinmo only has a function in CySCs and not in early cyst cells. In this second model, the existence is invoked of cofactors expressed only in CySCs that act in concert with Chinmo, thereby restricting Chinmo function only to CySCs (Flaherty, 2010).

Activation of the JAK/STAT signaling pathway in CySCs is sufficient to promote self-renewal of both CySCs and GSCs, indicating that CySCs can influence GSC maintenance (Leatherman, 2008). Although the mechanisms by which CySCs regulate GSC self-renewal have not yet been elucidated at the molecular level, two models have been proposed. Activated Stat92E, through its functional effectors, could (1) block CySC differentiation intrinsically, thus also inhibiting GSC differentiation at the same time, or (2) send one or more non-cell-autonomous signals from CySCs to GSCs, thus promoting GSC self-renewal. Either model would result in an increase in the number of CySCs and GSCs. In the first model, sustained activation of JAK/STAT signaling in CySCs allows these cells to continue proliferating. Therefore, they accumulate outside of the niche. Previous studies have shown that the mitoses of GSCs and CySCs must be linked in order to have the appropriate number of GSCs, and their progeny always encapsulated by two CySCs/cyst cells. In the second model, the self-renewal of GSCs depends on two independent signals: one is Upd sent from the niche, which activates Stat92E in adjacent GSCs, and the other is an unknown factor presumed to come from the neighboring CySCs. A similar situation occurs in female flies, where two signals are required for GSC maintenance. First, cap cells, which form the ovarian niche, produce Decapentaplegic (Dpp), which acts on GSCs by inhibiting the bam gene. Second, an Upd cytokine, produced by ovarian somatic support cells adjacent to the niche, acts on cap cells to increase Dpp production, thus influencing GSC self-renewal in a nonautonomous manner. It is currently unknown whether the nonautonomous signal from CySCs to GSCs in the testis is a secreted factor (Flaherty, 2010).

The results indicate that Chinmo has an important role in the self-renewal of CySCs, in the inhibition of CySC differentiation, and in the transduction of the nonautonomous signal from CySCs to GSCs. Furthermore, this expansion requires the BTB and ZF domains in Chinmo, suggesting that the molecular function of Chinmo in this process may be transcriptional repression and/or protein degradation. The fact that somatic misexpression of Chinmo, like that of Zfh1, can promote GSC self-renewal/expansion indicates that at least three factors play important roles in regulating stem cell self-renewal in a nonautonomous manner: activated Stat92E, Zfh1, and Chinmo. These observations raise many issues, such as the importance of JAK/STAT pathway activity in the soma, whether chinmo can bypass the requirement for Stat92E in CySCs, and the mechanism of nonautonomous self-renewal between adjacent stem cell populations. These issues need to be addressed at the molecular level in the future (Flaherty, 2010).

A protein BLAST search against the nonredundant mouse database identified mZFP509 as a potential Chinmo ortholog. mZFP509 is a 757 amino acid protein that has the same overall structure as Chinmo: an N-terminal BTB domain located between residues 20-120 separated from two C-terminal C2H2 zinc fingers by a stretch of ~400 amino acids. mZFP509 is 27% identical to Chinmo (with 36% identity in the BTB domain and 31% identity the ZF region) and is 83.7% identical to hZFP509. Microarray studies indicate that mzfp509 is enriched in normal and cancer stem cells. mzfp509 transcripts are present in mESCs but are substantially reduced during their differentiation. They are also significantly increased in PU.1-deficient preleukemic hematopoietic stem cells and normal mammary stem cells. These data suggest that ZFP509 and Chinmo are orthologs and that what is discovered about Chinmo in Drosophila may have a high probability of holding true for its mammalian counterpart. For example, blocking hZFP509 function may have therapeutic value in inhibiting cancer stem cells, thus offering better outcomes for human patients (Flaherty, 2010).

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. This study analyzed an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. The maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and evidence is presented that Zld binds to multiple non-canonical sites. A combination of in vitro binding experiments and bioinformatics analysis was used to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo (Struffi, 2011).

The experiments described in this study significantly refine understanding of how the eve 3+7 enhancer functions in the early embryo. In particular, it was shown that the maternal zinc-finger protein Zld is absolutely required for STAT-mediated enhancer activation, and that the gap proteins Kni and Hb establish stripe boundaries by directly binding to multiple sites within the enhancer (Struffi, 2011).

When first activated in late nuclear cycle 13, the minimal eve 3+7 enhancer drives weak stochastic expression in a broad central pattern, which refines in cycle 14 to a stripe that is about four nuclei wide. By contrast, stripe 7 expression, which is visible by enzymatic staining methods, is nearly undetectable using fluorescence in situ hybridization (Struffi, 2011).

Previous work showed that stripe 7 shares regulatory information with stripe 3 but is also controlled by sequences located between the minimal stripe 3+7 and stripe 2 enhancers, and possibly by sequences within and downstream of the stripe 2 enhancer. Thus, stripe 7 is unique among the eve stripes in that it is not regulated by a discrete modular element (Struffi, 2011).

Previous work showed that the terminal gap gene tailless (tll) is required for activation of eve 7. However, since the Tll protein probably functions as a dedicated repressor, it is likely that activation of eve 7 by Tll occurs indirectly, through repression of one or more repressors (Struffi, 2011).

The ubiquitous maternal protein Zld is required for the in vivo function of both the eve 3+7 and eve 2 enhancers, which are activated by the JAK-STAT pathway and Bicoid (Bcd), respectively. Zld was previously shown to bind to five sequence motifs that are over-represented in the regulatory regions of early developmental genes. Mutations of the single TAGteam site in the eve 3+7 enhancer caused a reduction in expression, but zld M- embryos, mutant for maternal zld expression, showed complete abolishment of eve 3+7-lacZ reporter gene expression. Also, the eve 2 enhancer, which does not contain any canonical TAGteam sites, is nonetheless inactive in zld M- embryos. This study showed that this enhancer contains at least four variants of the TAGteam sites, which suggests that Zld binding to non-canonical sites is crucial for its function in embryogenesis. ChIP-Chip data show that Zld binding extends throughout much of the eve 5' and 3' regulatory regions (Struffi, 2011).

The implication of such broad binding and the requirement for Zld for activation of two eve enhancers are consistent with its proposed role as a global activator of zygotic transcription. How might this work? One possibility is that there are cooperative interactions between Zld and the other activators of these stripes. A non-exclusive alternative is that Zld binding creates a permissive environment in broad regions of the genome, possibly by changing the chromatin configuration and making it more likely that the other activator proteins can bind. However, it is important to note that eve expression is not completely abolished in zld M- embryos, so at least some eve regulatory elements could function in the absence of Zld. Future experiments will be required to further characterize the role of Zld in the regulation of the entire eve locus (Struffi, 2011).

The genetic removal of kni causes a broad expansion of eve 3+7- lacZ expression in posterior regions of the embryo, and ectopic Kni causes a strong repression of both stripes. Interestingly, the posterior boundary of eve stripe 3 is positioned in regions with extremely low levels of Kni protein. If the stripe 3 posterior boundary is solely formed by Kni, the enhancer must be exquisitely sensitive to its repression, possibly through the high number of sites in the eve 3+7 enhancer. Previous attempts to mutate sites based on computational predictions failed to mimic the genetic loss of kni, so this study used a biochemical approach to identify Kni sites in an unbiased manner. EMSA analyses identified 11 Kni sites, and the PWM derived from these sites alone is very similar to the Kni matrix derived in a bacterial one-hybrid study. Thus, these studies provide biochemical support for the bacterial one-hybrid method as an accurate predictor of the DNA-binding activity of this particular protein (Struffi, 2011).

It was further shown that specific point mutations abolish binding to nine of the 11 sites, and when these mutations were tested in a reporter gene they caused an expansion that is indistinguishable from that detected in kni mutants. This result strongly suggests that Kni-mediated repression involves direct binding to the eve 3+7 enhancer, and that Kni alone can account for all repressive activity in nuclei that lie in the region between stripes 3 and 7. However, this work does not address the exact mechanism of Kni-mediated repression. The simplest possibility is that Kni competes with activator proteins for binding to overlapping or adjacent sites. This mechanism is considered unlikely because only one of the 11 Kni sites overlaps with an activator site. Also, the in vivo misexpression of a truncated Kni protein (Kni 1-105) that contains only the DNA-binding domain and the nuclear localization signal has no discernible effect on the endogenous eve expression pattern, whereas a similar misexpression of Kni 1-330 or Kni 1-429 strongly represses eve 3+7 (Struffi et al., 2004) (Struffi, 2011).

Whereas Kni-mediated repression forms the inside boundaries of the eve 3+7 pattern, forming the outside boundaries is dependent on Hb, which abuts the anterior boundary of stripe 3 and overlaps with stripe 7. Both stripes expand towards the poles of the embryo in zygotic hb mutants, and these expansions are mimicked by mutations in four or all nine Hb sites within the eve 3+7 enhancer. Further anterior expansions of the pattern are prevented by an unknown Bcd-dependent repressor (X) and the Torso (Tor)-dependent terminal system. Indeed, eve 3+7-lacZ expression expands all the way to the anterior tip in mutants that remove bcd and the terminal system (Struffi, 2011).

The mutational analyses suggest that Hb is a dedicated repressor of the eve 3+7 enhancer, and argue against a dual role in which high Hb levels repress, whereas lower concentrations activate, transcription. One caveat is that activation of the stripe might occur via maternal Hb in the absence of zygotic expression. However, triple mutants that remove zygotic hb, kni and tor, a terminal system component, show eve 3+7 enhancer expression that extends from ~75% embryo length (100% is the anterior pole) to the posterior pole. It is extremely unlikely that the maternal Hb gradient, which is not perturbed in this mutant combination, could activate expression throughout the posterior region. It is proposed that any activating role for Hb on this enhancer is indirect and might occur by repressing kni, which helps to define a space where the concentrations of both repressors are sufficiently low for activation to occur. kni expands anteriorly in hb mutants and is very sensitive to repression by ectopic Hb, consistent with an indirect role in activation. A similar mechanism has been shown to be important for the correct positioning of eve stripe 2. In this case, the anterior Giant (Gt) domain appears to be required for eve 2 activation, but it does so by strongly repressing Kr, thus creating space for activation in the region between Gt and Kr (Struffi, 2011).

The correct ordering of gene expression boundaries along the AP axis is crucial for establishing the Drosophila body plan. All gap genes analyzed so far seem to function as repressors that differentially position multiple boundaries. However, it is still unclear how differential sensitivity is achieved at the molecular level. Simple correlations of binding site number and affinity with boundary positioning cannot explain the exquisite differences in the sensitivity of individual enhancers, suggesting that they do more than 'count' binding sites and that specific arrangements of repressor and activator sites might control this process. The experiments described here better define the binding characteristics of both Hb and Kni and provide a firm foundation for future experiments designed to decipher the regulatory logic that controls differential sensitivity (Struffi, 2011).

An efficient approach to isolate STAT regulated enhancers uncovers STAT92E fundamental role in Drosophila tracheal development

The ventral veinless (vvl) and trachealess (trh) genes are determinants of the Drosophila trachea. Early in development both genes are independently activated in the tracheal primordia by signals that are ill defined. Mutants blocking JAK/STAT signaling at any level do not form a tracheal tree suggesting that STAT92E may be an upstream transcriptional activator of the early trachea determinants. To test this hypothesis STAT92E responsive enhancers activating the expression of vvl and trh in the tracheal primordia were sought. STAT92E regulated enhancers can be rapidly and efficiently isolated by focusing the analysis on genomic regions with clusters of putative STAT binding sites where at least some of them are phylogenetically conserved. Detailed analysis of a vvl early tracheal enhancer shows that non-conserved sites collaborate with conserved sites for enhancer activation. It was found that STAT92E regulated enhancers can be located as far 60 kb from the promoters. The results indicate that vvl and trh are independently activated by STAT92E which is the most important transcription factor required for trachea specification (Sotillos, 2010).

Mutations in the JAK/STAT pathway result in embryos only forming residual trachea fragments. This is caused by the abnormal activation of the early tracheal genes trh and vvl suggesting that the early trachea enhancers may be directly regulated by STAT92E in which case the trachea enhancers would be associated to STAT92E binding sites (Sotillos, 2010).

To test this the putative STAT92E binding sites were first localized in silico in the vvl 144 kb intergenic region. Then in vivo the enhancer activity was tested of regions that either (1) contain putative STAT92E sites that are conserved in several Drosophila species; (2) contain non-conserved putative STAT92E sites; or (3) contain no putative STAT92E sites. Of the 12 reporter lines made 10 have enhancer activity consistent with harboring embryonic vvl cis-regulatory elements. The expression of the enhancers either lacking STAT92E sites or containing non-conserved STAT92E sites is independent of JAK/STAT function. In contrast, of the seven vvl enhancers containing conserved sites, the expression of three of them required JAK/STAT regulation in the embryo. One of these enhancers drives expression in the hindgut and two are expressed in the trachea at stage 10. These results suggested that exclusively looking for conserved STAT92E sites would be sufficient to localize the STAT92E regulated sites, a prediction confirmed by isolating the trh gene tracheal enhancers. Although the presence of cryptic STAT binding sites that diverge from the ideal consensus could not be discarded in this analysis, such elements will probably have a minor contribution as ignoring their existence allowed finding of the main regulatory elements. Analysis of only eight fragments comprising a seventh of the trh locus was sufficient to find the early tracheal STAT92E responsive elements. This analysis revealed that the locus extends at least 40 kb upstream the trh promoter, with some enhancers located beyond the first predicted neighboring gene (Sotillos, 2010).

In both the trh and vvl genes it was found that other late tracheal enhancers are associated to STAT92E conserved sites suggesting that the STAT92E protein may be repeatedly used to control tracheal gene expression during development. This possibility is backed by the fact that upd is transcribed up to stage 13 and phosphorylated STAT92E can be detected in the trachea well after the stage 10 early specification stage. This late trachea specific expression of upd depends on trh suggesting that a feed-back loop maintains JAK/STAT activity during tracheal development (Sotillos, 2010).

It was confirmed that the expression of the vvl1+2 early tracheal enhancer depends on STAT92E by mutating its putative STAT92E binding sites. The results show that mutation of all three putative STAT92E sites in the vvl1+2 enhancer causes a severe loss of expression, indicating that a bona-fide direct enhancer was isolated. However, other possible direct enhancers like the vvlds1.5 hindgut element show an unexpected behavior. While vvlds1.5 perfectly recapitulates the vvl early hindgut activation its expression does not stop at stage 15, but keeps transcribing lacZ up to stage 17 well after the endogenous vvl gut transcription ceased. Despite this abnormal behavior, and pending direct site mutagenesis confirmation, it is believeed that these experiments show convincingly that the analysis of the genomic regions containing conserved STAT binding site clusters is an efficient way to quickly identify direct STAT92E regulated enhancers (Sotillos, 2010).

An important finding of this analysis has been the observation that STAT regulated enhancers can be tens of kilobases away from the transcriptional start of the target gene, even separated by another predicted gene. This indicates that STAT regulated enhancers can be functional at great distances and that search of STAT binding sites should not be restricted to the immediate vicinity of a gene (Sotillos, 2010).

Assuming there was no base content bias, the probability of finding in the genome either a 3n or a 4n STAT92E site by chance is (2 × 1/46) which is close to one site every 2 kb. In the 144 kb vvl genomic region there are 85 putative STAT92E sites, that is, 15 more than the expected 70 sites. Similarly, in the 70 kb trh locus there are 53 putative sites, 19 more than the expected 34 sites. The excess of sites found can be partially explained by selective pressure as there are 20 conserved sites in vvl. However, in the trh locus the 9 conserved STAT92E sites represent only half of the observed excess. An additional explanation for the excess STAT92E sites could be provided by the observation that in the regions were evidence was found for JAK/STAT regulation, there are clusters of conserved and non-conserved sites. It has been suggested that STAT site clustering helps forming tetramers that co-operatively increase STAT transcriptional output. Although STAT92E may form similar tetrameres in Drosophila, the distance of the clustered sites observed in this study is probably too large to allow tetramer formation and their existence must serve another purpose. When the vvl1+2 enhancer was subdivided it was observed that the distal non-conserved STAT92E site is dispensable for the enhancer function. However mutagenesis of the two conserved sites in vvl1+2 without mutating the distal non-conserved site has a mild decrease in the enhancer expression. Only when all three sites were mutated, including the non-conserved site, there is a strong effect on vvl1+2 expression. This shows that non-conserved sites may be functional in vivo even though they are not absolutely necessary. The results indicate that sites appearing distal to a STAT92E regulated enhancer may substitute for the proximal conserved sites, suggesting a way in which novel functional sites could eventually substitute conserved sites during evolution (Sotillos, 2010).

The crb and dome genes, which have been shown in vivo to be STAT92E targets, also show an accumulation of STAT92E sites in the introns where the enhancers localize. In the case of dome, five STAT92E sites cluster in a 700 bp fragment. Although only two of the five sites are conserved, and dissection of the enhancer showed that the conserved sites are crucial for JAK/STAT regulation, the non-conserved sites were also required for full enhancer expression. Therefore, clustered conserved and non-conserved STAT92E sites contribute to target gene regulation. It will be important to understand how STAT92E proteins binding to these distant sites interact with other transcriptional co-factors that presumably bind to the core enhancer (Sotillos, 2010).

The earliest genes activated specifically in the tracheal primordia are trh and vvl. Both genes have cross-regulatory interactions that help maintain each other's expression in the trachea, but their early activation is independent of each other. This study has localized the early trachea enhancers in trh and vvl and have shown that their activation in both cases depends on the JAK/STAT pathway making STAT92E the most important trachea activator. Stage 10 upd expression is consistent with a model where trh and vvl activation in the tracheae primordia is specified by a competitive interaction between the JAK/STAT and the WNT signaling pathways. STAT92E is probably acting with some other transcription factorS as inactivation of the JAK/STAT pathway does not result in a complete lack of expression of the enhancers. These other transcription factors would not necessarily have a restricted spatial expression as the precise positional activation of vvl could be provided by upd and wg. Although the early requirement of STAT92E for early tracheal specification precludes any studies at later stages, the maintained expression of upd in later tracheal development and the presence of STAT92E conserved sites associated to late tracheal enhancers suggest that the pathway is important for vvl and trh expression maintenance during tracheal development (Sotillos, 2010).

The observation that in crustaceans trh and vvl are co-expressed in the epipods that form the gills suggest that both genes where co-opted early in arthropod evolution to control the formation of the respiratory system (Franch-Marro, 2006). It would be interesting to find out if JAK/STAT is also required for trh and vvl expression in the crustaceans as that would suggest that the pathway had been adopted early in evolution fixing the regulation of both genes in the respiratory system (Sotillos, 2010).

Histone demethylase dUTX antagonizes JAK-STAT signaling to maintain proper gene expression and architecture of the Drosophila testis niche

Adult stem cells reside in microenvironments called niches, where they are regulated by both extrinsic cues, such as signaling from neighboring cells, and intrinsic factors, such as chromatin structure. This study reports that in the Drosophila testis niche an H3K27me3-specific histone demethylase encoded by Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (dUTX) maintains active transcription of the Suppressor of cytokine signaling at 36E (Socs36E) gene by removing the repressive H3K27me3 modification near its transcription start site. Socs36E encodes an inhibitor of the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. Whereas much is known about niche-to-stem cell signaling, such as the JAK-STAT signaling that is crucial for stem cell identity and activity, comparatively little is known about signaling from stem cells to the niche. The results reveal that stem cells send feedback to niche cells to maintain the proper gene expression and architecture of the niche. dUTX acts in cyst stem cells (CySCs) to maintain gene expression in hub cells through activating Socs36E transcription and preventing hyperactivation of JAK-STAT signaling. dUTX also acts in germline stem cells to maintain hub structure through regulating DE-Cadherin levels. Therefore, these findings provide new insights into how an epigenetic factor regulates crosstalk among different cell types within an endogenous stem cell niche, and shed light on the biological functions of a histone demethylase in vivo (Tarayrah, 2013).

This study identified a new epigenetic mechanism that negatively regulates the JAK-STAT signaling pathway in the Drosophila testis niche: the H3K27me3-specific demethylase dUTX acts in CySCs to remove the repressive H3K27me3 histone modification near the TSS of Socs36E to allow its active transcription. Socs36E acts upstream to suppress Stat92E activity and to restrict CySCs from overpopulating the testis niche. In addition, dUTX acts in CySCs to prevent hyperactivation of Stat92E in hub cells, which would otherwise ectopically turn on Zfh1 expression. When zfh1 cDNA was ectopically driven in hub cells using the upd-Gal4 driver, no obvious defect could be identified. Therefore, the biological consequence of ectopic Zfh1 expression in hub cells remains unclear. However, ectopic Zfh1 expression in hub cells and the overpopulation of Zfh1-expressing cells around the hub are two connected phenomena because both phenotypes are caused by loss of dUTX in CySCs (Tarayrah, 2013).

UTX also acts in GSCs to regulate DE-Cadherin levels to maintain proper GSC-hub interaction and normal morphology of the hub. It has been reported that differential expression of different cadherins causes cells with similar cadherin types and levels to aggregate. In wt testes, hub cells express higher levels of DE-Cadherin and therefore tightly associate with each other. Loss of dUTX in germ cells leads to higher levels of DE-Cadherin in GSCs, which probably allows them to intermingle with hub cells and causes disrupted hub architecture. It has also been demonstrated that the major role of JAK-STAT in GSCs is for GSC-hub adhesion, suggesting that the expression and/or activity of cell-cell adhesion molecules, such as DE-Cadherin, depends on JAK-STAT signaling. Therefore, the abnormal DE-Cadherin activity in GSCs in dUTX testis could also result from misregulated JAK-STAT signaling in the testis niche. dUTX is a new negative epigenetic regulator of the JAK-STAT signaling pathway (Tarayrah, 2013).

The JAK-STAT signaling pathway plays crucial roles in stem cell maintenance in many different stem cell types across a wide range of species. These studies identify the histone demethylase dUTX as a new upstream regulator of the JAK-STAT pathway, which directly controls the transcription of Socs36E. In addition to acting as an antagonist of JAK-STAT signaling, Socs36E has been reported to be a direct target gene of the Stat92E transcription factor (Terry, 2006). Therefore, increased Stat92E would be expected to upregulate Socs36E expression, but this was not observed in dUTX mutant testes. Instead, the data revealed that Socs36E expression decreased, whereas Stat92E expression increased, in dUTX testes, consistent with the hypothesis that Socs36E is a direct target gene of dUTX and acts upstream of Stat92E (Tarayrah, 2013).

Sustained activity of the JAK-STAT pathway in cyst cells has been reported to activate BMP signaling, which leads to GSC self-renewal outside the niche and gives rise to a tumor-like phenotype in testis. To examine BMP pathway activity, immunostaining experiments were performed using antibodies against phospho-SMAD (pSMAD), a downstream target of BMP signaling. No obvious difference was detected in the pSMAD signal between the dUTX testes and wt control, nor were any germline tumors detected in dUTX testes. It is speculated that germline tumor formation upon activation of the JAK-STAT pathway is secondary to the overproliferation of Zfh1-expressing cells, which was not observed in dUTX testes (Tarayrah, 2013).

This study also provides an example of the multidimensional cell-cell communication that takes place within a stem cell niche. Many studies of the stem cell niche have focused on understanding niche-to- stem cell signaling in controlling stem cell identity and activity. For example, in the Drosophila female GSC niche, Upd secreted from terminal filaments activates the JAK-STAT pathway in cap cells and escort cells, which subsequently produce the BMP pathway ligand Decapentaplegic (Dpp) to activate BMP signaling and prevent transcription of the differentiation factor bag-of-marbles (bam) in GSCs. In the Drosophila intestinal stem cell (ISC) niche, the visceral muscle cells underlying the intestine secrete Wingless to activate Wnt signaling and Upd to activate JAK-STAT signaling in ISCs, which are required to maintain ISC identity and activity (Tarayrah, 2013).

More studies have now revealed the multidirectionality of signaling within the stem cell niche. For example, in the Drosophila female GSC niche, GSCs activate Epidermal growth factor receptor (Egfr) signaling in the neighboring somatic cells, which subsequently represses expression of the glypican Dally, a protein required for the stabilization and mobilization of the BMP pathway ligand Dpp. Through this communication between GSCs and the surrounding somatic cells, only GSCs maintain high BMP signaling. The current studies establish another example of the multidimensional cell-cell communications that occur within the testis stem cell niche, where CySCs and GSCs have distinct roles in regulating hub cell identity and morphology (Tarayrah, 2013).

The data identified new roles of a histone demethylase in regulating endogenous stem cell niche architecture and proper gene expression. Previous studies have reported in vivo functions of histone demethylases in several model organisms. For example, mammalian UTX has been shown to associate with the H3K4me3 histone methyltransferase MLL2, suggesting its potential antagonistic role to the PcG proteins. The PcG proteins play a crucial role in Hox gene silencing in both Drosophila and mammals. Consistently, mammalian UTX has been reported to directly bind and activate the HOXB1 gene locus. In addition to antagonizing PcG function, H3K27me3 demethylases play crucial roles during development. For example, in zebrafish, inactivating the UTX homolog (kdm6al) using morpholino oligonucleotides leads to defects in posterior development, and in C. elegans the dUTX homolog (UTX-1) is required for embryonic and postembryonic development, including gonad development. Furthermore, loss of UTX function in embryonic stem cells leads to defects in mesoderm differentiation, and somatic cells derived from UTX loss-of-function human or mouse tissue fail to return to the ground state of pluripotency. These reports demonstrate that UTX is not only required for proper cellular differentiation but also for successful reprogramming. However, despite multiple reports on the in vivo roles of H3K27me3-specific demethylases, little is known about their functions in any endogenous adult stem cell system (Tarayrah, 2013).

Whereas mammals have multiple H3K27me3 demethylases, dUTX is the sole H3K27me3-specific demethylase in Drosophila. This unique feature, plus the well-characterized nature of Drosophila adult stem cell systems, make interpretation of the endogenous functions of histone demethylases in Drosophila unambiguous. Because mammalian UTX has been reported as a tumor suppressor, understanding the endogenous functions of dUTX in an adult stem cell system might facilitate the use of histone demethylases for cancer treatment. In summary, these results demonstrate that stem cells send feedback to the niche cells to maintain their proper gene expression and morphology. Furthermore, this feedback is regulated through the JAK-STAT signaling pathway, the activity of which is controlled by a chromatin factor, providing an example of crosstalk between these two regulatory pathways (Tarayrah, 2013).

CSN maintains the germline cellular microenvironment and controls the level of stem cell genes via distinct CRLs in testes of Drosophila melanogaster

Stem cells and their daughters are often associated with and depend on cues from their cellular microenvironment. In Drosophila testes, each Germline Stem Cell (GSC) contacts apical hub cells and is enclosed by cytoplasmic extensions from two Cyst Stem Cells (CySCs). Each GSC daughter becomes enclosed by cytoplasmic extensions from two CySC daughters, called cyst cells. CySC fate depends on an Unpaired (Upd) signal from the hub cells, which activates the Janus Kinase and Signal Transducer and Activator of Transcription (Jak/STAT) pathway in the stem cells. Germline enclosure depends on Epidermal Growth Factor (EGF) signals from the germline to the somatic support cells. Expression of RNA-hairpins against subunits of the COnstitutively Photomorphogenic-9- (COP9-) signalosome (CSN; see CSN5) in somatic support cells disrupted germline enclosure. Furthermore, CSN-depleted somatic support cells in the CySC position next to the hub had reduced levels of the Jak/STAT effectors Zinc finger homeotic-1 (Zfh-1) and Chronologically inappropriate morphogenesis (Chinmo). Knockdown of CSN in the somatic support cells does not disrupt EGF and Upd signal transduction as downstream signal transducers, phosphorylated STAT (pSTAT) and phosphorylated Mitogen Activated Protein Kinase (pMAPK), were still localized to the somatic support cell nuclei. The CSN modifies fully formed Cullin RING ubiquitin ligase (CRL) complexes to regulate selective proteolysis. Reducing cullin2 (cul2) from the somatic support cells disrupted germline enclosure, while reducing cullin1 (cul1) from the somatic support cells led to a low level of Chinmo. It is proposed that different CRLs enable the responses of somatic support cells to Upd and EGF (Qian, 2014).

Protein Interactions

The JAK/STAT signaling pathway plays important roles in vertebrate development and the regulation of complex cellular processes. Components of the pathway are conserved in Dictyostelium, Caenorhabditis, and Drosophila, yet the complete sequencing and annotation of the D. melanogaster and C. elegans genomes has failed to identify a receptor, raising the possibility that an alternative type of receptor exists for the invertebrate JAK/STAT pathway. domeless (dome) codes for a transmembrane protein required for all JAK/STAT functions in the Drosophila embryo. This includes its known requirement for embryonic segmentation and a newly discovered function in trachea specification. The DOME protein has an extracellular structure similar to the vertebrate cytokine class I receptors, although its sequence has greatly diverged. Like many interleukin receptors, DOME has a cytokine binding homology module (CBM) and three extracellular fibronectin-type-III domains (FnIII). Despite its low degree of overall similarity, key amino acids required for signaling in the vertebrate cytokine class I receptors are conserved in the CBM region. DOME is a signal-transducing receptor with most similarity to the IL-6 receptor family, but it also has characteristics found in the IL-3 receptor family. This suggests that the vertebrate families evolved from a single ancestral receptor that also gave rise to dome (Brown, 2001).

JAK/STAT signaling was first identified in vertebrates as mediating the response to some cytokines and growth factors. Ligand binding induces receptor homo- or hetero-dimerization and subsequent signal transduction. The receptors lack a tyrosine kinase domain but associate with cytoplasmic tyrosine kinases of the JAK family. After receptor dimerization, JAK phosphorylates a tyrosine residue on the receptor, and cytoplasmic STAT is recruited to the complex. JAK then phosphorylates STAT, which dimerizes, translocates to the nucleus, and induces gene transcription. In Drosophila, one JAK encoded by hopscotch (hop), one STAT encoded by stat92E, and one ligand encoded by unpaired (upd) have been identified, but no receptor has been found. Mutations for either hop, stat92E, or upd result in an identical, characteristic segmentation phenotype (Brown, 2001).

Mutations in stat92E affect the posterior spiracles, part of the respiratory apparatus of the larva. In a screen for P elements insertion mutations that give a phenotype similar to stat92E, domeless was identified. The six alleles, three strong (dome217, dome441, and dome468) and three weak (dome321, dome405, and dome367), all affect the shape of the posterior spiracles, with the strongest leading to a loss of the characteristic dome shape. Mobilization of the P element reverts both the lethality and the phenotype, confirming that the insertions cause the observed defects (Brown, 2001).

A database sequence search using DNA flanking the P elements identified an expressed-sequence tag (EST) encoding a putative transmembrane protein. The finding that expression of this cDNA rescues the dome spiracle phenotype confirms that this cDNA encodes the dome gene (Brown, 2001).

The 4.8 kb dome encodes a 1282 amino acid protein with a putative signal peptide of 23 amino acids and a transmembrane domain. The extracellular region contains five fibronectin-type-III (FnIII) domains, of which two have similarity to the cytokine binding module (CBM) found in the vertebrate cytokine receptor class I family. No invertebrate receptors of this family have been described, despite the sequencing projects in Drosophila and Caenorhabditis being complete. The vertebrate cytokine receptor family comprises more than 20 different receptors that signal through the JAK/STAT pathway. The CBM present in the vertebrate interleukin receptors is typically composed of two FnIII domains that contain a set of four conserved cysteine residues in the N-terminal domain and a WSXWS motif in the C-terminal domain. Dome contains these features, but the C-terminal domain of Dome has an incomplete WSXWS motif (NTXWS). Dome has 18% identity to LIFR and 26% identity to CNTFR and is within the typical range of sequence similarity limits for all cytokine receptors. Interestingly, Dome also has some characteristics of the IL-3 receptor family. These characteristics include an alternating region of hydrophobic residues (YXLXVRVR) in the CBM-C domain and the incomplete WSXWS motif, present only in IL-3Ralpha. The intracellular region of Dome is rich in both serine and threonine (16%) as well as proline (11%) and has an acidic region; features shared by the IL-2Rß receptor and GM-CSFR. Like other receptors of this class, Dome lacks a kinase domain. Although the sequence similarity of Dome with the vertebrate cytokine class I receptors is low, the shared characteristics suggest that domeless may encode the elusive Drosophila JAK/STAT receptor (Brown, 2001).

Tests were performed to see whether dome and stat92E interact genetically. Zygotic stat92E homozygotes have a very mild spiracle phenotype due to the persistence of maternally expressed RNA. Despite this, the weak dome367 phenotype is strongly enhanced by stat92E mutants, suggesting that both genes are in the same genetic pathway (Brown, 2001).

RNA in situ hybridization shows that, similar to hop (JAK) and stat92E, dome is expressed maternally. At later stages dome expression appears ubiquitous, although there is some variation in the levels of expression. At stage 11 the tracheal pits show more intense expression, whereas at stage 14, higher expression is detected in the posterior spiracles, gut, and head (Brown, 2001).

To determine the phenotype caused by eliminating domeless maternal and zygotic products, germ line clones were induced. Maternal and zygotic dome embryos have segmentation defects identical to those reported for mutations in the stat92E and hop (JAK) mutants. Defects include the deletion of the A5 and most of the A4 denticle belts, partial or total fusion of A6 to A7, and a variable reduction of the thoracic and the A8 segments. These phenotypes are also observed in Df(1)osUE69, which deletes the ligand, upd. The segmentation defects in stat92E, upd, and hop have been shown to be due to the abnormal expression of pair rule genes. In dome germ line clones, the expression of even-skipped is affected in stripes 3 and 5, as described for the other members of this pathway (Brown, 2001).

To further investigate whether dome has the genetic characteristics expected of the JAK/STAT receptor, dome interactions with upd, the known JAK/STAT ligand, were tested. To do this, advantage was taken of the fact that when the h-GAL4 line is used for ectopic expression of upd in the embryo, the result is abnormal head formation in 81% of the embryos. When upd is expressed ectopically in dome zygotic mutant embryos, this proportion is reduced to 16%. This result is consistent with dome being necessary to transduce the upd signal (Brown, 2001).

To find out if the intracellular domain of Dome is required for its function, UAS constructs were made in which the putative intracellular domain was deleted. One construct, UAS-domeDeltaCYT, contains the extracellular and transmembrane portion of the protein and should be membrane bound. The other, UAS-domeDeltaTMCYT, contains only the extracellular part and might be secreted. Neither of these proteins is able to rescue the dome zygotic phenotype, proving that the Dome intracellular domain is required for signal transduction. Because both forms still contain the cytokine binding domain, they must have the potential to titrate the ligand and act as signaling antagonists. This has been shown to be the case for the soluble form of gp130, the signal-transducing subunit of many vertebrate cytokine class I receptors. Consistent with this, when a maternal GAL4 was used for expression of UAS-domeDeltaTMCYT or UAS-domeDeltaCYT at early stages of development, approximately 50% of the larvae acquired segmentation defects. The most frequent defects were deletions and fusions of A4 and A5 segments, the segments more sensitive to loss of JAK/STAT function, but stronger defects were also observed. These phenotypes are increased if the mothers are also heterozygous for a hop allele, further proving the central role of dome in JAK/STAT signaling (Brown, 2001).

In embryos lacking both maternal and zygotic dome function, the trachea does not develop. Because such an extreme tracheal defect has not been described for other mutants of the pathway, whether the trachea is similarly affected in stat92E germ line clones was examined. In zygotic and maternal stat92E mutants, the trachea is mostly absent. This cannot be a result of the abnormal segmentation because the trachea forms in paternally rescued embryos that still have segment defects. The tracheal system forms from ten pairs of tracheal pits arising in segments from T2-A8. The pits can be identified at early stages by the expression of three genes: trachealess (trh), ventralveinless (vvl; also known as drifter), and knirps (kni). These three genes are activated in the trachea independently of one another. trh and vvl are then required for the expression of all known tracheal genes (except kni) and for their own maintenance from stage 13 onward. To discover at what stage of tracheal development the JAK/STAT pathway is required, the expression of trh, kni, and vvl was studied in dome or stat92E maternal and zygotic mutants. In both types of mutant embryos, neither trh nor kni is expressed, whereas early vvl expression is not affected. Becasue trh is essential for tracheal development, its loss from the tracheal pits is the likely cause for the tracheal defects observed. These results show that STAT92E is the earliest transcription factor required for trachea specification (Brown, 2001).

The evidence presented here indicates that domeless encodes a receptor of the Drosophila JAK/STAT pathway and shows that the pathway is conserved in invertebrates. The previous failure to detect the receptor was due to sequence similarity being restricted to a few critical amino acids in otherwise quite common protein domains. Sequence comparison with vertebrate receptors reveals that the structure and sequence of Dome are most similar to those of CNTFR and LIFR (of the IL-6 receptor family) but that Dome also has some characteristics of the IL-3 receptor family. This suggests that the vertebrate family of receptors evolved from a single ancestral receptor that also gave rise to dome. The identical nature of the mutant phenotypes of dome, hop, and stat92E suggests that Dome transduces all extracellular signals activating HOP and STAT. Vertebrate IL-6 receptors generally function as heterodimers, whereas some receptors, such as CNTFR, lack the intracellular transducing domain and act exclusively as the ligand binding partner, recruiting the signal-transducing component of the receptor complex. It is interesting to note that although the Dome intracellular domain is essential for signal transduction, Dome is most similar to CNTFR. The Drosophila genome appears to have no other protein with significant similarity to other receptor members. Dome might therefore form homodimers that can function both as ligand binding and signal transducers. However, given the low sequence conservation between dome and the vertebrate receptors, the possibility that Dome may form a complex with another yet-unidentified partner cannot be discarded. Future biochemical experiments should confirm if, as with the vertebrate receptors, Drosophila JAK binds directly to Dome (Brown, 2001).

The gp130-subfamily of receptors has no intrinsic tyrosine kinase domain, but is constitutively associated with tyrosine kinase JAKs. A tyrosine residue fitting a YXXQ consensus motif at the C terminus of the receptors provides a binding site for STAT. A C-terminal YXXQ sequence was found in Domeless/Mom, suggesting that Mom may bind Stat92E (Chen, 2002).

The physical interaction between Mom and Stat92E was directly assessed in cotransfection experiments. Cell lysates were prepared from S2 cell lines expressing V5-epitope-tagged Mom, Hop, and Stat92E with either Upd-V5 or vector alone. The lysates were immunoprecipitated using anti-Stat92E antibodies and then probed using anti-V5 antibodies. Mom coimmunoprecipitates with Stat92E only in the Upd-V5 and mom-V5 transfected cells. These data suggest that Stat92E binds to the activated Mom receptor (Chen, 2002).

In the mammalian system, JAK proteins are bound to monomeric cytokine receptors through the membrane-proximal domain. Signaling is triggered when cytokine binding induces receptor dimerization. This brings the receptor-associated JAK kinases into apposition, enabling them to transphosphorylate each other. The JAK kinases, now activated, phosphorylate a distal tyrosine on the receptor. This receptor phosphotyrosyl residue is subsequently recognized by the SH2 domain of the STAT proteins, drawing them into the receptor complex, where they are activated through phosphorylation on the tyrosine residue by JAKs (Chen, 2002).

To show Mom-dependent activation of the Hop/Stat92E pathway, the tyrosine phosphorylation of Mom, Hop, and Stat92E was examined. S2 cells were co-infected with V5-epitope-tagged Mom, Hop, and Stat92E with either Upd-V5 or vector alone. Anti-Stat92E immunoprecipitates were prepared and tested for reactivity with the anti-phosphotyrosine antibody 4G10. Whereas Upd, Mom, Hop, and Stat92E proteins are detectable in the transfected samples, increased tyrosine phosphorylation of Mom, Hop, and Stat92E is detected in immunoprecipitates prepared from Upd-V5- and Mom-V5-transfected cells. These data are consistent with the hypothesis that Mom is a receptor of Upd that activates the Hop/Stat92E signal transduction pathway (Chen, 2002).

In tissue culture cells, coexpression of marelle with hopscotch, the gene that codes for Drosophila JAK kinase, led to tyrosine phosphorylation of MRL (Yan, 1996a).

Transcriptional activation by (and therefore the physiologic impact of) activated tyrosine-phosphorylated STATs (signal transducers and activators of transcription) may be negatively regulated by proteins termed PIAS (protein inhibitors of activated stats), as shown by experiments with mammalian cells in culture. By using the genetic modifications in Drosophila, in vivo functional interaction of the Drosophila homologs stat92E and a Drosophila PIAS gene (dpias) have been demonstrated. A loss-of-function allele was used and dpias was conditionally overexpressed in JAK-STAT pathway mutant backgrounds. It is concluded that the correct dpias/stat92E ratio is crucial for blood cell and eye development (Betz, 2001).

By matching the available flanking sequence of the P element insertion of the stock l(2)03697 with the 5' untranslated region (UTR) of a cDNA highly homologous to the mammalian PIAS genes, a putative mutant allele of the Drosophila PIAS gene was identified. The P element insertion at the dpias locus (the dpias03697 allele) blocks all mRNA formation. Therefore, the dpias03697 allele constitutes a strong LOF or a null allele of the dpias gene (Betz, 2001).

Coimmunoprecipitation of mammalian PIAS and tyrosine-phosphorylated STATs has been established, and the interacting region of PIAS3 with STAT3 lies in the center of the molecule embracing a portion of a putative zinc finger domain. By using an in vitro protein association assay, it was found that a similar region of a dPIAS-GST fusion molecule binds to a FLAG-tagged STAT92E protein. The interaction depends on prior activation of the STAT92E protein brought about by the inhibition of tyrosine dephosphorylation with vanadate/peroxide, which was used because natural activation of STAT92E has not been accomplished in cell culture (Betz, 2001).

Because the dpias03697 allele is a homozygous lethal, genetic interaction crosses were designed in which flies heterozygous for the recessive dpias03697 allele were scored for the possible enhancement or suppression of known phenotypes in JAK-STAT pathway mutants. hopTum-l is a dominant hyperactive allele (increased HOP activity at elevated temperature) that causes tumor formation. This tumor formation, which is suppressed by stat92E LOF mutants, results from excessive proliferation of blood cells (plasmatocytes) that form melanotic abdominal tumors in larvae and pupae that can be scored in adults. At 25°C, 37% of heterozygous hopTum-l adult females had at least one abdominal tumor. Reduction of a negative activating regulator of this pathway should cause an increase in tumors. The percentage of flies with at least one tumor more than doubled in the hopTum-l/+;dpias03697/+ genotype compared with the progeny with two WT dpias alleles. Experiments on tumor frequency support the conclusion that dPIAS interacts negatively with the JAK-STAT pathway made overactive by hopTum-l: this leads to tumor formation. It is concluded that dPIAS decreases the transcriptional impact of the overactive STAT92E (Betz, 2001).

The role of dpias in eye development was examined because hypomorphic mutants of hop and os have small eyes. Two different lines, GMR-Gal4 and ey-Gal4, in which dpias overexpression depends on Gal4 activation at different times during eye development, were used. When the GMR-Gal4 line was used to drive UAS-dpias(537), no obvious effect on eye size or texture was observed. When UAS-dpias(537) was activated with the ey-Gal4 driver, eye size was severely reduced and the remaining small eye had a rough texture. A doubling of the transgene dosage further aggravated this phenotype and resulted in complete loss of the eyes in most of the surviving progeny. Because ey-Gal4 is active very early in eye development (before cellular differentiation) and GMR-Gal4 at later stages (during cellular differentiation), it is concluded that overexpression of dpias(537) has an effect primarily on cells in the early proliferating eye disc (Betz, 2001).

Whether this occurs because of a decreased activity of the JAK-STAT pathway was investigated. To this end Small-eyed UAS-dpias(537)/CyO;ey-Gal4 flies were crossed to a stock carrying a heat shock-inducible stat92E gene (hs-stat92E) and the progeny were raised under mild heat-shock conditions. A significant rescue of eye size and texture was observed only in progeny that carried the hs-stat92E transgene but not in genotypes without the hs-stat92E transgene segregating from the same cross. Moreover, a similar eye-size rescue effect was achieved by crossing the hopTum-l stock with small-eyed UAS-dpias(537)/CyO;ey-Gal4 flies, further bolstering the notion that activated STAT92E is required for eye development and that dPIAS counteracts the activated STAT92E (Betz, 2001).

The effect of replacing both WT copies of the dpias gene with the mutant dpias03697 alleles in eyes was examined. The yeast recombinase (flipase) system was used to generate clonal patches of mutant homozygous dpias03697 cells (from now on called dpias-/-) within heterozygous phenotypically WT flies. The lens structure completely fails to develop and is replaced by a heterogeneous bulged-out surface lacking bristles. Partially differentiated lenses surround the border of the clone (Betz, 2001).

The lack of, or abnormal differentiation of, lens structure observed on the surface of dpias-/- clones, in particular in the clonal border areas, appears to be phenotypically similar to Notch GOF phenotypes. Overexpression of activated Notch delays the differentiation of cone cells, the cells that secrete the lens material. Therefore, it is inferred that cone cell differentiation in surviving dpias-/- clones might be similarly affected (Betz, 2001).

Sections through dpias-/- clones reveal that cellular differentiation into photoreceptors and other cell types have failed, especially in the center of these clones. Along the clonal borders, partially differentiated ommatidia could be seen with incomplete sets of photoreceptors. In sections through dpias-/- clones, retinal cellular differentiation fails and is replaced by a heterogeneous cell mass. Other clones have apparently undergone either apoptotic or necrotic cell death, as indicated by frequent scars. Which of these diverse phenotypes might be caused by unopposed overactive STAT92E remains to be seen. It will be important to learn whether members of the mammalian PIAS genes are playing related roles in STAT-dependent tumor suppression, cell death, and differentiation (Betz, 2001).

Thus, with regard to eye development, a dramatic developmental role of dpias-stat92E interaction is found. Overexpression of dPIAS early (driven by ey-Gal4) aborts eyes, but loss of stat92E function later (by overexpression of dPIAS under the control of GMR-Gal4) has no apparent detrimental effect on cell growth or survival. Therefore, factors controlling stat92E function must be normally balanced in a critical time window in early eye development. Further increased expression of dpias or coupling with heterozygosity for the stat92E LOF allele stat06346 leads to transformation events with antennae frequently replacing eyes. LOF alleles of the Drosophila JAK-kinase hop of increasing severity cause the same sequence of increasing phenotypic abnormalities. Moreover, os1, a hypomorphic LOF allele of a JAK-STAT pathway ligand, results in small eyes. This phenotype can be partially suppressed and the eye size increased by reducing the dpias gene dosage, implying that with no transgenic intervention, dPIAS and STAT92E naturally interact in eye formation and eye determination (Betz, 2001).

The overgrowth of plasmatocytes and melanotic abdominal tumor formation caused by the hopTum-l allele presumably depends on too much activated STAT92E, because stat92E LOF mutants such as statHJ suppress tumor formation. By the same logic, it is inferred that dPIAS regulates the number of active STAT92E molecules, because increased dPIAS decreases tumor formation and decreased dPIAS increases tumor formation, indicating that HOP, STAT92E, and dPIAS act together in this pathway. This type of behavior -- genetic removal increasing tumorigenesis and overexpression reducing tumorigenesis -- is characteristic of genes in mammals that are labeled tumor-suppressor genes. By this definition, dpias would be a tumor suppressor. Recent widespread reports of persistently active STAT3 in a variety of human tumors and the demonstration of an engineered constitutively active STAT3 as an oncogene coupled with the present results predict that mutations in human PIAS3 might very well allow for persistent activation of STAT3, resulting in tumor formation. This interpretation is further supported by recent findings (Hari, 2001). Certain transheteroallelic dpias [Su(var)2-10] LOF alleles in otherwise genetically WT backgrounds caused melanotic tumors in third instar larvae (Betz, 2001).

Cyclin D-Cdk4 and Cyclin E-Cdk2 regulate the JAK/STAT signal transduction: Cdks physically interact with and phosphorylate STAT92E

The JAK/STAT signal transduction pathway regulates many developmental processes in Drosophila. However, the functional mechanism of this pathway is poorly understood. The Drosophila cyclin-dependent kinase 4 (Cdk4) 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. 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 (Chen, 2003).

In a large screen for autosomal P element-induced zygotic lethal mutations associated with specific maternal effect lethal phenotypes, a mutation, l(2)sh0671, located at 53C, was identified that showed a maternal effect segmentation phenotype. The phenotype is similar to the effect of loss of hop and stat92E gene activity during oogenesis. The P element, l(2)sh0671, was inserted into the second intron of the Cdk4 gene before the ATG translation initiation code (Chen, 2003).

The Cdk43 allele is an intragenic deletion of the Cdk4 gene, which eliminates the essential kinase domains. Cdk43 germline clone (GLC) phenotypes were examined; 70% of Cdk43 GLC embryos have strong 'hop-like' segmentation defects. As is the case with hop and stat92E embryos, the paternally rescued Cdk43 embryos show a consistent deletion of the fifth abdominal segment and the posterior mid-ventral portion of the fourth abdominal segment. Cdk43 null embryos have other defects, including reduction of the second thoracic and eighth abdominal denticle bands, fusion of the sixth and seventh bands, and head defects. The other 30% of embryos display other defects, such as poor cuticle development and dorsal open, that differ from alterations in components of the HOP/STAT92E pathway, suggesting that Cdk4 also functions in developmental processes other than the HOP/STAT92E pathway. The maternal effect phenotype for Cdk4 mutants is partially paternally rescuable. Expression of a Cdk4 cDNA encoding the full-length Cdk4 protein under the control of a heat-shock promoter in transgenic flies fully rescued the segmentation defects of Cdk43 GLC embryos at 25°C. Cdk4D175N is a dominant-negative form of Cdk4; the D175N mutation affects an aspartate residue that is required for the phosphotransfer reaction. Expression of the dominant-negative form of the cDNA under a maternal gene promoter generated the 'hop-like' segmentation defects. These data suggest that the observed segmentation defects are caused by disruption of the Cdk4 gene (Chen, 2003).

The similarity of the Cdk4 mutant phenotype to that of hop and stat92E suggests that these latter genes are involved in the same developmental process. A prediction of this hypothesis is that mutations in Cdk4 would affect the expression of segmentation genes in the same manner as hop and stat92E. The removal of either hop or stat92E activity is known to result in the stripe-specific loss of expression of several pair-rule genes. The enhancer elements responsible for control of the third stripe of eve expression have been mapped to a 500 bp element upstream of the eve transcriptional start site. A reporter gene construct containing a 5.2 kb eve promoter element driving lacZ shows expression of lacZ in eve stripes 2, 3, and 7. Removal of maternal activity of either hop or stat92E results in the loss of the third stripe from the reporter construct. Similarly, removal of maternal activity of Cdk4 also causes the specific loss of the third stripe, without affecting the second or seventh stripes (Chen, 2003).

The HOP/STAT92E pathway regulates tracheal formation through regulating trachealess (trh) gene expression in the embryo. It was reasoned that Cdk4 might also regulate tracheal formation. Tracheal formation was examined in wild-type, hop, and Cdk4 embryos by using an antibody [(mAb)2A12] that stains tracheal branches and trunks. In paternally rescued hop and cdk4 embryos, a similar defective tracheal system was formed that generally had several disruptions in the main trunk and several branches. These data suggest that Cdk4 regulates tracheal formation in a manner similar to the HOP/STAT92E signal transduction pathway (Chen, 2003).

To determine whether hop and Cdk4 genetically interact, a test was performed to see whether a reduction in the amount of maternal Cdk4 gene activity could enhance the maternal effect associated with a partial loss of function in the hop mutation. Embryos that are derived from mothers that carry GLCs of the hopmsv1 hypomorphic allele show weak segmentation defects, and many of them hatch. However, when these embryos are derived from females that also carry a single copy of Cdk43, they exhibit segmentation defects that are similar to embryos derived from females that lack all maternal hop activity, and none of them hatch. This result suggests that hop and Cdk4 act in concert to regulate embryonic segmentation (Chen, 2003).

Whether Cdk4 operates upstream or downstream of HOP was examined by testing whether the effect of a hyperactive hop allele could be negated by a reduction in the amount of Cdk4 gene activity. If Cdk4 is required for transducing the HOP signal, then reduction of Cdk4 should suppress a hop gain-of-function phenotype. The dominant temperature-sensitive hop allele, hopTum-l, was used for this experiment. When grown above 29°C, flies heterozygous for hopTum-l have reduced viability and the emerging adults develop melanotic tumors. Viability and formation of melanotic tumors at 29°C were compared in females heterozygous for hopTum-l and Cdk4 with females heterozygous only for hopTum-l. An improved survival rate was obtained by removing a single copy of Cdk4 in hopTum-l heterozygous females. However, the formation of melanotic tumors is affected less by removing a single copy of Cdk4 in hopTum-l heterozygous females (Chen, 2003).

To further examine the function of Cdk4 in the HOP/STAT92E signal transduction pathway, the genetic interactions of Cdk4 with hop and stat92E were tested in embryos. The hop (hopC111) and stat92E (stat92E6346) null embryos show a consistent deletion of the fifth abdominal segment and the posterior mid-ventral portion of the fourth abdominal segment, and none of them hatch. When HS-Cdk4 is ubiquitously expressed in hopC111 embryos, most embryos have complete fourth and fifth abdominal segments, and many of them hatch. Ubiquitous expression of Cdk4 has no effect in stat92E mutant embryos (Chen, 2003).

In mammals and Drosophila, Cdk4 forms a protein complex that regulates the cell cycle progression. The Cyclin D and Cdk4 complex (CycD-Cdk4) phosphorylates and releases RB from RB/E2F; free E2F then activates gene expression, including Cyclin E (CycE). Cyclin E and Cdk2 form a complex (CycE-Cdk2) and regulate the cell cycle at the G1-S transition point. To further examine relations between the HOP/STAT92E signal transduction pathway and cell cycle regulation, the genetic interaction of hop with CycE was tested. Like HS-Cdk4, HS-CycE rescues hopC111 embryo segmentation defects but has no effect on stat92E mutant embryos (Chen, 2003).

The viability and formation of melanotic tumors at 29°C were compared in females heterozygous for hopTum-l and CycE with females heterozygous only for hopTum-l. An improved survival rate was observed by removing a single copy of CycE in hopTum-l heterozygous females. As in the case of Cdk4, the formation of melanotic tumors is less affected by removing a single copy of CycE in hopTum-l heterozygous females. These results suggest that CycD-Cdk4 and CycE-Cdk2 complexes are members of the HOP/STAT92E signal transduction pathway and function downstream of the HOP tyrosine kinase and either upstream of or parallel to the STAT92E transcription factor (Chen, 2003).

Thus Cdk4 functions in the HOP/STAT92E pathway and regulates embryonic segmentation, tracheal formation, eye development, and melanotic tumor formation. Specific genetic interactions between Cdk4 and hop or stat92E mutations suggest that Cdk4 functions upstream of STAT and parallel to or downstream of the HOP tyrosine kinase. Furthermore, CycD-Cdk4 and CycE-Cdk2 bind and regulate STAT92E protein stability. These data demonstrate that, besides their role in regulating the cell cycle, CycD-Cdk4 and CycE-Cdk2 have a role in regulating cell fate determination and proliferation via STAT signaling (Chen, 2003).

STAT92E binds directly to the promoter of pair-rule genes and regulates their expression for segmentation. This occurs during the first 13 embryonic cell cycles, which are nearly synchronous and lack G1 and G2 gap phases. Obviously, the function of CycD-Cdk4 and CycE-Cdk2 is not to regulate the cell cycle during this period. The CycD-Cdk4 and CycE-Cdk2 complexes may regulate pair-rule gene expression through stabilizing STAT92E protein and increasing its transcription activity (Chen, 2003).

Expression of a kinase-impaired mutant form of Cdk4 (Cdk4D175N) using a maternal driver generates the 'hop-like' segmentation defects, indicating that the segmentation phenotype requires Cdk4 kinase activity. There is also a potential consensus sequence (SPVKR) of Cdk4/Cdk2 phosphorylation in the C terminus of STAT92E; however, mutation of S to A in the sequence does not significantly affect STAT92E activity in regulating gene expression, and attempts to detect STAT92E phosphorylation by Cdk4 have not been successful. CycD-Cdk4 and CycE-Cdk2 may simply bind and enrich nuclear STAT92E protein for its transcriptional activity. Regardless, the biochemical findings suggest that Cyclin-Cdk activity could affect STATs directly. STAT92E may have many transcriptional targets in the Drosophila genome, including genes involved in cell cycle regulation (such as RNrS). CycD-Cdk4 and CycE-Cdk2 can execute their versatile roles through regulating STAT92E protein. Cell cycle regulation may be one of their various functions (Chen, 2003).

A fraction of Cdk4 mutant embryos displays other defects that differ from alterations in components of mutations of the HOP/STAT92E pathway, suggesting that CycD-Cdk4 and CycE-Cdk2 have other targets besides STAT92E in Drosophila. RBF/E2F1 has been shown to be another target of the CycE-Cdk2 complex (Chen, 2003).

Excess HOP/STAT92E signaling induces cell overproliferation in the eye. Excess CycD-Cdk4 activity blocks differentiation and induces overgrowth in the eye. This study shows that excess HOP/STAT92E signaling can synergize with both CycD-Cdk4 and CycE-Cdk2 in melanotic tumors, but specifically synergizes with CycD-Cdk4, not CycE-Cdk2, to promote formation of an enlarged eye with extra ommatidia. Specifically, overexpression of either CycD-Cdk4 or CycE under the GMR-Gal4 driver significantly increased the fraction of S and G2 phase cells posterior to the MF at the expense of the G1-arrested cells. Both cyclins perturbed the normal program of cell cycle exit at differentiation. However, only CycD-Cdk4 appears to affect both cell cycle progression and cellular growth, whereas CycE affected only cell cycle progression (Chen, 2003).

The nucleosome remodeling factor (NURF) regulates genes involved in Drosophila innate immunity

The Drosophila nucleosome remodeling factor (NURF) is an ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF has the ability to alter chromatin structure and regulate transcription. Previous studies have shown that mutation of Drosophila NURF induces melanotic tumors, implicating NURF in innate immune function. This study shows that NURF mutants exhibit identical innate immune responses to gain-of-function mutants in the Drosophila JAK/STAT pathway. Using microarrays, a common set of target genes were identified that are activated in both mutants. In silico analysis of promoter sequences of these defines a consensus regulatory element comprising a STAT-binding sequence overlapped by a binding-site for a transcriptional repressor protein termed Ken and barbie, or Ken for short. Ken is an ortholog of the mammalian proto-oncogene Bcl6 and, like Bcl6, can down-regulate JAK/STAT target genes. NURF interacts physically and genetically with Ken. Chromatin immunoprecipitation (ChIP) localizes NURF to Ken-binding sites in hemocytes, suggesting that Ken recruits NURF to repress STAT responders. Loss of NURF leads to precocious activation of STAT target genes (Kwon, 2008).

Given the potential catastrophic effects of inappropriate activation of signaling cascades, it is essential that the gene targets of signaling pathways are maintained in a repressed state in the absence of activating ligand. It is assumed that packaging of DNA into nucleosomes, and positioning of nucleosomes over gene regulatory elements can block transcription. Members of the ISWI family of ATP-dependent chromatin remodeling enzymes are key regulators of nucleosome positioning and, this report has shown that NURF activity is required to maintain repression of JAK/STAT target genes. Repression by NURF is consistent with other studies of ISWI chromatin remodeling enzymes. For example, the yeast Isw2 remodeling complex is required for transcriptional repression. In humans, the Snf2h-containing chromatin remodeling complex NoRC slides nucleosomes to silence rRNA genes. More recently, ISWI in African trypanosomes has been demonstrated to silence variant surface glycoprotein gene expression sites (Kwon, 2008 and references therein).

Although a chromatin remodeling enzyme (SWI/SNF) is required for activation of STAT-inducible genes, this is the first report to implicate a chromatin remodeling enzyme in repression of JAK/STAT target genes. There is, however, evidence that covalent histone modification is involved in repression of JAK/STAT target genes. The co-repressor SMRT suppresses induction of STAT5 target genes. This suppression is blocked by the addition of the histone deacetylase inhibitor TSA, implying a chromatin component in repression. In Drosophila, mutations in the heterochromatin component HP1 have been shown to enhance tumor formation in hopTum gain-of-function JAK mutants, further implying a connection between chromatin, JAK/STAT and transcriptional repression (Kwon, 2008).

It cannot be excluded that some of the genes that show increases in expression in the Nurf301 and hopTum mutants may be indirect targets of NURF. Changes in the proportion of lamellocytes in these mutant backgrounds may affect transcription of some genes, for example the Drosophila β-integrin subunit mys. Nevertheless by ChIP it has been shown that NURF is located at the promoters of two potential targets, CG5791 and dei. Importantly, NURF-biding coincides with recognition sequences for STAT are overlapped by binding sites for the transcriptional repressor Ken. In addition, it was shown that NURF physically interacts with Ken, providing a means by which NURF can be recruited to JAK/STAT target genes (Kwon, 2008).

The data suggests a mechanism by which Ken represses transcription. It is proposed that in unstimulated conditions Ken binds to JAK/STAT target promoters and recruits NURF. NURF-mediated nucleosome-sliding then establishes a repressed chromatin configuration that blocks transcription, perhaps by positioning a nucleosome over the transcription start site. Upon stimulation Stat92E enters the nucleus, binds target promoters and, in addition to recruiting co-activators, displaces Ken and thus NURF. The promoter is switched from a repressive to active chromatin state, and transcription can occur. In NURF mutants, it is suggested that repressive nucleosome positions are either not established or maintained and, consequently, JAK/STAT targets are not silenced. As a result, transcription can occur in the absence of JAK/STAT activation (Kwon, 2008).

More than two decades ago, Travers and colleagues proposed that underlying DNA sequence can influence nucleosome positioning, with some sequences favoring, and others destabilizing nucleosomes. Recent computational analysis has revealed that sequences at yeast transcription start-sites encode nucleosomes that are intrinsically unstable. The NURF-related yeast Isw2 chromatin remodeling complex is able to override these refractory sequences, positioning nucleosomes over them, to block promoters. Interestingly, in ISW2 mutants, nucleosomes revert to thermodynamically favorable positions exposing the promoter. It is speculated that Drosophila transcription start-sites may similarly be refractory to nucleosomes. Normally, at JAK/STAT targets, NURF overrides these sequences but, in NURF mutants, these transcription start sites may similarly be exposed (Kwon, 2008 and references therein).

In the case of the innate immune system, prompt activation of signaling cascades such as the JAK/STAT pathway in response to pathogens are essential for survival. However, it is also paramount that in the absence of challenge the innate immune system be held in check or regulated, to prevent inappropriate damage. In humans chronic immune-mediated inflammatory conditions are characterized by the abnormal or continued episodic activation of these pathways leading to disease. Drosophila NURF has a vital function in preventing ectopic activation of the JAK/STAT pathway. In the absence of NURF, Drosophila develop an immune-mediated inflammatory syndrome -- melanotic tumors. Given the conservation of NURF between Drosophila and humans, it is tempting to speculate that human NURF may function to hold inflammatory pathways in check (Kwon, 2008).

Polarized subcellular localization of Jak/STAT components is required for efficient signaling: Par-3-dependent STAT apical localization

Three protein complexes control polarization of epithelial cells: the apicolateral Crumbs and Par-3 complexes and the basolateral Lethal giant larvae complex. Polarization results in the specific localization of proteins and lipids to different membrane domains. The receptors of the Notch, Hedgehog, and WNT pathways are among the proteins that are polarized, with subcellular receptor localization representing an important aspect of signaling regulation. For example, in the WNT pathway, differential DFz2 receptor localization results in activation of either the canonical or the planar polarity pathway. Despite the large body of research on the vertebrate JAK/STAT pathway, there are no reports indicating polarized signaling. By using the conserved Drosophila JAK/STAT pathway as a system, it was found that the receptor and its associated kinase are located in the apical membrane of epithelial cells. Unexpectedly, the transcription factor STAT is enriched in the apicolateral membrane domain of ectoderm epithelial cells in a Par-3-dependent manner. These results indicate that preassembly of STAT and the receptor/JAK complex to specific membrane domains is a key aspect for signaling efficiency. These results also suggest that receptor polarization in the ectoderm cell membrane restricts the cell's response to ligands provided by neighboring cells (Sotillos, 2008).

Besides setting up epithelial polarity, apicobasal complexes also modulate the subcellular compartmentalization or localized activation of various signaling molecules. The JAK/STAT signaling pathway is involved in processes ranging from immune response to organogenesis. In the vertebrate-signaling model, inactive STAT is shuttling from the cytoplasm to the nucleus. Ligand binding to the dimerized receptor results in the activation of JAK bound to the receptor. JAK phosphorylates itself and the receptor, creating docking sites for STAT. Inactive cytoplasmic STAT now binds to the phosphoreceptor/JAK complex, where it is phosphorylated by the kinase. Phosphorylated STAT is imported to the nucleus, where it activates the transcription of target genes. In contrast to vertebrates, in which the JAK/STAT core-signaling elements are highly redundant, the Drosophila pathway is composed of only three ligands, Unpaired (Upd), Unpaired2, and Unpaired3; one receptor, Domeless (Dome); one JAK, Hopscotch (Hop); and one transcription factor, STAT92E. Therefore, Drosophila was used as a model to investigate the polarization of the pathway (Sotillos, 2008).

dome, hop, and stat92E mRNAs are maternally provided and ubiquitously transcribed in the embryo. To analyze their protein subcellular localization, specific antibodies were used or functional tagged proteins were expressed by using UAS-dome, UAS-hop-Myc, and UAS-STAT92E-GFP. These constructs were expressed by using either mesodermal or ectodermal Gal4 drivers, and the subcellular localization of the proteins was analyzed, paying special attention to three organs where the endogenous ligand is expressed and the pathway is active: the posterior spiracles (ectodermal origin), the pharyngeal musculature (mesodermal), and the hindgut (an ectodermal tube surrounded by mesoderm) (Sotillos, 2008).

In the pharynx, as expected for a receptor, Dome localizes to the membrane, and does so in a dotted pattern that could correspond to endocytic vesicles. Hop-myc localizes to the cytoplasm, obscuring any membrane localization. This is due to the high levels of Hop-myc expressed, saturating the receptor binding sites and accumulating in the cytoplasm, as simultaneous coexpression of Hop-myc with the receptor relocates Hop to the membrane. This depends on the cytoplasmic domain of Dome, as it also occurs with a construct missing the extracellular domain but not with constructs missing the intracellular domain. STAT is detected in the cytoplasm and is more concentrated in the nuclei, as expected from the activation of the pathway in the pharynx. All of these observations agree with current knowledge of JAK/STAT activation based on vertebrate studies (Sotillos, 2008).

In contrast to the mesoderm, analysis of ectoderm cells shows a different picture. Both in the hindgut and the posterior spiracles, the Dome receptor localizes on the apical membrane. Hop is again cytoplasmic, but after coexpression with Dome both proteins localize to the apical membrane. Surprisingly, by using a specific antibody it was observed that STAT concentrates on the apical membrane of all embryonic ectodermal cells irrespective of the level of activation of the pathway. And, in cells in which the pathway is active, STAT also localizes to the nucleus. The signal detected by the antibody is specific; the same result by using a STAT-GFP fusion protein. STAT membrane localization is more prominent in cells in which the pathway is inactive; for instance, in the trunk epidermis or the spiracle after stage 15. This suggests that STAT translocates from the subapical membrane to the nucleus after pathway activation, returning to the membrane after inactivation (Sotillos, 2008).

To determine if STAT-GFP membrane localization is due to any other of the pathway's components, STAT-GFP localization was analyzed in upd, dome, or hop null mutants. STAT does not disappear from the membrane in a deficiency that removes all three Upd ligands. STAT membrane localization is not affected in null mutants for either dome or hop, demonstrating that apical STAT localization is independent of the pathway (Sotillos, 2008).

STAT localizes to the membrane domain in which the apical complexes are located. This, and the fact that STAT does not localize to the membrane in the mesoderm where Crb and Par-3 complexes are not formed, suggests the apical complexes could be recruiting STAT. To test this, different apical complex proteins were expressed in the mesoderm, and their capacity to modify STAT subcellular localization was studied. Neither the expression of Crb nor aPKC (another member of Par-3 complex) is able to translocate STAT to the membrane. In contrast, expression of Par-3 results in efficient membrane translocation of STAT and STAT-GFP. Moreover, STAT-GFP and Par-3 coimmunoprecipate from embryo extracts overexpressing STAT-GFP and par-3, pointing to Par-3 as the molecule responsible of STAT apical localization. In accordance, STAT-GFP is lost from the membrane in par-3 zygotic mutants, whereas in crb null mutants, where the polarity is highly compromised and Par-3 localization is severely affected, STAT remains in the membrane of cells only where Par-3 is still present. Similarly, in null aPKC embryos, STAT-GFP exclusively remains apical in cells in which Par-3 still localizes at the membrane. Thus, STAT recruitment is independent of Crb or aPKC and may directly depend on Par-3 (Sotillos, 2008).

To analyze if JAK/STAT polarization is functionally relevant, genetic interactions with polarity mutants were tested. Heterozygous polarity mutants or stat92E embryos are viable and normal. In contrast, embryos simultaneously heterozygous mutant for stat92E and either par-3, aPKC, or crb present phenotypes associated to JAK/STAT loss of function, including malformation of the posterior spiracles and abnormal segmentation. A specific readout of the pathway's activity was studied, analyzing the expression of a crb-spiracle enhancer that is directly activated by JAK/STAT (Lovegrove, 2006). The expression of this enhancer is severely reduced in zygotic par-3 mutants simultaneously heterozygous for stat92E, compared to its expression in heterozygous stat92E embryos or zygotic par-3 mutants. In contrast, the expression of the JAK/STAT independent ems-spiracle enhancer is not affected in the same genetic backgrounds. The capability of Par-3 to induce STAT membrane localization and the strong genetic interaction between stat92E and cell-polarity mutations indicate that the apical polarization of JAK/STAT components is required for full-signaling efficiency in the ectoderm (Sotillos, 2008).

Next, whether the apical localization of all JAK/STAT transducer components in the ectoderm results in signaling occurring exclusively through this membrane domain was tested. For this purpose the posterior hindgut, where JAK/STAT is required in the ectoderm and in the mesoderm surrounding it, was analyzed. Upd expressed from the most anterior ectodermal cells of the hindgut activates in the ectoderm ventral veinless (vvl) and upregulates in the mesoderm dome through the dome-MESO enhancer. Thus, vvl and the dome-MESO autoregulatory enhancer can be used as readouts for JAK/STAT activation in the different hindgut tissues (Sotillos, 2008).

If signaling in the ectoderm were transduced exclusively through the apical membrane, it would be expected that vvl activation on the hindgut would not be possible if Upd is presented from the basal side. To test this Upd was expressed either in the ectoderm or in the mesoderm, and its effect on vvl activation in the ectoderm was analyzed. As a positive control the expression of dome-MESO was analyzed. When expressed throughout the ectoderm, Upd induces ectopic expression of dome-MESO in the mesoderm and of vvl in the ectoderm, behaving as the endogenous Upd. In contrast, when Upd is expressed throughout the mesoderm, dome-MESO is ectopically activated, whereas vvl is not. The unresponsiveness of the ectoderm cells to Upd from the mesoderm is consistent with the endogenous receptor being apically localized in the hindgut ectoderm and, thus, unable to receive any mesoderm signal (Sotillos, 2008).

Many proteins involved in the establishment and maintenance of cell polarity also modulate signaling pathways by modifying or restricting the localization of their signaling components. Precise subcellular distribution may help the activation of the pathway or restrict its activity by sequestering key elements. This study has shown that in the epithelial cells the localization of JAK/STAT components is highly polarized. The apical restriction of the receptor can influence transduction, since only ligand presented to the apical side of the epithelium would be detected. This may be of relevance after septic injury, when circulating haemocytes secrete the Upd3 cytokine into the haemolymph. In this case, the secreted ligand would activate its targets in the fat body without stimulating the ectoderm epithelial cells, since the cell junctions efficiently block Upd diffusion to the apical side (Sotillos, 2008).

Par-3-dependent STAT apical localization is intriguing. The localization of STAT to the subapical membrane seems important for signal transduction, since mutations reducing the amount of cell polarity proteins enhance stat loss of function phenotypes and reduce the activation of direct pathway targets. It is proposed that in ectodermal cells, where the receptor and the kinase locate apically, the existence of a subapical pool of STAT facilitates its rapid translocation to the activated receptor, increasing signaling efficiency. Future research should resolve whether this is achieved simply by the increased local concentration of apical STAT facilitating receptor binding or if there exists some dedicated machinery to translocate STAT from the subapical region to the active receptor similar to the one involved in nuclear import. It is interesting to note that crb expression is upregulated by JAK/STAT signaling in the follicle cells and in the posterior spiracles. Since Crb helps maintaining Par-3 in the apical membrane, upregulation of crb by STAT might increase apical Par-3, reinforcing signal transduction by increasing the apical concentration of STAT (Sotillos, 2008).

There are few reports of polarized vertebrate JAK/STAT signaling. However, analysis of the subcellular localization of two IL-6 receptors in MDCK epithelial cells has shown that gp130 localizes basolaterally and CNTF-R apically. Also, in the mammary glands, the IL-4Ra receptor is localized apically in luminal cells during gestation and lactation. Recently, activated STAT3 has been transiently detected at the membrane in the nascent cell-cell contacts of squamous cell carcinoma of the head and neck. In vertebrates the Par-3 complex functions as a regulator of junction biogenesis. It will be interesting to investigate whether Par-3 also mediates the localization of STAT3 in the membrane. The results suggest that JAK/STAT polarization in epithelia may be a general feature (Sotillos, 2008).

Sumoylation of Drosophila transcription factor STAT92E

STAT92E is an essential transcription factor in Drosophila for the development of several organs and the immune system. The JAK/STAT pathway employs different evolutionary conserved regulatory mechanisms to control biological processes. Numerous transcription factors in both mammals and invertebrates have been shown to be either activated or inhibited by a covalent modification with a small ubiquitin-like modifier (Sumo). This study show that Drosophila STAT92E is modified by Sumo at a single lysine residue 187 in S2 cells. Mutation of Lys187 increases the transcriptional activity of STAT92E, thus suggesting that sumoylation of STAT92E has a repressive role in the regulation of the JAK/STAT pathway in Drosophila (Grönholm, 2010).

Removal of endogenous Sumo E3 ligase dPIAS by dsRNA has been shown to increase the STAT92E activity on TotM promoter approximately to the same level as K187R mutation in STAT92E, suggesting that sumoylation of STAT92E is involved in dPIAS-mediates inhibition of STAT92E. The mechanisms of how sumoylation is affecting STAT92E are presently unknown, but several possible mechanisms can be envisioned. The sumoylation site Lys187 is localized in the coiled coil domain, which in the mammalian system is involved in nuclear transport of STATs. The coiled coil domain of STATs is composed of 4 α-helixes that are pointing out from the DNA-bound STAT dimer, forming a hydrophilic surface able to interact with other molecules. Thus, sumoylation of Lys187 may interrupt the interaction between STAT92E and its transcriptional coregulators or the proteins involved in its nuclear translocation. Alternatively, sumoylation may lead to the recruitment of histone deacetylases to the promoter or allow the interaction with a transcription repression complex similarly to Drosophila Sp3. The effect of sumoylation on DNA-binding properties of STAT92E was not analyzed, but the coiled coil domain is not contacting DNA, suggesting that direct effects upon the promoter-binding activity are less likely (Grönholm, 2010).

Transcription co-activator SAYP mediates the action of STAT activator

Jak/STAT is an important signaling pathway mediating multiple events in development. This study describes participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly (Vorobyeva, 2009) and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to developmental defects similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT (Panov, 2012).

This study analyzed the role of co-activator SAYP in the Jak/Stat pathway in Drosophila and found that SAYP interacts with STAT and mediates its activation potential. SAYP operates as a component of large protein complex BTFly, which also contains Brahma and TFIID, and mediates subsequent recruitment of PolII onto the promoter. The results of polytene chromosome staining and measurement of multiple STAT-dependent genes testify to genome-wide cooperation of STAT and SAYP in gene activation (Panov, 2012).

The activation domain of STAT interacts with the SAY-PHD fragment of SAYP within the BTFly, and the STAT-BTFly association is not mediated by Brahma or TFIID. It is noteworthy that the interacting fragment of SAYP belongs to its conserved core, which is also found in vertebrate homologues of SAYP. Therefore, the above interaction may also take place in other species (Panov, 2012).

This finding broadens the known spectrum of transcription factors mediating the effect of STAT on gene expression. Such a diversity of cooperating factors appears to provide a basis for the specificity and strength of Jak/Stat-driven response in different cell types. In particular, BTFly may serve for rapid induction of transcription, as proposed previously. Indeed, the positive effect of SAYP content on short-term induction after STAT activation was observed. SAYP is also important for PolII stalling, which may provide for the precision of responses of target genes in the signaling pathway. One more point of interest is the putative role of SAYP in coordinating the crosstalk between Jak/Stat and other signaling pathways, which follows from the fact that SAYP is also involved in the ecdysone cascade and probably in some other cascades (Panov, 2012).

Mutation of both STAT and SAYP leads to formation of excess numbers of ovarian follicular cells and ectopic wing veins. SAYP is abundant in cells with high proliferative potential, and its mutation results in overproliferation of polar cells in embryos. Therefore, SAYP may participate in regulation of proliferation of certain cell types. Interestingly, PHF10-a vertebrate homologue of SAYP-is required for proliferation of stem/progenitor cells (Lessard, 2007) and fibroblasts (Banga, 2009). On the other hand, it has been shown that STAT also has a role in proliferation and growth control. Therefore, it appears that SAYP and STAT are jointly involved in regulation of proliferation and differentiation of certain cell types during metazoan development (Panov, 2012).

Src kinases mediate the interaction of the apical determinant Bazooka/PAR3 with STAT92E and increase signalling efficiency in Drosophila ectodermal cells

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). This study found 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. It is believed that the requirement of Src proteins for STAT92E apical localization is mediated through Baz, since can Src can be co-precipitated 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 (Sotillos, 2013).

In vertebrates, there is strong evidence showing that, besides JAK, the Src kinases can activate STAT signalling. First, STAT3 and STAT5 are crucial downstream factors in Src-induced transformation. Second, Src kinase activation has been shown to result in STAT tyrosine phosphorylation. By contrast, in Drosophila the available data suggest that the involvement of Src in STAT92E activation is probably marginal. This is shown by the fact that in Drosophila the phenotypes of null stat92E alleles are very similar to those of mutants in which the function of the JAK kinase, the receptor or of all ligands, is abolished, indicating that STAT92E activation via alternative kinases occurs in a minority of tissues. One of the few cases reported of JAK-independent STAT92E activation occurs during the proliferation and migration of the embryo pole cells where STAT92E is activated by the Ras/Raf pathway downstream of the Torso tyrosine kinase receptor. The study of mutations in the C-terminal Src kinase (Csk), a negative regulator of Src signalling, offers indirect evidence to suggest that Src may activate STAT92E in the Drosophila eye. Eyes that lack Csk are larger than normal, a phenotype also observed when the canonical JAK/STAT pathway is ectopically activated during eye development. Moreover, Csk clones exhibit higher levels of STAT92E expression, which has been interpreted as being due to the induction of a STAT92E positive-feedback loop. However, these results do not clarify whether Src-induced STAT92E activation is due to direct STAT92E phosphorylation or caused by indirect regulation (Sotillos, 2013).

In the ectoderm, where the JAK/STAT pathway is highly polarized, efficient signalling requires STAT92E apical localization achieved through interaction with the membrane-associated apical polarity protein Baz (Sotillos, 2008). This study obtained several pieces of evidence showing that Src is also required for the correct STAT92E membrane localization and signalling. First, although heterozygous stat92E or Src mutant embryos are normal, double heterozygous stat92E and Src mutant embryos display phenotypes that resemble a partial JAK/STAT signalling failure. Second, the expression of a direct target of STAT92E is downregulated in a Src mutant background. Third, decrease of Src gene activity affects STAT92E localization to the membrane of epithelial cells or to the membrane of mesodermal cells expressing Baz. Fourth, in S2R+ cultured cells STAT92E co-localizes with Baz only when co-expressed with Src42A or Src64B. The data also show that tyrosine 711 is not required for membrane localization, demonstrating that this function of Src is independent of STAT92E activation (Sotillos, 2013).

Although it was possible to co-precipitate Baz with STAT92E and Baz with Src42A, co-precipitation of Src42A with STAT92E was not achieved. This suggests that the interaction between Src42A and STAT92E is either too labile to be detected or that Src modifies Baz and that this allows the recruitment of STAT92E to Baz. The data also suggest that the kinase activity of Src42A is dispensable for STAT92E-Baz interactions, as a kinase-dead isoform of Src42A is able to rescue membrane localization in Src mutant backgrounds to the same level that a constitutively activated form can. Although a direct activation of STAT92E in response to Src and growth factors was possible in the minority of cell types, the genetic interactions do not reveal any phenotype apart from those of the canonical JAK/STAT pathway (Sotillos, 2013).

In addition to the parallel dimers formed by SH2 interaction with phosphor-Tyrosine in activated STATs (Mohr, 2012), vertebrate STAT proteins have been shown to form 'inactive' antiparallel dimers mediated by the region that includes the N-terminal and the coiled-coil domains (Mao, 2005; Neculai, 2005; Ota, 2004). In Drosophila, active phosphorylated STAT92E also forms parallel homodimers through an SH2-phosphotyrosine interaction. This study suggests that STAT92E may also form inactive homodimers mediated through the N-terminal coiled-coil domain. Thus, formation of STAT92E dimers prior to pathway activation could be an ancestral STAT characteristic, reinforcing Drosophila as a model for studying vertebrate STAT signalling (Sotillos, 2013).

Previous papers have described the requirement of a STAT92E-Baz interaction for efficient JAK/STAT signalling (Sotillos, 2008). This study has uncovered the domains in both molecules involved in this physical interaction. In the case of STAT92E the region was narrowed down to the transactivation (TA) domain of the protein: only the most C-terminal part of the molecule is able to localize to the apical membrane cortex on its own and to co-precipitate with Baz. Moreover, when the TA domain is removed, the C-terminal domain is unable to localize to the cell membrane where Baz is located and reduces its ability to co-precipitate with Baz, probably owing to the loss of binding to the N-terminal part of Baz. However, this construct is still able to bind to the C-terminal part of Baz, indicating that another domain in this fragment is also involved in this interaction. In vertebrates, the TA domain has been shown to be crucial for the regulation of the activity of STAT through the interaction with several proteins. This study has added a new function to this domain as a mediator of the interaction of STAT92E with Ba (Sotillos, 2013).

Baz is a scaffolding protein that is able to interact with various proteins and lipids through different regions. Interaction with STAT92E requires both the Baz N-terminal region (1-317) that includes the oligomerization domain and the C-terminal region (1048-1464) that includes the phosphatidylinositol-binding site. Given that both Baz N- and C-terminal domains are conserved, and that STAT92E TA domain is conserved in vertebrate STATs, it is possible that the PAR3-STAT interaction is a conserved feature of JAK/STAT signalling. S2 cell and embryo experiments show a requirement of Src in the STAT92E-Baz interaction. Paradoxically, it is possible to precipitate STAT92E in the absence of Src using the N- and the C-terminal fragments of Baz in vitro. Since in vitro Baz fragments are being used that most probably have a different conformation from the full-length protein, this paradox can be explained if, in vivo, Src function was required to change the conformation of Baz or to displace another protein that interferes with the binding, events that would not take place in the in vitro binding (Sotillos, 2013).

In summary, these results show that Src42A and Src64B are required redundantly in the ectoderm to allow STAT92E to bind to Baz. This interaction leads to the priming of JAK/STAT signalling by concentrating inactive STAT92E dimers apically near the polarized receptor kinase complex, contributing in this way to the efficient canonical JAK/STAT signalling. Although it cannot be completely discarded that future studies in Drosophila may find some specific cell type in which direct STAT92E activation by Src kinases exists, the data indicate that, in general, there is no direct STAT92E activation by Src in Drosophila. It is speculated that the existence of complexes where STAT, PAR3 and Src interact might have allowed the evolution of STAT-activation shortcuts that in vertebrates would have led to Src directly phosphorylating STAT in tyrosine 700 without the intervention of JAK or the canonical receptors. Considering the relevance of these proteins in development and disease, future studies should address whether apical STAT localization through PAR3-Src activity is also functioning in the vertebrate lineage (Sotillos, 2013).

PI3K signaling and Stat92E converge to modulate glial responsiveness to axonal injury

Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. This study shows glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. Evidence is provided for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. It is proposed that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury (Doherty, 2014: PubMed).

Combining genetic perturbations and proteomics to examine kinase-phosphatase networks in Drosophila embryos

Connecting phosphorylation events to kinases and phosphatases is key to understanding the molecular organization and signaling dynamics of networks. This study has generated a validated set of transgenic RNA-interference reagents for knockdown and characterization of all protein kinases and phosphatases present during early Drosophila melanogaster development. These genetic tools enable collection of sufficient quantities of embryos depleted of single gene products for proteomics. As a demonstration of an application of the collection, multiplexed isobaric labeling was used for quantitative proteomics to derive global phosphorylation signatures associated with kinase-depleted embryos to systematically link phosphosites with relevant kinases. This strategy uncovers kinase consensus motifs and prioritizes phosphoproteins for kinase target validation. This approach was validated by providing auxiliary evidence for Wee kinase-directed regulation of the chromatin regulator Stonewall. Further, it was shown how correlative phosphorylation at the site level can indicate function, as exemplified by Sterile20-like kinase-dependent regulation of Stat92E (Sopko, 2014).


STAT/marelle: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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