BIOLOGICAL OVERVIEW

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 hop^Tuml allele of Drosophila JAK. In hop^Tuml 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 hop^Tuml 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).

Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis

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).

JAK/STAT autocontrol of ligand-producing cell number through apoptosis

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).

Actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in Drosophila melanogaster

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 (Srinivasan, 2016).

Trauma, burns, ischemia, strenuous exercise, all induce a sterile inflammatory response. It is likely that this response evolved to clear cell debris, promote tissue repair and maintain tissue sterility but, if uncontrolled, it can lead to (aseptic) shock and, in some cases, death. The prevailing notion is that sterile inflammation is initiated by pro-inflammatory signals that are released by damaged cells. These include intracellular components that are exposed when cells lose their membrane integrity, such as ATP, uric acid, RNA and DNA, collectively known as damage-associated molecular patterns (DAMPs). The universe of DAMPs and their receptors, as well as the mechanisms regulating DAMP responses, remains underexplored. This is partly because early research in this area was tainted by issues of microbial contamination and because immunologists have often focussed on sterile inflammation from the narrow perspective of adaptive immunity. However, it is probable that responses to DAMPs, like responses to microbes, pre-date the vertebrate evolution of T and B cells and have an early metazoan origin, much like the clearance of dead cells. Therefore, the study of invertebrate responses to DAMPs could offer a different perspective into the induction of sterile inflammation, akin to how research into insect immunity to infection led to the identification of Toll signalling and paved the way to the discovery of an analogous pathway in vertebrates (Srinivasan, 2016).

The immune system of Drosophila melanogaster has been widely studied in the context of infection. It consists of a cellular and a humoural arm, in addition to cell-intrinsic antiviral RNAi responses. The cellular arm is made up of three macrophage-like types of cells, collectively termed haemocytes. The humoural immune response relies on antimicrobial peptides (AMPs) that are synthesised in the fat body (the fly equivalent of the liver) and then secreted into the haemolymph to provide systemic protection from bacteria and fungi. The production of AMPs is regulated by two different pathways. The Toll pathway is activated by peptidoglycan fragments of Gram-positive bacteria, fungal β-glucans, and pathogen-derived protease activity in the haemolymph. The Imd pathway is activated by peptidoglycan fragments from Gram-negative bacteria. Activation of either pathway results in the translocation of distinct NF-κB family transcription factors into the nucleus and the subsequent synthesis of AMPs best suited to neutralise the type of microorganism detected. A third pathway contributing to Drosophila humoural immunity involves Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling. In contrast to the Toll and Imd pathway, the JAK/STAT pathway has not yet been shown to be directly induced by sensors of invading microorganisms. However, it has been implicated in resistance to as well as tolerance to viral infections. Notably, the JAK/STAT pathway is activated by different types of stresses (e.g. heat, mechanical pressure, oxidative stress or UV irradiation). All of these insults likely result in cell death suggesting the possibility that JAK/STAT pathway activation might be triggered by DAMPs rather than microbes (Srinivasan, 2016).

The JAK/STAT pathway is elicited by cytokines of the Unpaired (Upd) family -- Upd1, Upd2 and Upd3 -- all of which serve as ligands for the only JAK/STAT-coupled receptor in Drosophila, Domeless (dome). The binding of Upds induces Domeless dimerization and activation of a single JAK (termed Hopscotch). Activated Hopscotch proteins phosphorylate one another allowing for recruitment of the single Drosophila STAT family transcription factor, STAT92E. The latter is then phosphorylated by Hopscotch, resulting in dimerisation and translocation into the nucleus. STAT92E dimers bind to the promoters of their target genes including, amongst others, ones encoding proteins involved in viral resistance, as well as proteins of the Turandot family such as Turandot M (TotM). The exact function of Turandot family proteins is not known but they have been controversially argued to be linked to stress resistance. Besides a role in host defence, the JAK/STAT pathway has also been linked to energy metabolism and regenerative processes, for example in the gut. The involvement of JAK/STAT signalling in regeneration is particularly interesting given the role of DAMPs in contributing to tissue repair (Srinivasan, 2016).

Previously work has identified DNGR-1 (also known as CLEC9A) as a vertebrate-restricted innate immune receptor dedicated to DAMP recognition (Sancho, 2009). DNGR-1 is phosphorylated by Src family kinases and then signals via Syk although it does not induce inflammation. Rather, DNGR-1 is expressed by dendritic cells (DCs) and signals to favour cross-presentation of antigens from dead cells, contributing to CD8+ T cell responses to cytopathic infections and, possibly, tumours. The DAMP recognised by DNGR-1 is F-actin, the polymer of G-actin that provides higher eukaryotic cells with structural integrity (Ahrens, 2012; Zhang, 2012). Actin is an ideal DAMP given that it is extremely conserved (90% identity between yeast and humans) and highly abundant and ubiquitous within all eukaryotic cells but absent from extracellular fluids. It was therefore hypothesised that released actin constitutes an evolutionarily-conserved DAMP whose detection might involve a signalling pathway conserved from flies to mammals. This would be analogous to the conservation of the Toll signalling pathway (albeit not the upstream receptors) in the Drosophila and vertebrate response to fungi and bacteria. This study shows systemic administration of actin to Drosophila selectively triggers a JAK/STAT response and that this requires the fly homologues of Src and Syk. The data therefore reveal an evolutionarily-conserved tyrosine kinase-based pathway for recognising damage through sensing of released or exposed actin (Srinivasan, 2016).

Dysregulated and/or chronic inflammation, often of sterile origin, is increasingly recognised as a contributing factor to a vast range of human diseases, from cancer to neurodegeneration. Furthermore, because injury and infection often overlap, understanding of immunity necessitates a consideration of the interplay between the processes that detect pathogen invasion and those that sense tissue damage. The study of invertebrate responses to DAMPs might therefore lead to a new understanding of sterile inflammation and the identification of conserved elicitors, detectors and signaling pathways that are utilised across evolution to detect loss of cell integrity (Srinivasan, 2016).

Previous work has shown that actin, one of the most abundant and conserved proteins in eukaryotic cells, acts as a DAMP in mouse and humans, binding to DNGR-1, a Src and Syk-coupled dead cell receptor expressed on DCs. This study provides evidence that actin is also a DAMP in Drosophila melanogaster, triggering a response that, like in vertebrates, requires Syk and Src family kinases. The presence of extracellular actin in the haemolymph of Drosophila elicits a reaction in the fat body via Shark and Src42A, whose activation depends on reactive oxygen species (ROS) generated by the NADPH oxidase Nox. Consistent with these data, ROS generation by NADPH oxidases is a highly conserved response to wounding and has been shown to directly activate Lyn/Src42A in zebrafish and Drosophila through oxidation of a single redox-sensitive cysteine residue (Srinivasan, 2016).

In contrast to DNGR-1 dependent recognition, the fly response to extracellular actin is elicited equally by G- and F-actin, does not require phagocytes but the fat body and its function is not to prime adaptive immunity, which is absent in invertebrates. Rather, it is coupled to production of Upd3 cytokine, which acts in an autocrine and paracrine manner to induce Domeless signalling via STAT and to cause the induction of STAT-responsive genes, the products of which are released into the haemolymph. This systemic inflammatory-like response involving cytokine amplification and the fat body is reminiscent of the acute phase response in mammals, which can be triggered by infection or trauma and leads to the production of cytokines such as IL-6 that act on the liver (mammalian equivalent of the fat body) to cause production of acute phase proteins. These are secreted into the plasma to regulate multiple processes such as host defence, coagulation, vascular permeability and metabolism (Medzhitov, 2010). Similarly, the Drosophila fat body response to actin results in secretion into the haemolymph of proteins that may regulate multiple aspects of fly physiology that coordinately impact resistance or tolerance to insult. However, it is important to note that while some components of the extracellular actin-sensing circuitry are conserved between flies and mammals (Shark, Src42A and ROS), others are not (DNGR1, cross-presentation, dendritic cells). These differences suggest that DAMPs can be more conserved than their receptors or the responses they evoke. This is akin to pathogen-associated molecular patterns (PAMPs) such as, for example, lipopolysaccharide (LPS), a hallmark of Gram-negative bacteria. The sensing of LPS is conserved in plants, protists and animals, but the relevant receptors and subsequent responses diverge depending on the host. Similarly, peptidoglycans and β-glucans are used in both flies and mammals to signify bacterial or fungal presence, yet are detected by different receptors that, nevertheless, can couple to conserved signalling pathways (Srinivasan, 2016).

The JAK/STAT pathway in Drosophila can be induced by mechanical pressure, heat shock, dehydration, cytopathic infection, septic wounds and other traumas. How such seemingly disparate stimuli trigger a single pathway is puzzling. However, a common denominator in all these settings is cell death and it has been speculated that STAT activation might therefore occur in response to DAMP release. The current data support that notion and suggest that actin is a potent DAMP for triggering the JAK/STAT pathway. Notably, pathogen infection in Anopheles gambiae and Drosophila melanogaster has been shown to lead to the release of actin into the haemolymph, where it can act as an antibacterial or antiparasitic agent. Therefore, actin release may serve as a two-pronged defense mechanism, both directly as an antimicrobial and indirectly by activating a systemic JAK/STAT response (Srinivasan, 2016).

The role of the systemic JAK/STAT response is unclear at present. Despite being commonly used as a marker of STAT activation, the function of Tot and Tep proteins in Drosophila is unknown. Nevertheless, genetic loss-of-function studies have implicated JAK/STAT signaling in resistance and/or tolerance to viral, bacterial and parasitoid infections. Furthermore, the JAK/STAT pathway has a well-established role in maintenance of fly intestinal homeostasis, both at steady state and following infection or injury. Given these precedents, attempts were made to investigate the role of the inducible actin-triggered JAK/STAT circuit by injecting actin into flies prior to challenge with viruses (Flock house virus, Drosophila C virus, Sindbis virus and Cricket paralysis virus) or bacteria (Erwinia carotovora, Escherichia coli, Micrococcus luteus and Listeria monocytogenes) but failed to find an effect on either resistance or tolerance to infection. Similarly, in models of stress or injury (starvation, heat shock, irradiation, paraquat feeding and a recently-described model of concussion), no evidence was found of protection or susceptibility afforded by actin pre-injection. Finally, no effect was found of actin injection on fat body metabolism. The failure to find a system in which prior upregulation of STAT target genes by exogenous actin leads to a difference in outcome is a current experimental limitation. However, it might reflect the fact that STAT activation is already induced to sufficient levels in those models in response to actin released from dying cells. Consistent with this notion, septic injury was observed to lead to a rapid increase in actin levels within the haemolymph. In such a situation, additional induction of the STAT pathway by actin pre-injection may not confer additional protection or tolerance. Reinforcing this notion is a recent study showing that loss of basal Diedel levels leads to reduced tolerance to Sindbis virus, yet the upregulation of Diedel levels that takes place during infection is itself dispensable. Unfortunately, loss-of-function experiments to assess the effect of released actin on different challenges are not feasible because actin is essential for viability. Surrogate loss-of-function experiments, such as examining the role of Nox and Src42A or Shark in the fat body in the context of infection or injury, have not been reported and their interpretation is complicated by the pleiotropic effects of those proteins. Nevertheless, the finding that actin is released into the haemolymph upon septic injury and that this induces JAK/STAT activation dependent on fat body expression of Src42A and Nox may suggest that previous reports of septic injury-induced STAT activation can be partially ascribed to extracellular actin (Srinivasan, 2016).

The identity of the putative receptor that recognises extracellular actin in Drosophila remains unknown. The requirement for Upd3 rules out the possibility that actin serves as a direct ligand for Domeless, a conclusion further supported by the fact that actin does not induce TotM upregulation in various Drosophila cell lines that respond to Upd cytokines in vitro. Therefore, the simplest interpretation of the data is that Upd3 is synthesised by fat body cells that detect extracellular actin via a sensor(s) that couple(s) to a Nox-Src42A-Shark cascade. By analogy with other receptors that engage a Syk-dependent pathway, that sensor might be an ITAM- or hemITAM-bearing receptor or one that associates in trans with an ITAM-containing signalling chain. Interestingly, in Drosophila responses to wounding and in the clearance of axonal debris and neuronal cell corpses, one such receptor is Draper, a member of the Nimrod family and orthologue of C. elegans Ced1. Draper contains an ITAM that is phosphorylated by Src42A. However, Draper was found to be dispensable for TotM induction in response to actin injection. Similarly, no role was found for Nimrod C1, C4 and the scavenger receptor CD36. Whether these data indicate the activity of an unknown receptor, multiple redundant receptors or an indirect sensing mechanism, akin to the activation of the vertebrate NLRP3 receptor, will need to be investigated (Srinivasan, 2016).

In sum, these data suggest that extracellular actin released by dead cells induces a response in Drosophila that requires signalling in the fat body via the non-receptor tyrosine kinase, Shark, and the Src family kinase, Src42A. This pathway leads to production of Upd cytokines that act in an autocrine and paracrine manner to induce Domeless signalling via STAT and cause induction of STAT-responsive genes. Thus, the presence of actin in the extracellular space triggers a response previously associated with wounding and dead cell clearance, indicating that actin exposure acts as an ancient sign of tissue damage and that actin constitutes an evolutionarily-conserved DAMP. The notion that actin exposure can act as a universal sign of cell damage might apply more generally to other cytoskeletal proteins (Srinivasan, 2016).

Germline soma communication mediated by gap junction proteins regulates epithelial morphogenesis

Gap junction (GJ) proteins, the primary constituents of GJ channels, are conserved determinants of patterning. Canonically, a GJ channel, made up of two hemi-channels contributed by the neighboring cells, facilitates transport of metabolites/ions. This study demonstrates the involvement of GJ proteins during cuboidal to squamous epithelial transition displayed by the anterior follicle cells (AFCs) from Drosophila ovaries. Somatically derived AFCs stretch and flatten when the adjacent germline cells start increasing in size. GJ proteins, Innexin2 (Inx2) and Innexin4 (Inx4), functioning in the AFCs and germline respectively, promote the shape transformation by modulating calcium levels in the AFCs. These observations suggest that alterations in calcium flux potentiate STAT activity to influence actomyosin-based cytoskeleton, possibly resulting in disassembly of adherens junctions. These data have uncovered sequential molecular events underlying the cuboidal to squamous shape transition and offer unique insight into how GJ proteins expressed in the neighboring cells contribute to morphogenetic processes (Sahu, 2021).

Morphogenesis involves complex cellular interactions, regulated via short- or long-range influences, which operate both at the local as well as global level. Interactions between different cell types have acquired extra-ordinary significance during proper execution of complex morphogenetic processes including organogenesis. Interacting cellular partners of heterogenous origins participate in generation of a number of coordinated cues. Such cues either biochemical or mechanical in nature, operate both in localized and widespread manner to ultimately orchestrate the formation of simple as well as complex multicellular assemblies. Variety of signals that control the process, and underlying mechanisms that synthesize as well as propagate such signals are being investigated in different developmental contexts (Sahu, 2021).

This study demonstrates that Inx2, a gap junction protein, controls cuboidal to squamous cell shape transition in the follicular epithelium. Effect of Inx2 is mediated by its ability to regulate STAT activity, which in turn can influence the cytoskeleton. Supporting the conclusion, changes in STAT or alterations in the cytoskeletal machinery components, can either enhance or mitigate the phenotypic consequences induced by the loss of Inx2. Importantly, Inx2 engineers this shape transition likely by acting as a component of a calcium channel. It seems to achieve this by partnering with Zpg, another gap junction protein, belonging to the same family (Sahu, 2021).

Gap junction proteins have been shown to be required in a number of different biological processes including establishment of epithelial integrity and morphogenesis. For example, Inx2 has been shown to control formation of proventriculus, during foregut formation in the flies. Previous reports have also suggested that Inx2 can form homomeric as well as heteromeric gap junction channels. However, the molecular mechanism of Inx2 mediated epithelial morphogenesis has remained elusive (Sahu, 2021).

This study advances mechanistic understanding of how the gap junction channels contribute to morphological changes in the epithelial cells. Though calcium has been implicated in regulation of cell shape transition in various developmental contexts, the precise molecular mechanism underlying calcium dependent cell shape transition has not been documented. These findings demonstrate that calcium dependent JAK-STAT signaling regulates Myosin distribution to engineer cell shape transition. Interestingly previous work has shown that the same molecular cassette is used for proper specification of BCs. Together these data suggest that Inx2-zpg mediates the calcium flux in the follicle cells, which probably regulates JAK-STAT signaling in a context specific manner to achieve diverse outcomes (Sahu, 2021).

A heterotypic combination of Inx2-Zpg gap junction channel has been reported to regulate germline-soma interaction during early Drosophila spermatogenesis. In addition, heterotypic combination between Inx2 with other somatic gap junction protein, Inx3 has been reported in developing Drosophila embryo during the regulation of epithelial morphogenesis. This study has assayed the ability of a putative Inx2-Zpg heterotypic GJ channel responsible for cell shape transition underlying egg chamber morphogenesis. The results suggest that via Zpg, nurse cells induce the follicle cells to elevate free calcium levels as germline specific depletion of Zpg affects the absolute intensity and duration of calcium flux in the follicle cells. It is thus believed that optimum amount of calcium is critical for mediating proper shape transition of AFCs from cuboidal to squamous fate. Although, the nurse cells exhibit high level of GCaMP6 fluorescence, signal transmission to the adjacent follicle cells is relatively infrequent. This observation suggests that the transmission of the signal via Inx2-Zpg is spatially and temporally calibrated during oogenesis. Nurse cells are relatively of a larger size and, as such a direct physical contact may be established between a single nurse cell with a number of follicle cells via formation of a heterotypic GJ channel(s). Contact dependent assembly of Inx2-Zpg channel likely confers selectivity on the release and strength of the calcium flux. Taken together these data are consistent with the calcium flux being a crucial player during this process while the precise identity of the signal, remains to be determined. Whether calcium itself is the elusive 'signal' needs to be investigated further. It is possible that the 'signal' is more complex and consists of several components with calcium being one of the pieces of the puzzle. In such a scenario, future experiments ought to focus on uncovering the identity of the other components of the signal. It will also be important to determine as to how the non-autonomous 'signal' secreted by the nurse cells, is conveyed to the somatic neighbors to modulate the calcium flux. Follicle cells express many gap junction proteins including Inx2 on their cell surface and are competent to communicate with one another. Thus, possible contribution of neighboring follicle cells, not in direct contact with the nurse cells, may also be an important determinant (Sahu, 2021).

Recently product of tao gene has been shown to regulate follicle cell flattening by modulating the removal of lateral membrane component Fas2 by endocytosis. On similar lines, the current data shows that Inx2 permits the disassembly, and perhaps also the disappearance of adherens junction component DE-cadherin from cuboidal cells to facilitate the follicle cell stretching. Since calcium is known to regulate the rate of endocytosis, it is proposed that calcium may regulate the levels of DE-Cadherin via endocytosis to mediate the shape change in the AFCs. Partial rescue of inx2 phenotype in the heterozygous shg mutant background suggests that the accumulation of DE-Cadherin is indeed functionally connected to the phenotype. Further experiments will help reveal mechanistic connection between calcium flux and adherens junction assembly and its influence on cell shape transitions (Sahu, 2021).

Epidermal stratification during development of mammalian skin depends on cell division, collective migration and controlled differentiation. Together these three processes are responsible for the basal to squamous fate transformation. It is reported that this fate change is also accompanied by increase in the levels of GJ proteins and inter- as well as intracellular calcium. The results have implicated combined involvement of GJ proteins and calcium flux in mediating cell shape changes. It will be worthwhile to examine if similar mechanisms are employed by the differentiating basal cells. Intriguingly, several skin disorders have been linked to mutations in connexins (vertebrate counterparts of GJ proteins). These results thus provide a readily accessible genetic platform to compare and contrast the molecular mechanisms underlying epithelial cell shape transitions in diverse cellular contexts (Sahu, 2021).

GENE STRUCTURE

The mrl transcript is 4.0 kb, composed of seven exons. The first exon is 200 bp consisting of 5' untranslated DNA and separated from the remaining exons by approximately 7 kb (Hou, 1996).

PROTEIN STRUCTURE

Amino Acids - 761

Structural Domains

MRL contains a src homology2-like domain and a DNA binding motif (Hou, 1996 and Yan, 1996a).

date revised: 19 June 2024

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