Gene name - Signal-transducer and activator of transcription protein at 92E
Synonyms - D-STAT, marelle (mrl)
Cytological map position - 92E2
Symbol - Stat92E
Genetic map position -
Classification - STAT homolog - src homology 2 domain
Cellular location - cytoplasmic and nuclear
|Recent literature||Katsuyama, T., Comoglio, F., Seimiya, M., Cabuy, E. and Paro, R. (2015). During Drosophila disc regeneration, JAK/STAT coordinates cell proliferation with Dilp8-mediated developmental delay. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25902518
Regeneration of fragmented Drosophila imaginal discs occurs in an epimorphic manner involving local cell proliferation at the wound site. After disc fragmentation, cells at the wound site activate a restoration program through wound healing, regenerative cell proliferation, and repatterning of the tissue. However, the interplay of signaling cascades driving these early reprogramming steps is not well-understood. This study profiled the transcriptome of regenerating cells in the early phase within 24 h after wounding. JAK/STAT signaling was found to become activated at the wound site and to promote regenerative cell proliferation in cooperation with Wingless (Wg) signaling. In addition, the expression of Insulin-like peptide 8 (dilp8), which encodes a paracrine peptide to delay the onset of pupariation, was found to be controlled by JAK/STAT signaling in early regenerating discs. These findings suggest that JAK/STAT signaling plays a pivotal role in coordinating regenerative disc growth with organismal developmental timing.
|Monahan, A. J. and Starz-Gaiano, M. (2015). Socs36E limits STAT signaling via Cullin2 and a SOCS-box independent mechanism in the Drosophila egg chamber. Mech Dev [Epub ahead of print]. PubMed ID: 26277564
The Suppressor of Cytokine Signaling (SOCS) proteins are critical, highly conserved feedback inhibitors of signal transduction cascades. The family of SOCS proteins is divided into two groups: ancestral and vertebrate-specific SOCS proteins. Vertebrate-specific SOCS proteins have been heavily studied as a result of their strong mutant phenotypes. However, the ancestral clade remains less studied, a potential result of genetic redundancies in mammals. Use of the genetically tractable organism Drosophila melanogaster enables in vivo assessment of signaling components and mechanisms with less concern about the functional redundancy observed in mammals. This study investigated how the SOCS family member Suppressor of Cytokine Signaling at 36E (Socs36E) attenuates Jak/STAT activation during specification of motile border cells in Drosophila oogenesis. Socs36E genetically interacts with the Cullin2 (Cul2) scaffolding protein. Like Socs36E, Cul2 is required to limit the number of motile cells in egg chambers. Loss of Cul2 in the follicle cells significantly increased nuclear STAT protein levels, which resulted in additional cells acquiring invasive properties. Further, reduction of Cul2 suppressed border cell migration defects that occur in a Stat92E-sensitized genetic background. These data incorporated Cul2 into a previously described Jak/STAT-directed genetic regulatory network that is required to generate a discrete boundary between cell fates. It was also found that Socs36E is able to attenuate STAT activity in the egg chamber when it does not have a functional SOCS box. Collectively, this work contributes mechanistic insight to a Jak/STAT regulatory genetic circuit, and suggests that Socs36E regulates Jak/STAT signaling via a Cul2-dependent mechanism, as well as by a Cullin-independent manner, in vivo.
|Saadin, A. and Starz-Gaiano, M. (2016). Identification of novel regulators of the JAK/STAT signaling pathway that control border cell migration in the Drosophila ovary. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27175018
The JAK/STAT signaling pathway is an essential regulator of cell migration both in mammals and fruit flies. Cell migration is required for normal embryonic development and immune response but can also lead to detrimental outcomes, such as tumor metastasis. A cluster of cells termed "border cells" in the Drosophila ovary provides an excellent example of a collective cell migration, in which two different cell types coordinate their movements. Border cells arise within the follicular epithelium and are required to invade the neighboring cells and migrate to the oocyte to contribute to a fertilizable egg. Multiple components of the STAT signaling pathway are required during border cell specification and migration; however, the functions and identities of other potential regulators of the pathway during these processes are not yet known. To find new components of the pathway that govern cell invasiveness, 48 predicted STAT modulators were knocked down using RNAi expression in follicle cells, and defective cell movement was assayed. Seven of these regulators were shown to be involved in either border cell specification or migration. Examination of the epistatic relationship between candidate genes and Stat92E reveals that the products of two genes, protein tyrosine phosphatase 61f (ptp61f) and brahma (brm), interact with Stat92E during both border cell specification and migration.
|Tamori, Y., Suzuki, E. and Deng, W. M. (2016). Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment. PLoS Biol 14: e1002537. PubMed ID: 27584724
Malignant tumors are caused by uncontrolled proliferation of transformed mutant cells that have lost the ability to maintain tissue integrity. Although a number of causative genetic backgrounds for tumor development have been discovered, the initial steps mutant cells take to escape tissue integrity and trigger tumorigenesis remain elusive. This study shows through analysis of conserved neoplastic tumor-suppressor genes (nTSGs) in Drosophila wing imaginal disc epithelia that tumor initiation depends on tissue-intrinsic local cytoarchitectures, causing tumors to consistently originate in a specific region of the tissue. In this "tumor hotspot" where cells constitute a network of robust structures on their basal side, nTSG-deficient cells delaminate from the apical side of the epithelium and begin tumorigenic overgrowth by exploiting endogenous JAK/STAT signaling activity. Conversely, in other regions, the "tumor coldspot" nTSG-deficient cells are extruded toward the basal side and undergo apoptosis. When the direction of delamination is reversed through suppression of RhoGEF2, an activator of the Rho family small GTPases, and JAK/STAT is activated ectopically in these coldspot nTSG-deficient cells, tumorigenesis is induced. These data indicate that two independent processes, apical delamination and JAK/STAT activation, are concurrently required for the initiation of nTSG-deficient-induced tumorigenesis. Given the conservation of the epithelial cytoarchitecture, tumorigenesis may be generally initiated from tumor hotspots by a similar mechanism.
|Verghese, S. and Su, T. T. (2016). Drosophila Wnt and STAT define apoptosis-resistant epithelial cells for tissue regeneration after irradiation. PLoS Biol 14: e1002536. PubMed ID: 27584613
Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. This study has identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. These findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue.
|Srinivasan, N., Gordon, O.,
Ahrens, S., Franz, A., Deddouche, S., Chakravarty, P., Phillips, D.,
Yunus, A.A., Rosen, M.K., Valente, R.S., Teixeira, L., Thompson, B.,
Dionne, M.S., Wood, W., Reis, E. and Sousa, C. (2016). Actin
is an evolutionarily-conserved damage-associated molecular pattern that
signals tissue injury in Drosophila melanogaster. Elife
[Epub ahead of print]. PubMed ID: 27871362
Damage associated molecular patterns (DAMPs) are released by dead cells and can trigger sterile inflammation and, in vertebrates, adaptive immunity. Actin is a DAMP detected in mammals by the receptor, DNGR-1, expressed by dendritic cells (DCs). DNGR-1 is phosphorylated by Src-family kinases and recruits the tyrosine kinase Syk to promote DC cross-presentation of dead cell-associated antigens. This study reports that actin is also a DAMP in invertebrates that lack DCs and adaptive immunity. Administration of actin to Drosophila melanogaster triggers a response characterised by selective induction of STAT target genes in the fat body through the cytokine Upd3 and its JAK/STAT-coupled receptor, Domeless. Notably, this response requires signalling via Shark, the Drosophila orthologue of Syk, and Src42A, a Drosophila Src-family kinase, and is dependent on Nox activity. Thus, extracellular actin detection via a Src-family kinase-dependent cascade is an ancient means of detecting cell injury that precedes evolution of adaptive immunity.
|Pan, C., Wang, W., Yuan, H., Yang, L., Chen, B., Li, D. and Chen, J. (2017). The immediate early protein WSV187 can influence viral replication via regulation of JAK/STAT pathway in Drosophila. Dev Comp Immunol 72: 89-96. PubMed ID: 28232015
The world production of shrimp is seriously affected by the white spot syndrome virus (WSSV). Viral immediate-early (IE) genes encode regulatory proteins critical for the viral lifecycle. In spite of their importance, only five out of the 21 identified WSSV IE genes are functionally characterized. This paper reports the use of Drosophila melanogaster as a model to explore the role of WSSV IE gene wsv187. In vivo expression of WSV187 in transgenic flies show WSV187 localized in the cytoplasm. Overexpression of wsv187 results wing defects consistent with phenotypes observed in JAK/STAT exacerbated flies. After artificial infection of the DCV virus, the flies expressing wsv187 showed a lower viral load, a higher survival rate and an up-regulated STAT92E expression. These data demonstrate wsv187 plays a role in the controlling of virus replication by activating host JAK/STAT pathway.
|Tsurumi, A., Zhao, C. and Li, W. X. (2017). Canonical and non-canonical JAK/STAT transcriptional targets may be involved in distinct and overlapping cellular processes. BMC Genomics 18(1): 718. PubMed ID: 28893190
In addition to a canonical pathway that uses the phosphorylated form of the STAT transcription factor, a non-canonical JAK/STAT pathway involving heterochromatin formation by unphosphorylated STAT was recently uncovered. This study has used the simple Drosophila system in which the non-canonical pathway was initially characterized, to compare putative canonical versus non-canonical transcriptional targets across the genome. Microarray expression patterns were analyzed of wildtype, Jak gain- and loss-of-function mutants, as well as the Stat loss-of-function mutant during embryogenesis, since the contribution of the canonical signal transduction pathway has been well-characterized in these contexts. Previous studies have also demonstrated that Jak gain-of-function and Stat mutants counter heterochromatin silencing to de-repress target genes by the non-canonical pathway. Compared to canonical target genomic loci, non-canonical targets were significantly more associated with sites enriched with heterochromatin-related factors (p = 0.004). Furthermore, putative canonical and non-canonical transcriptional targets identified displayed some differences in biological pathways they regulate, as determined by Gene Ontology (GO) enrichment analyses. Canonical targets were enriched mainly with genes relevant to development and immunity, as expected, whereas the non-canonical target gene set mainly showed enrichment of genes for various metabolic responses and stress response, highlighting the possibility that some differences may exist between the two loci. Canonical and non-canonical JAK/STAT genes may regulate distinct and overlapping sets of genes and may perform specific overall functions in physiology. Further studies at different developmental stages, or using distinct tissues may identify additional targets and provide insight into which gene targets are unique to the canonical or non-canonic pathway.
|Verghese, S. and Su, T. T. (2017). STAT, Wingless, and Nurf-38 determine the accuracy of regeneration after radiation damage in Drosophila. PLoS Genet 13(10): e1007055. PubMed ID: 29028797
Regeneration in Drosophila larval wing imaginal discs has been studied after damage by ionizing radiation. Faithful regeneration was detected that restored a wing disc and abnormal regeneration that produced an extra wing disc. A sequence of changes in cell number, location and fate is described that occur to produce an ectopic disc. A group of cells was identified that not only participate in ectopic disc formation but also recruit others to do so. STAT92E (Drosophila STAT3/5) and Nurf-38, which encodes a member of the Nucleosome Remodeling Factor complex, oppose each other in these cells to modulate the frequency of ectopic disc growth. The picture that emerges is one in which activities like STAT increase after radiation damage and fulfill essential roles in rebuilding the tissue. But such activities must be kept in check so that one and only one wing disc is regenerated.
The JAK/STAT pathway pathway is involved in communicating extracellular events on an intracellular level: signals must be carried across the cell membrane, through the cytoplasm and finally into the nucleus. Receptors that function through Janus kinase (JAK) proteins do not themselves have kinase activity, but rely on the kinase activity of the JAK family to transduce their signals into the cell.
Two laboratories have simultaneously described STAT, referred to here as marelle, the Drosophila homolog for mammalian STAT (signal transducer and activator of transcription) proteins. One characterized a maternal lethal effect that exhibited a segmentation phenotype similar to the effect of the loss of hopscotch. hopscotch is the Drosophila homolog of the mammalian JAK family kinases. The second lab had been searching for a Drosophila homolog for the mammalian STAT gene (Hou, 1996 and Yan, 1996a). Discovery of marelle completes the identification of the JAK-STAT pathway in the fly.
The mammalian janus family tyrosine kinases (JAK kinases) associate with the intracellular domains of particular cytokine receptors, and become activated following ligand-induced assembly of receptor subunits at the cell surface. The STATs are a family of src homology 2 proteins that are cytoplasmic transcription factors that serve to transduce signals from the activated JAK kinases to the nucleus. The src homology 2 domain serves to dock STAT proteins to phosphorylated substrates. STAT proteins are phosphorylated in turn. When activated by tyrosine phosphorylation, STAT proteins undergo dimerization. They translocate to the nucleus and promote transcriptional activation of cytokine inducible genes (Schindler, 1995). The identity of possible Drosophila cytokines, if there are any in this system, is not known.
Drosophila STAT, named marelle (the French term for "hopscotch"), is present in the egg at the time of fertilization and is expressed early in development. It is transcribed initially in a pair-rule striped pattern and later in a 14 striped segment polarity pattern. MRL binds to and regulates the even-skipped stripe 3 promoter, and regulates the pair rule gene runt (Hou, 1996 and Yan, 1996).
Polarity of the Drosophila compound eye is established at the level of repeating multicellular units (known as ommatidia), which are organized into a precise hexagonal array (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation). The adult eye is composed of ~800 ommatidia, each of which forms one facet. Sections through the eye reveal that each ommatidium contains eight photoreceptor cells in a stereotypic trapezoidal arrangement that has two mirror-symmetric forms: a dorsal form present above the dorsoventral (DV) midline, and a ventral form below. An axis of mirror-image symmetry runs along the DV midline and is known as the equator. By analogy to the terrestrial equator, the extreme dorsal and ventral points of the eye are referred to as the poles. Differentiation of ommatidia begins during the third instar larval stage when a furrow moves from posterior to anterior over the epithelium of the eye imaginal disc. Each ommatidial unit is born as a bilaterally symmetrical cluster of photoreceptor precursors, that is polarized on its anteroposterior axis. The clusters then become polarized on the DV (or equatorial-polar) axis, by the process of proto-ommatidium rotation via two 45° steps away from the DV midline, forming the equator. It has been suggested that the direction of this rotation is a consequence of a gradient of positional information emanating from either the midline or the polar regions of the disc (Zeidler, 1999 and references).
A number of recent studies have shed light on some of the mechanisms involved in the positioning of the equator on the DV midline of the eye imaginal disc. It is now clear that a critical step is the activation of Notch activity in a line of cells along the midline, and that this localized activation of Notch is a consequence of the restricted expression of the fringe (fng) gene product in the ventral half of the disc and the homeodomain transcription factor Mirror (Mirr) in the dorsal half of the disc. Furthermore, an important role for Wingless (Wg) in polarity determination on the DV axis has been demonstrated. Wg is a secreted protein (and the founder member of the Wnt family of morphogens) that is expressed at the poles of the eye disc. Wg has been shown to act as an activator of mirr expression; increasing the levels of Wg expression in the eye disc shifts mirr expression and the position of the equator ventrally, whereas reduction of wg function shifts mirr expression dorsally. Additionally, it has been shown convincingly that a gradient of Wg signaling across the DV axis of the eye disc regulates ommatidial polarity such that the lowest point of Wg signaling coincides with the equator (Zeidler, 1999 and references).
The JAK/STAT pathway is central to the establishment of planar polarity during Drosophila eye development. A localized source of the pathway ligand, Unpaired/Outstretched, present at the midline of the developing eye, is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror (Zeidler, 1999).
The data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localized activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, since fng loss of function clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity. Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals (most likely detected by the receptor Frizzled) that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal (Zeidler, 1999 and references).
Loss of function (LOF) clones for mutations in the Drosophila JAK and STAT homologs were generated by the FLP/FRT system. Tangential sections through LOF clones of both hop and stat alleles show a regular array of ommatidia containing a wild-type complement of correctly differentiated and correctly positioned photoreceptor cells. Thus, the JAK/STAT pathway is not absolutely required for imaginal disc cell proliferation, cell fate specification, or differentiation. Mutant clones are, however, associated with stereotyped defects in ommatidial polarity (Zeidler, 1999).
A large proportion of hop LOF clones result in polarity defects in which ommatidia straddling the polar boundary of the clone exhibit inverted DV polarity. The phenotype is strongest in larger clones and in clones in which the polar boundary runs parallel to the equator. Typically, one or two ommatidial rows are inverted, with the strongest phenotype observed showing about five inverted rows. Mutant ommatidia in the center of the clone and on the equatorial margin of the clone show a normal orientation. Both totally mutant ommatidia adjacent to the polar boundary and chimeric ommatidia comprising both wild-type and mutant cells on the clonal border can assume an inverted fate. Occasional inversions are observed in clusters immediately outside the clone in which all of the photoreceptors are wild type. LOF hop clones examined in third instar imaginal discs show the same phenotype (Zeidler, 1999).
The downstream pathway component STAT was also tested by inducing clones of stat92E alleles. These give qualitatively identical phenotypes to hop clones, but at a lower penetrance. The frequency with which inversions are recovered is increased in a genetic background heterozygous for hop, demonstrating that removal of a single copy of hop can sensitize the pathway to loss of stat92E. The weak nature of the stat92E phenotype would appear to indicate that the stat92E gene product is only partially required to transduce the hop-mediated signal. Although unexpected, this finding is consistent with previous evidence that more than one STAT homolog exists in flies, and suggests that they act semiredundantly in ommatidial polarity determination. Thus, the juxtaposition of wild-type cells and cells unable to transduce the JAK/STAT signal can generate ectopic axes of ommatidial mirror-image symmetry that resemble the normal equator (Zeidler, 1999).
As LOF JAK/STAT clones result in ectopic axes of ommatidial symmetry, the effects of ectopic activation of the pathway were examined by misexpression of the pathway ligand Upd/Outstretched. GOF Upd clones were generated by a combination of the FLP/FRT cassette, such that Upd is expressed in discrete groups of marked cells in the developing eye. This results in inversion of ommatidial polarity in the wild-type tissue on the equatorial side of the clone, with the greatest effect observed in clones closer to the poles of the disc. Taken together, these LOF and GOF results indicate that JAK/STAT function across the DV axis of the eye disc is necessary for the normal establishment of a single axis of ommatidial mirror-image symmetry along the DV midline, and is sufficient to define ectopic axes of mirror-image symmetry (Zeidler, 1999).
An interesting aspect of the original P-element-mediated insertional mutation in the stat92E locus (stat92E06346) is the lacZ expression pattern produced by this enhancer detector. Eye discs from larvae carrying this insertion (subsequently referred to as stat92E-lacZ) show a gradient of lacZ activity that is highest at the poles and decreases to a low point at the DV midline. Increased expression is also seen in the ocellar spot region, and, independently, in many of the macrophage-like blood cells often associated with the eye imaginal disc complex. However, in situ hybridization experiments undertaken with probes specific for the stat92E transcript show ubiquitous expression of stat92E mRNA in third instar eye discs, suggesting that this enhancer detector might only report a subset of stat92E transcript expression (Zeidler, 1999).
An intriguing possibility was that stat92E-lacZ expression might be related to JAK/STAT pathway activity. stat92E-lacZ staining was therefore examined in larvae carrying the constitutively active hopTuml allele of Drosophila JAK. In hopTuml eye discs with uniformly increased JAK/STAT activity, the overall level of lacZ activity is consistently lower than in discs from wild-type siblings stained in parallel. Additional experiments show that the level of stat92E-lacZ expression is inversely proportional to the level of JAK/STAT pathway activation: High activation produced by Upd expression abolishes stat92E-lacZ activity; moderate activation produced by the hopTuml allele gives reduced activity, whereas cells in which there is no JAK/STAT signaling (such as hop clones) show maximal levels of stat92E-lacZ activity. Comparing the results of these experiments with the endogenous pattern of stat92E-lacZ staining in the eye disc, it is concluded that JAK/STAT activity must be highest at the DV midline (where stat92E-lacZ activity is lowest) and low at the poles (where stat92E-lacZ activity is upregulated to levels similar to those seen in hop clones) with a gradient of JAK/STAT activity present between these extremes (Zeidler, 1999).
Given the role of Upd in restricting mirr expression, one possible mechanism by which JAK/STAT LOF clones might induce ectopic axes of mirror-image symmetry would be through the generation of ectopic boundaries of mirr expression. The expression of mirr-lacZ was examined in hop clones. Many clones lying both dorsally and ventrally were examined in eye discs, and in no case was an alteration in mirr-lacZ expression observed. Additionally, hundreds of adults carrying mirr-lacZ were examined, in which hop clones had been induced, and, again, in no case was a change in mirr-regulated white+ expression observed (Zeidler, 1999).
Thus, ommatidial polarity inversions generated by hop clones are mirr independent. It is therefore concluded that the process of midline equator definition by dorsally restricted mirr expression and the regulation of ommatidial polarity by the JAK/STAT pathway are separable processes. It is also noted that these results suggest that Upd might act independently of Hop to regulate mirr expression (Zeidler, 1999).
The ommatidial polarity phenotype produced by removal of JAK activity in mosaic clones has a number of important features: (1) the phenotype observed is an inversion of ommatidial polarity in which either the dorsal rotational form is seen in the ventral hemisphere of the eye or vice versa; (2) the phenotype is only observed on the polar boundary of the mosaic tissue; (3) the strength of the phenotype (in terms of the number of inverted ommatidia seen) is dependent on the size and shape of the clone; (4) the phenotype is cell nonautonomous as either fully mutant, fully wild-type, or as mosaic clusters that can manifest the phenotype (Zeidler, 1999).
From these characteristics, the following can be deduced: the nonautonomy of the phenotype produced by removal of the autonomously acting pathway component JAK, and its dependence on clone size and shape, suggests that JAK/STAT affects ommatidial polarity via a secreted downstream signal (which subsequently will be referred to as a second signal, most likely detected by Frizzled). The direction of the nonautonomy (only in a polar direction) and the strict DV nature of the polarity inversions indicates that this second signal must be graded in its activity along the DV axis, with a change in direction of the gradient at the equator. The direction of this gradient would then be the instructive cue to which ommatidia respond when rotating to establish their mature polarity (Zeidler, 1999).
The simplest model would be that there is a single second signal secreted from the equator, which is downstream of mirr/fng/Notch, and that Wg and Upd/JAK/STAT feed into this pathway upstream of Notch. This is consistent with the roles of Wg and Upd as regulators of mirr expression and, thus, in positioning the endogenous equator. However, it is not consistent with the observed ommatidial polarity inversions produced in the eye field both dorsally and ventrally by Wg-pathway and JAK/STAT-pathway LOF and GOF clones. These phenotypes indicate that second-signal concentration is dependent on Wg pathway and JAK/STAT pathway activity across the whole of the eye field, and thus the second signal cannot be only secreted from the DV midline as a consequence of localized Notch activation. It is conceivable that Notch is activated on the polar boundary of JAK/STAT LOF clones, but in this context the only known mechanism of Notch activation is via mirr/fng interactions, and this possibility has been ruled out (Zeidler, 1999).
Instead, the data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localize activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, becausefng LOF clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity (Zeidler, 1999).
Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal. A number of observations provide a great deal of support for such a model. (1) It is consistent with the known timing of the events involved. The requirement for fng function has been shown to lie between late first instar and mid second instar, which coincides with the first appearance of high levels of Upd expression at the optic stalk. However, the ommatidia are not formed (and thus do not respond to the polarity signal) until the start of the third instar, a stage when localized Upd expression still persists. Furthermore, extracellular Upd protein can be seen in a concentration gradient many cell diameters from the optic stalk at the early third instar stage, consistent with Upd being at least partly responsible for setting up the long-range gradient of JAK/STAT activity across the DV axis of the eye disc that is revealed by the stat92E-lacZ reporter. (2) This model does not require that a single source of second signal secreted by a narrow band of cells at the equator should be capable of determining ommatidial polarity across the whole of the DV axis of the disc during the third instar stage of development. Instead, the band of activated Notch at the equator would serve to draw a straight line between the fields of dorsally and ventrally polarized ommatidia, and need only secrete a localized source of second signal to polarize ommatidia in this region. Further from the equator, the opposing gradients of Upd and Wg signaling would provide a robust mechanism for maintenance of correct ommatidial polarity across the DV axis. Conversely, without the mirr/fng/Notch mechanism to draw a straight line, it would be impossible to imagine how Upd at the posterior margin and Wg at the poles alone could provide the perfectly straight equator that is ultimately formed. (3) The phenotypes that are observed are consistent with multiple competing mechanisms responsible for determining ommatidial polarity. When inversions of ommatidial polarity are induced by generating hop clones or ectopically expressing Upd, straight equators are not produced, such that two cleanly abutting fields of dorsal and ventral ommatidia are produced. Instead, there is usually some confusion of ommatidial identities as if they might be receiving conflicting signals. Additionally, when upd activity is removed from the optic stalk, an equator still forms (albeit at the ventral edge of the disc), but some ommatidia dorsal to the equator still adopt a ventral fate as if the determination of ommatidial polarity is less robust in the absence of Upd (Zeidler, 1999).
Cell competition is a conserved mechanism that regulates organ size and shares properties with the early stages of cancer. In Drosophila, wing cells with increased Myc or with optimum ribosome function become supercompetitors that kill their wild-type neighbors (called losers) up to several cell diameters away. This study reports that modulating STAT activity levels regulates competitor status. Cells lacking STAT become losers that are killed by neighboring wild-type cells. By contrast, cells with hyper-activated STAT become supercompetitors that kill losers located at a distance in a manner that is dependent on hid but independent of Myc, Yorkie, Wingless signaling, and of ribosome biogenesis. These results indicate that STAT, Wingless and Myc are major parallel regulators of cell competition, which may converge on signals that non-autonomously kill losers. As hyper-activated STATs are causal to tumorigenesis and stem cell niche occupancy, these results have therapeutic implications for cancer and regenerative medicine (Rodrigues, 2012).
This study establishes a role for JAK/STAT signaling in cell competition between somatic cells that contribute to the adult organism. Wing disc cells lacking Stat92E activity suffer from competitive stress exerted by their wild-type neighbors and undergo apoptosis. However, when these same cells are placed with growth-disadvantaged cells (i.e. M/+), they are viable. This context-dependent behavior of cells (i.e. viable when homotypic but disadvantaged when in apposition to more robust cells) is a hallmark of cell competition. Interestingly, the growth of Stat92E clones can be rescued by inhibition of apoptosis. By contrast clones lacking Myc or ribosomal genes such as Rpl135 cannot grow even when death is inhibited. This may represent an important difference between activated Stat92E and Myc function in losers (Rodrigues, 2012).
Despite these differences, activated Stat92E does in fact share distinguishing features of cell competition with Myc: winners with activated Stat92E (1) induce programmed cell death in losers at a distance and (2) require hid to do so. In addition, the observation that cells null for Myc can be at least partially rescued by autonomous activation of Stat92E is noteworthy because autonomous expression of Yki does not rescue these cells. It is proposed that the partial rescue of null Myc cells by activated Stat92E is probably not due to an increase in ribosome activity or in expression of Myc target genes that drive ribosome assembly as activated Stat92E does not induce a subset of ribosomal genes during third instar (Rodrigues, 2012).
This study demonstrates that the JAK/STAT pathway plays an obligate role in growth of all cells in the young wing disc (30-48 hours AED). During this period of exponential growth, it was shown that imaginal cells lacking Stat92E are less competitive and are subjected to stress imposed by their wild-type neighbors and they are ultimately killed by hid-dependent apoptosis. No regional effects of Stat92E were observed at early time points: Stat92E clones grew poorly regardless of their position on the AP and DV axes when induced at 30 or 36 hours AED. The results from the Stat92E clonal analyses presented in this study are consistent with but stronger than those published by another group (Mukherjee, 2005). This discrepancy may be due to their use of a weaker allele Stat92E06346 (Rodrigues, 2012).
This study demonstrates that cells with activated Stat92E also achieve supercompetitor status and induce death of their wild-type neighbors up to several cell diameters away, which is similar to the non-autonomous death of wild-type cells induced by Myc or Wg supercompetitors. These results strongly suggest that non-autonomous cell death is a key feature of cell competition in response to local cellular differences in either STAT activity, Wg signaling or Myc. Moreover, it has been demonstrated that, like Myc, cells with activated Stat92E require the pro-apoptotic gene hid to kill surrounding neighbors and achieve supercompetitor status. Although these results suggest a link between Stat92E and Myc, surprisingly no link was found between JAK/STAT signaling and Myc mRNA or Myc protein or in targets of the Hippo pathway. Furthermore, no regulation of Wg signaling was found by activated STAT and no effect was found of Wg on STAT activity. Taken together, these results strongly suggest that activated STAT functions in parallel to Yki, Myc and Wg in growth and cell competition (Rodrigues, 2012).
Differences in ribosome activity between winners and losers appear to be crucial to Myc- and Minute-induced cell competition and may also be required by Myc for its supercompetitor activity. Activated STAT does not increase expression of an important set of ribosome biogenesis genes during late larval stages. It is conceivable that JAK/STAT signaling might affect other ribosomal aspects not tested in this study. Assuming a similar relationship exists at earlier larval stages - when Stat92E is required for clonal growth - the model is favored that STAT-dependent cell competition is largely independent of de novo ribosome biogenesis. This would represent an important difference between JAK/STAT and Myc- or Minute-dependent cell competition. Regardless, the results at the very least suggest the presence of multiple sensors of competitive situations and indicate that the way cells compare their fitness with one another is more complex than previously believed. Indeed, Myc- and ribosome-independent supercompetition appears to be a newly emerging paradigm in the field. In conclusion, this study found that differences in Stat92E activity reveal differences in cellular fitness that are in large part unrelated to Myc, ribosome biogenesis, Hippo, Wg or Dpp signaling activity. Moreover, given the conservation between the components of the Drosophila and mammalian JAK/STAT signaling pathway, these findings lead the way for further investigation of cell competition in mammals (Rodrigues, 2012).
During development, specific cells are eliminated by apoptosis to ensure that the correct number of cells is integrated in a given tissue or structure. How the apoptosis machinery is activated selectively in vivo in the context of a developing tissue is still poorly understood. In the Drosophila ovary, specialised follicle cells [polar cells (PCs)] are produced in excess during early oogenesis and reduced by apoptosis to exactly two cells per follicle extremity. PCs act as an organising centre during follicle maturation as they are the only source of the JAK/STAT pathway ligand Unpaired (Upd), the morphogen activity of which instructs distinct follicle cell fates. This study shows that reduction of Upd levels leads to prolonged survival of supernumerary PCs, downregulation of the pro-apoptotic factor Hid, upregulation of the anti-apoptotic factor Diap1 and inhibition of caspase activity. Upd-mediated activation of the JAK/STAT pathway occurs in PCs themselves, as well as in adjacent terminal follicle and interfollicular stalk cells, and inhibition of JAK/STAT signalling in any one of these cell populations protects PCs from apoptosis. Thus, a Stat-dependent unidentified relay signal is necessary for inducing supernumerary PC death. Finally, blocking apoptosis of PCs leads to specification of excess adjacent border cells via excessive Upd signalling. These results therefore show that Upd and JAK/STAT signalling induce apoptosis of supernumerary PCs to control the size of the PC organising centre and thereby produce appropriate levels of Upd. This is the first example linking this highly conserved signalling pathway with developmental apoptosis in Drosophila (Borensztejn, 2013).
A role for STAT in cell death and survival has been clearly documented in mammals, and depending on which of the seven mammalian Stat genes is considered and on the cellular context, both pro- and anti-apoptotic functions have been characterised. In the Drosophila developing wing, phosphorylated Stat92E has been shown to be necessary for protection against stress-induced apoptosis, but not for wing developmental apoptosis. This study provides evidence that Upd and the JAK/STAT pathway control developmental apoptosis during Drosophila oogenesis (Borensztejn, 2013).
This study demonstrated that the JAK/STAT pathway ligand, Upd, and all components of the JAK/STAT transduction cascade (the receptor Dome, JAK/Hop and Stat92E) are involved in promoting apoptosis of supernumerary PCs produced during early oogenesis. It is argued that The JAK/STAT pathway is essential for this event for several reasons. Indeed, in the strongest mutant context tested, follicle poles containing large TFC and PC clones homozygous for Stat92E amorphic alleles, almost all of these (95%) maintained more than two PCs through oogenesis. Also, RNAi-mediated reduction of upd, dome and hop blocked PC number reduction and deregulated several apoptosis markers, inhibiting Hid accumulation, Diap1 downregulation and caspase activation in supernumerary PCs. Altogether, these data, along with what has already been shown for JAK/STAT signalling in this system, fit the following model. Upd is secreted from PCs and diffuses in the local environment. Signal transduction via Dome/Hop/Stat92E occurs in nearby TFCs, interfollicular stalks and PCs themselves, leading to specific target gene transcription in these cells, as revealed by a number of pathway reporters. An as-yet-unidentified Stat92E-dependent pro-apoptotic relay signal (X) is produced in TFCs, interfollicular stalks and possibly PCs, which promotes supernumerary PC elimination via specific expression of hid in these cells, consequent downregulation of Diap1 and finally caspase activation. An additional cell-autonomous role for JAK/STAT signal transduction in supernumerary PC apoptosis of these cells is also consistent with, though not demonstrated by, the results (Borensztejn, 2013).
Relay signalling allows for spatial and temporal positioning of multiple signals in a tissue and thus exquisite control of differentiation and morphogenetic programmes. In the Drosophila developing eye, the role of Upd and the JAK/STAT pathway in instructing planar polarity has been shown to require an as-yet-uncharacterised secondary signal. In the ovary, the fact that JAK/STAT-mediated PC apoptosis depends on a relay signal may provide a mechanism by which PC apoptosis and earlier JAK/STAT-dependent stalk-cell specification can be separated temporally (Borensztejn, 2013).
Although neither the identity, nor the nature, of the relay signal are known, it is possible to propose that the signal is not likely to be contact-dependent, and could be diffusible at only a short range. Indeed, Stat92E homozygous mutant TFC clones in contact with PCs, as well as those positioned up to three cell diameters away from PCs, are both associated with prolonged survival of supernumerary PCs, whereas clones further than three cell diameters away from PCs are not. In addition, fully efficient apoptosis of supernumerary PCs may require participation of all surrounding TFCs, stalk cells and possibly PCs, for production of a threshold level of relay signal. In support of this, large stat mutant TFC clones are more frequently associated with prolonged survival of supernumerary PCs, and the effects of removing JAK/STAT signal transduction in several cell populations at the same time are additive. Interestingly, the characterisation of two other Drosophila models of developmental apoptosis, interommatidial cells of the eye and glial cells at the midline of the embryonic central nervous system, also indicates that the level and relative position of signals (EGFR and Notch pathways) is determinant in selection of specific cells to be eliminated by apoptosis (Borensztejn, 2013).
The results indicate that only the supernumerary PCs respond to the JAK/STAT-mediated pro-apoptotic relay signal, whereas two PCs per pole are always protected. Indeed, this study found that overexpression of Upd did not lead to apoptosis of the mature PC pairs and delayed rather than accelerated elimination of supernumerary PCs. Recently, it was reported that selection of the two surviving PCs requires high Notch activation in one of the two cells and an as-yet-unknown Notch-independent mechanism for the second cell. Intriguingly, expression of both Notch and Stat reporters is dynamic in PC clusters and PC survival and death fates are associated with respective activation of the Notch and JAK/STAT pathways. However, this study found that RNAi-mediated downregulation of upd did not affect either expression of Notch or that of two Notch activity reporters. Therefore, JAK/STAT does not promote supernumerary PC apoptosis by downregulating Notch activity in these cells. Identification of the relay signal and/or of Stat target genes should help further elucidate the mechanism underlying the induction of apoptosis in selected PCs (Borensztejn, 2013).
Interfollicular stalk formation during early oogenesis has been shown to depend on activation of the JAK/STAT pathway. The presence of more than two PCs during these stages may be important to produce the appropriate level of Upd ligand to induce specification of the correct number of stalk cells. Later, at stages 7-8 of oogenesis, correct specification of anterior follicle cell fates (border, stretch and centripetal cells) depends on a decreasing gradient of Upd signal emanating from two PCs positioned centrally in this field of cells. Attaining the correct number of PCs per follicle pole has been shown to be relevant to this process and border cells (BC) specification seems to be particularly sensitive to the number of PCs present. Previously work has shown apoptosis of supernumerary PCs is physiological necessary for PC organiser function, as blocking caspase activity in PCs such that more than two PCs are present from stage 7 leads to defects in PC/BC migration and stretch cell morphogenesis. This study now shows that the excess PCs produced by blocking apoptosis lead to increased levels of secreted Upd and induce specification of excess BCs compared with the control, and these exhibit inefficient migration. These results indicate that reduction of PC number to two is necessary to limit the amount of Upd signal such that the correct numbers of BCs are specified for efficient migration to occur. Taken together with the role shown for Upd and JAK/STAT signalling in promoting PC apoptosis, it is possible to propose a model whereby Upd itself controls the size of the Upd-producing organising centre composed of PCs by inducing apoptosis of supernumerary PCs. Interestingly, in the polarising region in the vertebrate limb bud, which secretes the morphogen Sonic Hedgehog (Shh), Shh-induced apoptosis counteracts Fgf4-stimulated proliferation to maintain the size of the polarising region and thus stabilise levels of Shh. It is likely that signal autocontrol via apoptosis of signal-producing cells will prove to be a more widespread mechanism as knowledge of apoptosis control during development advances (Borensztejn, 2013).
MRL contains a src homology2-like domain and a DNA binding motif (Hou, 1996 and Yan, 1996a).
date revised: 15 NOV 97
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