STAT/marelle


DEVELOPMENTAL BIOLOGY

Embryonic

Unfertilized eggs have a maternally deposited Marelle mRNA. At the blastoderm stage, zygotic expression starts with a broad central domain showing 7 stripes in a pair rule pattern. Clusters of cells in both anterior and posterior terminal regions express mrl at approximately 3.5 hours. At germ band extension, a segmental pattern of 14 stripes appears restricted to mesodermal tissue, with an anterior boundary corresponding to the anterior engrailed stripe. After germ band retraction, mrl is expressed mainly in hindgut and foregut and gonadal precursors (Yan, 1996a).

The JAK/STAT pathway mediates cytokine signaling in mammals and is involved in the function and development of the hematopoietic and immune systems. To investigate the biological functions of the JAK/STAT pathway during Drosophila development, the tissue-specific localization of the tyrosine-phosphorylated, or activated form of Drosophila STAT, STAT92E, was examined. During Drosophila embryonic development STAT92E activation is prominently detected in multiple tissues and in different developmental stages. These tissues include the tracheal pits, elongating intestinal tracks, and growing axons. stat92E mutants are defective in tracheal formation, hindgut elongation, and nervous system development. Conversely, STAT92E overactivation caused premature development of the tracheal and nervous systems, and over-elongation of the hindgut. These results suggest that STAT activation is involved in proper differentiation and morphogenesis of multiple tissues during Drosophila embryogenesis (Li, 2003b).

At stage 14, STAT92E activation is detectable in the embryonic nervous system, with prominent antibody labeling of the axon fibers of the ventral nerve cord. To test the possibility that STAT92E activation might be involved in the development and/or function of the nervous system, axon development was examined in both the central and peripheral nervous system (CNS and PNS) of stat92E mutant embryos. The monoclonal antibodies BP102 and 22C10 were used; BP102 specifically labels both longitudinal connectives and commissures of the CNS and 22C10 recognizes the microtubule associated Futsch protein in cell body and axons of all PNS as well as a subset of CNS neurons. In stat92Emat-zyg- embryos, the organization of the CNS was grossly disrupted and some segments of the CNS were completely missing, Similar defects, though to a lesser extent, were also found in stat92Emat-zyg+ embryos, in which the segmentation defects were less severe. The CNS defects, namely, gaps in the longitudinal tracks and missing commissures, could be explained by an early requirement for STAT92E in segmentation and/or neuronal cell fate determination and are consistent with the finding that hop mutant embryos exhibit gaps in the ventral nerve cord. However, because STAT92E activation was seen in axons, long after cell fate determination, it is speculated that lack of STAT92E activation might additionally cause defects in the growth and organization of axonal projections (Li, 2003b).

To determine whether STAT92E activation plays a role in axonal growth, a few identifiable mAb22C10-positive CNS neurons were examined in stat92Emat-zyg+ embryos, in which the segmentation defects were less severe, presumably due to paternal rescue. The CNS neurons prominently stained by mAb22C10 include the anterior and posterior corner cells (aCC and pCC) that project axons laterally and the ventral unpaired median neurons (VUM), which send axons that bifurcate at the anterior commissure. In stage 15 stat92Emat-zyg+ and a small number of stat92Emat+zyg- embryos, these neurons were present, but often failed to grow axons. These neurons start to grow axons in stage 13 wild-type embryos. In contrast, in stage 13 stat92Emat-zyg+ embryos, it was found that the aCC, pCC, and VUM neurons were born but failed or were delayed in sending axons. Therefore, it is concluded that a failure of axonal growth contributes to the CNS defects exhibited by stat92E mutants (Li, 2003b).

The role of STAT92E in PNS development was studied; consistent with the defects exhibited by CNS neurons, many PNS neurons fail to extend their axons in stat92Emat-zyg+ embryos. The stat92Emat-zyg- embryos exhibit much more severe phenotypes and many neurons are absent, presumably resulting from a multitude of developmental defects. Since some PNS neurons in the same embryos were able to extend their axons, it is unlikely that the failure of these neurons to extend axons was due to premature cessation of development of stat92Emat-zyg+ embryos. Furthermore, in a small number of the stage 15 stat92Emat+zyg- embryos, which do not exhibit any segmentation defects, the neurons in one or two dorsal clusters did not grow axons. Conversely, when STAT92E is overactivated, such as in hopGOF embryos, PNS neurons prematurely differentiate and grow axons. In wild-type embryos, PNS neurons start to appear during stage 13 and do not fully differentiate until stage 15. In hopGOF mutants, in contrast, many of them (17/68) developed their PNS earlier. A few exhibited nearly fully differentiated PNS as early as stage 12. No 22C10-positive PNS neurons were detectable in wild-type embryos at this stage. These results suggest that STAT92E activation is necessary for axonal extension and, when overactivated, it can also promote axonal growth (Li, 2003b).

A common denominator for the diverse tissues and processes in which STAT92E activation was detected appears to be cell movement. In tracheal development, the tracheal pits form by the invagination of a group of predetermined epidermal cells and the elongation and migration of these cells forms a network of tracheal branches in the absence of further cell division. During hindgut elongation, cells rearrange without mitosis to form a thin, long tubule. Axon growth during the development of the nervous system represents a type of cell movement that involves a dramatic increase of cell membrane-based and actin-rich projections. Interestingly, the mysterious extra-embryonic migratory cells can extend long cellular projections, resembling neurons or fibroblasts in morphology. Finally, STAT92E activation was detected in cells that are involved in shape changes during gastrulation, including those in the dorsal folds, cephalic furrows, invaginating posterior midgut rudiment and hindgut primordium. In addition to cell shape changes, guided and/or invasive cell migrations, as represented by the behaviors of the ovarian border cells and primordial germ cells, are also key features of morphogenetic movements essential for animal development. The invasive migration of both types of cells has been shown to require STAT92E activation. Taken together, these observations seem to suggest that STAT activation may be fundamental to cell movements and shape changes. However, it is also noted that not all tissues that undergo morphogenesis exhibit prominent pSTAT92E staining, and therefore the requirement for STAT92E activation in these tissues was not investigated. The tissues or biological processes that were not affected by the stat92E mutation or not investigated in this study include but were not limited to dorsal closure, mesoderm formation and migration. Based on tissue and developmental stage-specific detection of STAT92E activation and phenotypic analyses, it is concluded that STAT92E activation is involved in at least a subset of morphogenetic movements during Drosophila embryogenesis. It would be interesting to investigate to what extent STAT activation is involved in morphogenesis in general and whether and how STAT activation collaborates with other signaling pathways to regulate morphogenetic movements (Li, 2003b).

Although STAT92E is prominently activated in a number of tissues undergoing morphogenetic changes and a mutation of stat92E exhibited developmental defects in these tissues, a role of STAT92E activation in the initial specification of the cell types that constitute these tissues cannot be ruled out. This may be particularly true for the trachea and nervous system. Ideally, to distinguish the role of STAT92E activation in cell fate specification versus differentiation, mosaic analysis of marked mutant cells lacking STAT92E would be preferable. The 'mosaic analysis with a repressible cell marker' (MARCM) technique was used to study the function of STAT92E in the embryonic PNS development by generating positively marked stat92E zygotic null neurons. Unfortunately, this effort is hindered by the abundant maternal contribution of the stat92E gene product and no defects could be found in dendrites or axons of single stat92Ezyg- mutant PNS neurons in mosaic animals. Based on the finding that activated STAT92E is detected in postmitotic neurons, and that nearly all stat92Emat-zyg+ mutant embryos and a small number of neurons in stat92Emat+zyg- mutant embryos fail to extend their axons properly, it is proposed that the axonal phenotype is at least in part due to loss of STAT92E function in postmitotic neurons (Li, 2003b).

It has recently been shown that oriented cell rearrangement and hindgut elongation require localized JAK/STAT signaling. Consistent with the results from analysis of stat92E mutants, the current study demonstrated that mutants of a number of Hop/STAT92E pathway components exhibit shorter and wider hindgut, possibly as a result of defective cell rearrangement. However, in the previous study, activating the Hop/STAT92E pathway in the hindgut has effects that are identical to a lack of STAT92E activation. This is in contrast to results of gain-of-function experiments. hopGOF embryos have phenotypes that are the opposite of loss-of-function mutants, namely longer hindgut and other internal tubule structures. The latter would be expected if overactivating the STAT92E pathway promotes hindgut elongation. The previous result was interpreted to suggest that spatially restricted activation of the Hop/STAT92E pathway is necessary for hindgut elongation, whereas high-level uniform activation of this pathway is not compatible with oriented cell rearrangement. A plausible explanation to account for the different outcomes of the two sets of gain-of-function experiments is that the expression levels of these gain-of-function molecules are critical for the oriented cell rearrangement. Modestly higher levels of Hop/STAT92E signaling, while preserving the endogenous signaling gradient, may promote hindgut elongation, whereas too high levels of signaling could abolish the gradient along the gut and prevent oriented cell rearrangement (Li, 2003b and references therein).

Embryonic development involves a series of programmed morphogenetic movements that include bending, folding, and invaginating of epithelial tissues in order to form various organs as well as the final shape of the body. Cell migration, shape changes, and rearrangements underlie most of the morphogenetic movements essential for gastrulation and organogenesis during Drosophila embryonic development. Although it has been shown that actin-based cytoskeletal reorganization plays a crucial role in cell shape changes, little is known about the signaling pathways that trigger these morphogenetic movements during embryogenesis. The identification of STAT as a potential regulator of morphogenetic movements posed an interesting question regarding the involvement of STAT activation in the transcriptional regulation of genes required for these movements. Indeed, STAT92E has been shown to be involved in the transcriptional activation of many signaling molecules as well as key transcription factors. A recent systematic search for STAT target genes has revealed a plethora of genes that might be directly activated by STAT92E, among which are those involved in the regulation of cytoskeletal movements and actin reorganization. Elucidation of the target genes of STAT and the extracellular signals that lead to STAT activation should shed light on the molecular mechanisms that govern morphogenetic movements. It remains to be elucidated whether STAT92E directly activates these genes or whether it acts in collaboration with yet unidentified signaling pathways (Li, 2003b).

Finally, the JAK/STAT signaling pathway has also been extensively studied in model organisms other than Drosophila, and a general role of this pathway in morphogenesis and cell movement is beginning to emerge. For instance, in Dictyostelium discoideum, Dd-STATa is required for cell movement in the prestalk region in response to cAMP signals through a unique mechanism. In Zebrafish, inhibition of JAK/STAT signaling slows cell intercalation movement during gastrulation. In the mouse, STAT3 deficiency results in early embryogenesis and gastrulation defects and compromises cell migration in keratinocytes. Therefore, it appears that the function of STAT activation in morphogenetic movements is not limited to Drosophila, but likely applies to animal development in general (Li, 2003b and references therein).

STAT is an essential activator of the zygotic genome in the early Drosophila embryo

In many organisms, transcription of the zygotic genome begins during the maternal-to-zygotic transition (MZT), which is characterized by a dramatic increase in global transcriptional activities and coincides with embryonic stem cell differentiation. In Drosophila, it has been shown that maternal morphogen gradients and ubiquitously distributed general transcription factors may cooperate to upregulate zygotic genes that are essential for pattern formation in the early embryo. This study shows that Drosophila STAT (STAT92E) functions as a general transcription factor that, together with the transcription factor Zelda, induces transcription of a large number of early-transcribed zygotic genes during the MZT. STAT92E is present in the early embryo as a maternal product and is active around the MZT. DNA-binding motifs for STAT and Zelda are highly enriched in promoters of early zygotic genes but not in housekeeping genes. Loss of Stat92E in the early embryo, similarly to loss of zelda, preferentially down-regulates early zygotic genes important for pattern formation. STAT92E and Zelda synergistically regulate transcription. It is concluded that STAT92E, in conjunction with Zelda, plays an important role in transcription of the zygotic genome at the onset of embryonic development (Tsurumi, 2011).

This study describes a bioinformatics approach to investigating the mechanisms controlling transcription of the zygotic genome that occurs during the MZT; STAT92E was identified as an important general transcription factor essential for up-regulation of a large number of early 'zygotic genes'. The role of STAT92E was described in controlling transcription of a few representative early zygotic genes, such as dpp, Kr, and tll, that are important for pattern formation and/or cell fate specification in the early embryo. These studies suggest that STAT92E cooperates with Zelda to control transcription of many 'zygotic genes' expressed during the MZT. While STAT mainly regulates transcription levels, but not spatial patterns, of dpp, tll, and Kr, and possibly also other 'zygotic genes', Zelda is essential for both levels and expression patterns of these genes (Tsurumi, 2011).

The transcriptional network that controls the onset of zygotic gene expression during the MZT has remained incompletely understood. It has been proposed that transcription of the zygotic genome depends on the combined input from maternally derived morphogens and general transcription factors. The former are distributed in broad gradients in the early embryo and directly control positional information (e.g., Bicoid, Caudal, and Dorsal), whereas the latter are presumably uniformly distributed regulators that augment the upregulation of a large number of zygotic genes. Other than Zelda, which plays a key role as a general regulator of early zygotic expression, the identities of these general transcriptional activators have remained largely elusive. It has been shown that combining Dorsal with Zelda- or STAT-binding sites supports transcription in a broad domain in the embryo. The demonstration of STAT92E as another general transcription factor sheds light on the components and mechanisms of the controlling network in the early embryo. Moreover, STAT92E and Zelda may cooperate to synergistically regulate zygotic genes. The results thus validate the bioinformatics approach as useful in identifying ubiquitously expressed transcription factors that may play redundant roles with other factors and thus might otherwise be difficult to identify (Tsurumi, 2011).

The conclusion that STAT92E is important for the levels but not the spatial domains of target gene expression in the early embryo is consistent with several previous reports. It has been shown that in Stat92E or hop mutant embryos, expression of eve stripes 3 and 5 are significantly reduced but not completely abolished. In addition, JAK/STAT activation is required for the maintenance of high levels, but not initiation, of Sxl expression during the MZT. Moreover, it has previously been shown that STAT92E is particularly important for TorsoGOF-induced ectopic tll expression but not essential for the spatial domains of tll expression in wild-type embryos under normal conditions. On the other hand, Zelda may be important for both levels and spatial patterns of gene expression. This idea is consistent with the finding that Zelda-binding sites are enriched in both promoter and promoter-distal enhancers regions, whereas STAT-binding sites are enriched in promoter regions only. It has been reported that pausing of RNA polymerase II is prominently detected at promoters of highly regulated genes, but not in those of housekeeping genes. In light of these results that STAT and Zelda sites are highly enriched in the early zygotic gene promoters, it is suggested that these transcription factors might contribute to chromatin remodeling that favors RNA polymerase II pausing at these promoters (Tsurumi, 2011).

Finally, the MZT marks the transition from a totipotent state to that of differentiation of the early embryo. As a general transcription factor at this transition, STAT, together with additional factors (such as Zelda), is important for embryonic stem cell differentiation. Further investigation is required to understand the molecular mechanism by which STAT and Zelda cooperate in controlling zygotic transcription in the early Drosophila embryo. Moreover, it would be interesting to investigate whether STAT plays similar roles in embryonic stem cell differentiation in other animals (Tsurumi, 2011).

Somatic control of germline sexual development is mediated by the JAK/STAT pathway

Germ cells must develop along distinct male or female paths to produce the sperm or eggs required for sexual reproduction. In both mouse and Drosophila, sexual identity of germ cells is influenced by the sex of the surrounding somatic tissue, but little is known about how the soma controls germline sex determination. This study shows that the JAK/STAT pathway provides a sex-specific signal from the soma to the germline in the Drosophila embryonic gonad. The somatic gonad expresses a JAK/STAT ligand, unpaired (upd), in a male-specific manner, and activates the JAK/STAT pathway in male germ cells at the time of gonad formation. Furthermore, the JAK/STAT pathway is necessary for male-specific germ cell behavior during early gonad development, and is sufficient to activate aspects of male germ cell behavior in female germ cells. This work provides direct evidence that the JAK/STAT pathway mediates a key signal from the somatic gonad that regulates male germline sexual development (Wawersik, 2005).

While investigating communication between the somatic gonad and germline, the JAK/STAT pathway was found to be specifically activated in male, but not female, germ cells. In Drosophila, JAK/STAT signaling is initiated when an UPD or UPD-like ligand binds a transmembrane receptor (Domeless), activating the JAK Hopscotch (HOP), which phosphorylates the STAT92E transcription factor. STAT activation has been shown to regulate stat gene expression and can induce upregulation of the STAT92E protein, which can be used as an assay for JAK/STAT pathway activation. STAT92E is upregulated specifically in male, but not female germ cells at the time of gonad formation. This reflects male-specific activation of the JAK/STAT pathway since (1) the activated form of STAT92E (phospho-STAT92E) is also detected in only male germ cells, and (2) JAK activity is necessary and sufficient for STAT92E expression. Expression of a JAK inhibitor, Socs36E, results in loss of STAT92E expression in male germ cells and expression of constitutively active JAK (hopTumL) induces STAT92E in female germ cells. The male-specific activation of STAT92E at this time is distinct from STAT92E activation in germ cells in the early embryo, which is not sex-specific and is regulated by the MAP kinase pathway (Wawersik, 2005).

It was also found that STAT92E expression in male germ cells is dependent on their association with the somatic gonad. STAT92E is not detected in germ cells that are migrating to the gonad, but is detected in male germ cells after they contact the somatic gonad. STAT92E expression is greatly reduced or absent in eya mutants, where somatic gonad identity is initiated, but not maintained. Furthermore, STAT92E is not detected in germ cells found outside the gonad in wild type embryos or in mis-localized germ cells in wunen and HMG-CoA reductase mutants which lack guidance cues that target germ cells to the somatic gonad. However, in these same mutants, STAT92E is detected in the few germ cells that contact the somatic gonad in male embryos (Wawersik, 2005).

STAT92E expression in the germline is dependent on the sex of the surrounding soma. When XX (normally female) germ cells were present in a soma that was masculinized by expression of the male form of the somatic sex determination gene doublesex (dsx), germ cells now expressed STAT92E. dsx does not play an autonomous role in germ cells themselves, indicating that STAT92E induction in these embryos is caused by masculinization of the soma. Conversely, when the somatic gonad of an XY (normally male) embryo is feminized by expression of the sex determination gene transformer (tra) in the mesoderm, but not germ cells, STAT92E expression is no longer observed in XY germ cells. Taken together, these data indicate that the male somatic gonad is necessary and sufficient to activate the JAK/STAT pathway in either XX or XY germ cells (Wawersik, 2005).

Consistent with this, it was found that the JAK/STAT ligand, upd, is expressed specifically in the male, but not female, somatic gonad. Expression of STAT92E in male germ cells was no longer detected in embryos in which upd and two homologs, upd2 and upd3, are deleted [Df(os1a]. Since male germ cells from embryos mutant for upd alone still express STAT92E, JAK/STAT activation in the germline may be regulated redundantly by upd and one or more of its homologs. In addition, expression of upd in either the mesoderm or germ cells is sufficient to induce STAT92E expression in XX germ cells. Expression of upd2 or upd3 is also capable of inducing STAT92E in germ cells (Wawersik, 2005).

upd is also important for embryonic patterning and somatic sex determination. Interestingly, upd promotes female identity in the soma, but promotes male development in the germline. To verify that the effects of upd on the germline are not indirectly caused by other effects of upd, indicators of embryonic segmentation (Engrailed), somatic sex determination (Sex lethal), somatic gonad identity (Eyes absent), and somatic gonad sexual identity (Sox100B) were examined. Df(os1a) hemizygous male embryos exhibit segmentation defects as expected, but form gonads that express normal somatic and sex-specific markers. Embryos ectopically expressing upd are normal in all respects examined (Wawersik, 2005).

Whether activation of the JAK/STAT pathway by the male somatic gonad regulates male-specific development of germ cells was examined. In adult testes, the JAK/STAT pathway is required for maintenance of germline stem cells, making it difficult to assess the role of this pathway on male germ cell identity at this stage. Instead, germ cells were examined during embryogenesis and early larval stages, when germ cell development first becomes sexually dimorphic. In the mouse, the earliest manifestation of sex determination in the germline is differential regulation of the germline cell cycle by the soma. In Drosophila, germ cells undergo 1-2 divisions after their formation, but are arrested in the cell cycle during germ cell migration and only resume division shortly after the gonad has formed. Since larval testes contain more germ cells than larval ovaries, whether proliferation is regulated differently in male and female germ cells was examined. Indeed, sex-specific analysis of a mitotic marker (phosphohistone-H3) in the germline indicates that germ cell proliferation is entirely male-specific during early stages of gonad development. Furthermore, male-specific germ cell division is dependent on the male somatic gonad. Male germ cells do not proliferate in eya mutants that lack the somatic gonad, or in lost germ cells within wunen mutant embryos. XX germ cells in a masculinized soma (dsxD/ dsx1) proliferate, while XY germ cells in a feminized soma (UAS-traF; twist-Gal4) do not. Thus, the pattern of germ cell proliferation correlates exactly with activity of the JAK/STAT pathway in germ cells (Wawersik, 2005).

To assess whether JAK/STAT signaling regulates male-specific germ cell division, embryos lacking zygotic Stat92E activity were examined and a dramatic decrease was observed in male germ cell proliferation. Similar reductions in germ cell proliferation are observed in the upd/upd-like mutant (Df(os1a)) and in embryos where the JAK inhibitor Socs36E is expressed in germ cells. Thus, JAK/STAT activity is required within germ cells for proper male-specific germ cell division in the gonad. Expression of upd in the germline is sufficient to induce proliferation in female germ cells. Thus, the JAK/STAT pathway can induce XX germ cells to exhibit this male-specific germ cell behavior (Wawersik, 2005).

Whether the JAK/STAT pathway regulates other aspects of male germ cell development was examined. male germline marker-1 (mgm-1) is a lacZ enhancer trap line that is expressed in male germ cells, but not female germ cells, and therefore is a marker for male germ cell identity. Inhibiting the JAK/STAT pathway by removing zygotic Stat92E activity does not affect mgm-1 expression in the embryo, which is as expected since initial mgm-1 expression is dependent on germ cell autonomous cues. However, removal of zygotic Stat92E activity completely abolished mgm-1 expression in first instar larvae. In wild-type first instar male larvae, mgm-1 expression is observed in most germ cells, which are likely to be developing male germline stem cells and spermatogonia. No mgm-1 expression is observed in Stat92E-mutant larvae, and β-galactosidase expression is only observed in the soma, not the germline, in the pattern expected from the Stat92E P element allele. In an experiment where 25% of larvae were expected to be both male and contain the mgm-1 enhancer trap, 23.2% (n=262) of wild type larvae exhibited mgm-1 expression in the germ cells, while no Stat92E mutant larvae exhibited germ cell mgm-1 expression; this is significantly different from wild type siblings. Thus, Stat92E mutants exhibit a strong effect on male germline development, and some male germline cell types are either missing, or have an altered identity (Wawersik, 2005).

Finally, the extent to which activation of the JAK/STAT pathway can masculinize female germ cells was assessed. Female germ cells expressing upd are not expected to be fully masculinized because, although a male-specific signal is being activated, these germ cells are otherwise still in a female somatic environment and retain female germ cell autonomous cues. Indeed, such embryos give rise to fertile adult females, indicating that at least some germ cells retain, or revert back to, a female identity. This may be due, in part, to the failure of the upd construct to be expressed in the adult female germline. However, upd is sufficient to induce male-specific gene expression in embryonic XX germ cells. While mgm-1 is normally expressed only in germ cells in males, mgm-1 was expressed in all embryos when upd was ectopically expressed. In addition, two new male germline markers, disc proliferation abnormal (dpa) and minichromosome maintenance 5 (mcm5), were identified, that can also be induced by upd. Whereas these genes are normally expressed in germ cells only in males, female embryos exhibit germ cell expression of these genes when upd is ectopically expressed. In an experiment where only 50% of embryos are expected to express ectopic upd in the germline, 32.5% of female embryos expressed dpa and 21.3% expressed mcm5. Therefore, upd expression is sufficient to activate male-specific gene expression in female germ cells (Wawersik, 2005).

These data indicate that the JAK/STAT pathway mediates a critical signal from the male somatic gonad that is required for male germ cell development. This signal likely acts together with male germ cell autonomous cues to promote male germline identity and spermatogenesis. This signal is also sufficient to activate the male pattern of proliferation and gene expression in female germ cells, even when these germ cells retain female germ cell autonomous cues and are present in an otherwise female soma. It will be very interesting in the future to identify additional (e.g. female) somatic signals, along with germ cell autonomous cues, and to assess the relative contribution of these factors to proper germline sexual development. Since one of the earliest aspects of sex-specific germ cell behavior in both Drosophila and mouse is the regulation of the germline cell cycle by the somatic gonad, it will be of further interest to determine if the somatic signals operating in Drosophila play a similar role in germline sex determination in mammals (Wawersik, 2005).

Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche

In the Drosophila testis, germline stem cells (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells, termed the hub, which produce a variety of growth factors contributing to the niche microenvironment that regulates both stem cell pools. This study shows that CySC but not GSC maintenance requires Hedgehog (Hh) signalling in addition to Jak/Stat pathway activation. CySC clones unable to transduce the Hh signal are lost by differentiation, whereas pathway overactivation leads to an increase in proliferation. However, unlike cells ectopically overexpressing Jak/Stat targets, the additional cells generated by excessive Hh signalling remain confined to the testis tip and retain the ability to differentiate. Interestingly, Hh signalling also controls somatic cell populations in the fly ovary and the mammalian testis. These observations might therefore point towards a higher degree of organisational homology between the somatic components of gonads across the sexes and phyla than previously appreciated (Michel, 2012).

Hh thus provides a niche signal for the maintenance and proliferation of the somatic stem cells of the testis. CySCs that are unable to transduce the Hh signal are lost through differentiation, whereas pathway overactivation causes overproliferation. Hh signalling thereby resembles Jak/Stat signalling via Upd. Partial redundancy between these pathways might explain why neither depletion of Stat activity nor loss of Hh signalling causes complete CySC loss (Michel, 2012).

This study has shown that loss of Hh signalling in smo mutant cells blocks expression of the Jak/Stat target Zfh1, whereas mutation of ptc expands the Zfh1-positive pool. Overexpression of Zfh1 or another Jak/Stat target, Chinmo, is sufficient to induce CySC-like behaviour in somatic cells irrespective of their distance. By contrast, Hh overexpression in the hub using the hh::Gal4 driver only caused a moderate increase in the number of Zfh1-positive cells relative to a GFP control. Ectopic Hh overexpression in somatic cells under c587::Gal4 control increased this number further. However, unlike in somatic cells with constitutively active Jak/Stat signalling, the additional Zfh1-positive cells remained largely confined to the testis tip, although their average range was increased threefold. Thus, Hh appears to promote stem cell proliferation, in part, also independently of competition (Michel, 2012).

It is tempting to speculate that further stem cell expansion is limited by Upd range. Consistently, cells with an ectopically activated Jak/Stat pathway remain undifferentiated, whereas ptc cells can still differentiate. Future experiments will need to formally address the epistasis between these pathways. However, the observations already show that Hh signalling influences expression of the bona fide Upd target gene zfh1, and therefore presumably acts upstream, or in parallel to, Upd in maintaining CySC fate (Michel, 2012).

In addition, the reduction in GSC number following somatic stem cell loss implies cross-regulation between the different stem cell populations that presumably involves additional signalling cascades, such as the EGF pathway (Michel, 2012).

In recent years, research has focused on the differences between the male and female gonadal niches. This paper instead emphasizes the similarities: in both cases, Jak/Stat signalling is responsible for the maintenance and activity of cells that contribute to the GSC niche, and Hh signalling promotes the proliferation of stem cells that provide somatic cells ensheathing germline cysts. In the testis, both functions are fulfilled by the CySCs, whereas in the ovary the former task is fulfilled by the postmitotic escort stem cells/escort cells and the latter by the FSCs. Finally, male desert hedgehog (Dhh) knockout mice are sterile. Dhh is expressed in the Sertoli cells and is thought to primarily act on the somatic Leydig cells. However, the signalling microenvironment of the vertebrate spermatogonial niche is, as yet, not fully defined. Future experiments will need to clarify whether these similarities reflect convergence or an ancestral Hh function in the metazoan gonad (Michel, 2012).

Hedgehog is required for CySC self-renewal but does not contribute to the GSC niche in the Drosophila testis

The Drosophila testis harbors two types of stem cells: germ line stem cells (GSCs) and cyst stem cells (CySCs). Both stem cell types share a physical niche called the hub, located at the apical tip of the testis. The niche produces the JAK/STAT ligand Unpaired (Upd) and BMPs to maintain CySCs and GSCs, respectively. However, GSCs also require BMPs produced by CySCs, and as such CySCs are part of the niche for GSCs. This study describes a role for another secreted ligand, Hedgehog (Hh), produced by niche cells, in the self-renewal of CySCs. Hh signaling cell-autonomously regulates CySC number and maintenance. The Hh and JAK/STAT pathways act independently and non-redundantly in CySC self-renewal. Finally, Hh signaling does not contribute to the niche function of CySCs, as Hh-sustained CySCs are unable to maintain GSCs in the absence of Stat92E. Therefore, the extended niche function of CySCs is solely attributable to JAK/STAT pathway function (Amoyel, 2013).

This study has shown that Hh from the Drosophila testis niche is a self-renewal factor for CySCs and that Hh signaling does not contribute to the role of CySCs as a niche for GSCs. This supports the model that the Hh and JAK/STAT pathways act independently within CySCs. The results therefore confirm those recently reported by another group (Michel, 2012), who showed that Hh regulates CySC self-renewal, and extend their results by demonstrating the genetic independence of Hh and the other pathway (i.e. JAK/STAT) that is crucial in CySC function (Amoyel, 2013).

It is notable that two signals regulate CySC self-renewal but only JAK/STAT signaling contributes to the GSC niche. Moreover, despite the drastic reduction in CySCs in hhts2 testes (from ~36 in controls to ~8), GSCs do remain in hh mutant animals albeit at reduced numbers. The reduction in GSCs in hh mutants is not due to changes in the size of the hub. These data suggest that most CySCs are dispensable for their niche function and that only a few BMP-producing CySCs are needed to maintain GSC self-renewal. This raises the question as to whether, in a wild-type animal, there are distinct populations of CySCs, some with activated Stat92E that produce BMPs and act as a niche for GSCs, and others with activated Hh signaling that participate only in self-renewal and the production of cyst progeny. This is consistent with the fact that, despite the presence of ~36 Zfh1-positive CySCs, elevated Stat92E is only seen in a few CySCs. However, it is also conceivable that all Zfh1-positive CySCs are equivalent and that high Stat92E correlates, for instance, with a specific phase of the cell cycle, such as the repositioning of the spindle during anaphase that brings the nucleus of the CySC closer to the hub interface and might expose that CySC to more Upd ligand. This possibility implies a much more dynamic stem cell niche for the GSCs than has been previously appreciated (Amoyel, 2013).

The results indicate that the Hh and JAK/STAT pathways act mostly in parallel, although activating Hh may delay the differentiation of CySCs that are deficient for JAK/STAT pathway components. It is unclear why the CySC would require both signaling inputs to be maintained. However, it should be noted that these inputs contribute different information, as JAK/STAT signaling imparts niche potential, and Hh signaling additionally ensures that the right number of CySCs are present and provide cyst cells for normal spermatogonial development. Future work will establish whether self-renewal in CySCs depends on two sets of genes controlled separately by the Hh and JAK/STAT pathways or whether they converge on the same targets. The first possibility is supported by the fact that Hh does not contribute to the niche function of STAT in CySCs, indicating that different targets (presumably BMPs) are regulated differently (Amoyel, 2013).

One consequence of this work is to lead to a reevaluation of the differences between male and female gonad development in Drosophila. Indeed, Hh signaling is an essential regulator of the self-renewal and the number of follicle stem cells, the offspring of which carry out a comparable function to cyst cells by ensheathing germ line cysts. In the ovary, as in the testis, JAK/STAT signaling in somatic cells is required for the maintenance of GSCs via BMP production. However, in the ovary, the escort cells and cap cells are the JAK/STAT-responsive niche cells, implying that CySCs in the male gonad fulfill the function of two cell types in the female gonad and require both the signals used in the female to do so. Finally, the data evoke the interesting possibility that Hh has a conserved ancestral role in male gonads. Mutation in one of the three mammalian hh homologs, desert hedgehog (Dhh), causes male sterility and a loss of somatic support cells called Leydig cells. However, the cellular niche for spermatogenesis in mammals is less well understood than in Drosophila and it remains to be established whether the Hh pathway orchestrates similar cellular functions (Amoyel, 2013).

The Drosophila BCL6 homolog Ken and Barbie promotes somatic stem cell self-renewal in the testis niche

Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the JAK-STAT pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somatic cyst stem cells (CySCs). This study found that ken and barbie (ken) is autonomously required for the self-renewal of CySCs but not GSCs. Furthermore, Ken misexpression in the CySC lineage induces the cell-autonomous self-renewal of somatic cells as well as the nonautonomous self-renewal of germ cells outside the niche. Thus, Ken, like Stat92E and its targets ZFH1 and Chinmo, is necessary and sufficient for CySC renewal. However, ken is not a JAK-STAT target in the testis, but instead acts in parallel to Stat92E to ensure CySC self-renewal. Ken represses a subset of Stat92E targets in the embryo suggesting that Ken maintains CySCs by repressing differentiation factors. In support of this hypothesis, it was found that the global JAK-STAT inhibitor Protein tyrosine phosphatase 61F (Ptp61F) is a JAK-STAT target in the testis that is repressed by Ken. Together, this work demonstrates that Ken has an important role in the in the inhibition of CySC differentiation. Studies of ken may inform understanding of its vertebrate orthologue B-Cell Lymphoma 6 (BCL6) and how misregulation of this oncogene leads to human lymphomas (Issigonis, 2012).

This study demonstrates that ken, the orthologue of the human oncogene BCL6, plays a novel and crucial role in adult stem cell maintenance. Furthermore, the data show that ken is sufficient to promote the self-renewal of CySCs outside of their normal niche, which in turn drives the nonautonomous self-renewal of GSCs. This is consistent with previous studies, which have shown that hyperactivation of JAK-STAT signaling or misexpression of the Stat92E targets ZFH1 or Chinmo are sufficient to induce ectopic CySCs and GSCs. This work also reveals a previously unappreciated role for Stat92E in the Drosophila testis- transcriptional repression of target genes (Issigonis, 2012).

This study demonstrates the importance of ken in maintaining CySC fate. The only three genes other than Stat92E currently known to be necessary and sufficient for CySC self-renewal are ken, zfh1, and chinmo. Remarkably, all three genes are known to behave as transcriptional repressors. Furthermore, both ken and chinmo encode proteins that share the same overall domain structure: an N-terminal BTB domain and C-terminal DNA-binding zinc fingers. The Drosophila genome encodes 32 BTB-ZF proteins, so it would be interesting to see whether other BTB-ZF proteins are also sufficient to induce ectopic CySCs and GSCs when expressed in the CySC lineage. BTB-ZF proteins regulate many important biological processes such as cell survival and differentiation and generally behave as transcriptional repressors. Therefore, it is clear that transcriptional repression plays a critical role in regulating CySC fate (Issigonis, 2012).

It will be interesting to learn whether Ken, ZFH1, and Chinmo each control a distinct set of genes, or whether some of their targets are co-regulated. Both ZFH1 and BCL6, the mammalian homolog of Ken, are known to interact with the corepressor CtBP (C-terminal binding protein). Furthermore, heterodimerization between different BTB-ZF family members has been shown to occur. Since the transcriptional repressors Ken, ZFH1, and Chinmo have similar loss-of-function phenotypes (CySC loss) and gain-of-function phenotypes (ectopic CySCs), it seems likely that identifying their common targets will lead to identification of key effectors required to promote CySC self-renewal. An important parallel can be drawn to studies on embryonic stem (ES) cells which demonstrate that the main ES cell self-renewal factors OCT4, SOX2, and NANOG promote stem cell fate by transcriptionally repressing genes required for differentiation. Interestingly, OCT4, SOX2, and NANOG have been shown to co-occupy a number of target genes. Mapping Ken as well as ZFH1 and Chinmo to their binding sites within CySCs will reveal how these transcriptional regulators behave to promote self-renewal and block differentiation (Issigonis, 2012).

Previous studies have uncovered the dependence of the germ cells on CySCs for their self-renewal and on cyst cells for their proper differentiation. However, further investigation is required to elucidate the mechanisms by which ectopic CySCs are induced, and how this consequently leads to GSC self-renewal. It is unknown whether blocking differentiation in CySCs is sufficient to stall GSCs in an undifferentiated state or whether CySCs send a signal to neighboring germ cells causing them to self-renew. This work and previous studies have begun to uncover the regulatory network comprised of transcription factors and chromatin remodelers (Cherry, 2010) in CySCs. In order to understand how these transcriptional regulatory networks control the decision between stem cell fate versus differentiation in CySCs, and how CySC self-renewal promotes GSC identity, one must identify the downstream target genes of these critical transcriptional regulators (Issigonis, 2012).

Previous work from several labs has shown the importance of JAK-STAT activity for the maintenance of both CySCs and GSCs. In CySCs, JAK-STAT signaling promotes stem cell identity by activating the transcription of self-renewal factors, and in GSCs, pathway activation primarily regulates their adhesion to the hub. However, attenuation of JAK-STAT signaling is critical as well; expression of the Stat92E target Socs36E in CySCs is necessary to create a negative feedback loop that prevents CySCs from activating Stat92E at aberrantly high levels and consequently outcompeting neighboring GSCs (Issigonis, 2009). Therefore, differentially fine-tuning the overall global levels of JAK-STAT pathway activation in the two stem cell types is essential. But how do the stem cells precisely regulate which JAK-STAT targets are activated in the appropriate cell lineage? For example, even though the JAK-STAT pathway is activated in both CySCs and GSCs (albeit at different levels), the target genes zfh1 and Socs36E are expressed in the CySCs but not the GSCs. It is possible that distinct STAT targets respond to different thresholds of STAT activation. Furthermore, certain co-activators or co-repressors may be uniquely expressed or may function exclusively in one cell lineage and not the other. For example, ZFH1 is only expressed in CySCs and is required for their maintenance. On the other hand, Chinmo is expressed in both GSCs and CySCs, but functions solely in the latter stem cell population for their maintenance. Ken is enriched in the testis apex, and similar to the transcriptional repressorsZFH1 and Chinmo, is required in CySCs, but not GSCs. However, in the testis, ken is not a target of the JAK-STAT pathway, unlike zfh1 and chinmo. It is worth noting that although their loss-of-function phenotypes are similar, ken mutant CySC clones are lost more slowly than stat92E, zfh1, or chinmo mutant CySCs. One reason for this difference may be attributed to the fact that the available ken alleles are not null. However, it is also possible that genes such as zfh1 and chinmo may have stronger loss-of-function phenotypes because they play a primary role in CySC maintenance whereas Ken may perform secondary functions such as fine-tuning the transcriptional output of the JAK-STAT pathway. The Drosophila testis niche presents a unique opportunity to study how a single signaling pathway regulates two different stem cell populations within a niche via (1) differential regulation of global antagonists (i.e. Socs36E), (2) activation of a distinct set of target genes exclusively in one stem cell type (i.e. zfh1), and (3) differential regulation by transcriptional repressors (i.e. ken and chinmo) (Issigonis, 2012).

An interesting discovery from this study is that Stat92E represses the expression of Ptp61F. STATs were originally discovered as activators of gene transcription in response to interferons. Recently, however, increasing evidence indicates that in addition to their more familiar and well-documented role as transcriptional activators, STATs can also behave as functional repressors in an indirect manner (via STAT-induced activation of a repressor) or directly (through interactions with DNA methyltransferases, histone deacetylases, or heterochromatin proteins) (Issigonis, 2012).

In Drosophila, JAK-STAT pathway activation is known to upregulate the transcription of some targets, while repressing others. However, how a transcription factor such as Stat92E can stimulate the expression of individual genes while inhibiting others that have potentially conflicting roles is not well understood. The Drosophila testis provides a good model system to study this problem; Stat92E is required for the self-renewal of CySCs, presumably by positively regulating genes required for stem-cell identity while repressing those which would lead to opposite fates (i.e. differentiation). The current results indicate that Ptp61F is negatively regulated by JAK-STAT signaling in the testis since the activation of JAK-STAT leads to a dramatic decrease in Ptp61F expression. Since Ptp61F expression was quickly downregulated in hs-upd testes after a single heat-shock pulse, it is thought that Stat92E may be directly repressing Ptp61F transcription instead of activating the expression of a Ptp61F repressor. Support for this comes from work performed in an ex vivo system using Drosophila haemocyte-like cells to identify JAK-STAT targets. Upd or HopTumL stimulation of these haemocyte-like cells leads to a significant increase in the transcript levels of the 'immediate-early' JAK-STAT target Socs36E, which responds within two hours of pathway activation. These observations were recapitulated in vivo, since a robust increase in Socs36E expression levels was observed in response to a heat-shocking protocol in hs-upd testes. Similarly, the rapid response seen in Ptp61F expression levels upon JAK-STAT pathway activation may reflect a direct repression of this target as opposed to a secondary effect. Future studies will address the mechanism by which Stat92E represses the JAK-STAT inhibitor Ptp61F to promote CySC self-renewal (Issigonis, 2012).

While the mechanism by which Ken represses JAK-STAT targets is currently unknown, clues to how Ken may be behaving can be drawn from its orthologue BCL6, which interacts with chromatin modifiers such as SMRT, mSIN3A, N-CoR, BcoR, and histone deacetylases (HDACs). This suggests that Ken may be acting through these partners to block transcriptional activation through chromatin modification. Another possibility is that Ken directly blocks Stat92E from binding to and transcriptionally activating expression of target genes. Furthermore, since Stat92E can either activate or repress expression of targets, it is also possible that Ken behaves as a Stat92E co-repressor. Any of these non-exclusive possibilities will further understanding of how a signaling pathway is able to transcriptionally activate different target genes in different cell types and stages of development as opposed to eliciting the indiscriminate activation of all possible target genes at once (Issigonis, 2012).

Chromosomal rearrangements and point mutations that lead to the misregulation of BCL6 occur frequently in human lymphomas. Furthermore, constitutive overexpression of BCL6 in mice promotes the development of lymphomas. BCL6 has been shown to repress differentiation of B-cells and mammary cells. This study has found that Ken plays an analogous role in repressing differentiation of CySCs in the Drosophila testis. Future studies on Drosophila Ken and its targets will further understanding of the mammalian oncogene BCL6 (Issigonis, 2012).

The adult Drosophila gastric and stomach organs are maintained by a multipotent stem cell pool at the foregut/midgut junction in the cardia (proventriculus)

Stomach cancer is the second most frequent cause of cancer-related death worldwide. Thus, it is important to elucidate the properties of gastric stem cells, including their regulation and transformation. To date, such stem cells have not been identified in Drosophila. Using clonal analysis and molecular marker labeling, this study has identified a multipotent stem-cell pool at the foregut/midgut junction in the cardia (proventriculus). Daughter cells migrate upward either to anterior midgut or downward to esophagus and crop. The cardia functions as a gastric valve and the anterior midgut and crop together function as a stomach in Drosophila; therefore, the foregut/midgut stem cells have been named gastric stem cells (GaSC). JAK-STAT signaling regulates GaSC proliferation, Wingless signaling regulates GaSC self-renewal, and hedgehog signaling regulates GaSC differentiation. The differentiation pattern and genetic control of the Drosophila GaSCs suggest the possible similarity to mouse gastric stem cells. The identification of the multipotent stem cell pool in the gastric gland in Drosophila will facilitate studies of gastric stem cell regulation and transformation in mammals (Singh, 2011).

This study has identified multipotent gastric stem cells at the junction of the adult Drosophila foregut and midgut. The GaSCs express the Stat92E-GFP reporter, wg-Gal4 UAS-GFP, and Ptc, and are slowly proliferating. The GaSCs first give rise to the fast proliferative progenitors in both foregut and anterior midgut. The foregut progenitors migrate downward and differentiate into crop cells. The anterior midgut progenitors migrate upward and differentiate into midgut cells. However, at this stage because of limited markers availability and complex tissues systems at cardia location, it is uncertain how many types of cells are produced and how many progenitor cells are in the cardia. Clonal and molecular markers analysis suggest that cardia cells are populated from gastric stem cells at the foregut/midgut (F/M) junction; however, it cannot be ruled out that there may be other progenitor cells with locally or limited differential potential that may also take part in cell replacement of cardia cells. Nevertheless, the observed differentiation pattern of GaSCs in Drosophila may be similar to that of the mouse gastric stem cells. Gastric stem cells in the mouse are located at the neck-isthmus region of the tubular unit. They produce several terminally differentiated cells with bidirectional migration, in which upward migration towards lumen become pit cells and downward migration results in fundic gland cells (Singh, 2011).

Three signal transduction pathways differentially regulate the GaSC self-renewal or differentiation. The loss of JAK-STAT signaling resulted in quiescent GaSCs; that is, the stem cells remained but did not incorporate BrdU or rarely incorporated BrdU. In contrast, the amplification of JAK-STAT signaling resulted in GaSC expansion (Singh, 2011).

These observations indicate that JAK-STAT signaling regulates GaSC proliferation. In contrast, the loss of Wg signaling resulted in GaSC loss, while the amplification of Wg resulted in GaSC expansion, indicating that Wg signaling regulates GaSC self-renewal and maintenance. Finally, the loss of Hh signaling resulted in GaSC expansion at the expense of differentiated cells, indicating that Hh signaling regulates GaSC differentiation. The JAK-STAT signaling has not been directly connected to gastric stem cell regulation in mammal. However, the quiescent gastric stem cells/progenitors are activated by interferon γ (an activator of the JAK-STAT signal transduction pathway), indicating that JAK-STAT pathways may also regulate gastric stem cell activity in mammals. Amplification of JAK-STAT signaling resulted in expansion of stem cells in germline, posterior midgut and malpighian tubules of adult Drosophila. In the mammalian system, it has been reported that activated STAT contributes to gastric hyperplasia and that STAT signaling regulates gastric cancer development and progression. Wnt signaling has an important function in the maintenance of intestinal stem cells and progenitor cells in mice and hindgut stem cells in Drosophila, and its activation results in gastrointestinal tumor development. Tcf plays a critical role in the maintenance of the epithelial stem cell. Mice lacking Tcf resulted in depletion of epithelial stem-cell compartments in the small intestine as well as being unable to maintain long-term homeostasis of skin epithelia. A recent study even demonstrates that the Wnt target gene Lgr5 is a stem cell marker in the pyloric region and at the esophagus border of the mouse stomach. Further, it has been found that overactivation of the Wnt signaling can transform the adult Lgr5+ve stem cells in the distal stomach, indicating that Wnt signaling may also regulate gastric stem cell self-renewal and maintenance in the mammal. Sonic Hedgehog (Shh) and its target genes are expressed in the human and rodent stomach. Blocking Shh signaling with cyclopamine in mice results in an increase in the cell proliferation of gastric gland, suggesting that Shh may also regulate the gastric stem cell differentiation in mice. These data together suggest that the genetic control of the Drosophila GaSC may be similar to that of the mammalian gastric stem cells (Singh, 2011).

The potential GaSCs niche. In most stem cell systems that have been well characterized to date, the stem cells reside in a specialized microenvironment, called a niche.66 A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The niche stromal cells often secrete growth factors to regulate stem cell behavior, and the stem cell niche plays an essential role in maintaining the stem cells, which lose their stem-cell status once they are detached from the niche (Singh, 2011).

Loss of the JAK-STAT signaling results in the GaSCs being quiescent; the stem cells remain but do not proliferate or rarely proliferate. The Dome receptor is expressed in GaSCs, while the ligand Upd is expressed in adjacent cells. Upd-positive hub cells function as a germline stem cell niche in the Drosophila testis. Further, thia study demonstrated that overexpression of upd results in GaSC expansion, suggesting that the Upd-positive cells may function as a GaSC niche. Furthermore, while Stat92E-GFP expression is regulated by the JAK-STAT signaling in other systems, its expression at the F/M junction seems independent of the JAK-STAT signaling because Stat92E-GFP expression is not significantly disrupted in the Stat92Ets mutant flies, suggesting that the GaSCs may have unique properties (Singh, 2011).

The stomach epithelium undergoes continuous renewal by gastric stem cells throughout adulthood. Disruption of the renewal process may be a major cause of gastric cancer, the second leading cause of cancer-related death worldwide, yet the gastric stem cells and their regulations have not been fully characterized. A more detailed characterization of markers and understanding of the molecular mechanisms control gastric stem cell behavior will have a major impact on future strategies for gastric cancer prevention and therapy. The information gained from this report may facilitate studies of gastric stem cell regulation and transformation in mammals (Singh, 2011).

PERK limits Drosophila lifespan by promoting intestinal stem cell proliferation in response to ER stress

Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. This study shows that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK is also specifically activated in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response becomes deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. These studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPRER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan (Wang, 2015).

Progressive decline of proliferative homeostasis in high-turnover tissues is a hallmark of aging, resulting in cancers and degenerative diseases. This is of particular relevance in barrier epithelia, such as the intestinal epithelium, where homeostatic tissue renewal has to be balanced with acute regenerative episodes in response to acute damage or infection. Accordingly, the control of intestinal stem cell (ISC) proliferation has to integrate endogenous control mechanisms with stress and inflammatory signals that promote mitogenic activity of these cells. How cellular stress responses of intestinal epithelial cells (IECs) and intestinal stem cells (ISCs) coordinate and maintain such regenerative processes is a critical question that will provide insight into the etiology of pathologies ranging from inflammatory bowel diseases (IBDs) to colorectal cancers (Wang, 2015).

Long-term homeostasis of the intestinal epithelium is significantly impacted by ER stress. In mouse models for IBDs, ER stress is increased in the intestinal epithelium, and genetic conditions that impair protein folding capacity in the ER of IECs result in complex cell-autonomous and non-autonomous activation of stress signaling pathways, triggering inflammatory conditions similar to IBDs. Recent studies in mice suggest that the UPRER may also influence regenerative processes in the gut directly, as it is engaged in cells transitioning from a stem-like state into the transit amplifying state in the small intestine of mice. In flies, ER stress promotes ISC proliferation, and increased ER stress across the intestinal epithelium is associated with age-related dysplasia in this tissue (Wang, 2014). The downstream signaling mechanisms promoting ISC proliferation in response to ER stress remain unclear (Wang, 2015).

Three highly conserved UPRER sensors coordinate the cell-autonomous response to ER stress: PERK, the transcription factor ATF6, and the endoribonuclease IRE1. IRE1 promotes splicing of the mRNA encoding the transcription factor Xbp1 (see Drosophila Xbp1), PERK phosphorylates and inhibits the translation initiation factor 2 alpha (eIF2α) (Heijmans, 2013; Shi, 1998; Harding, 1999), and ER stress-induced cleavage of ATF6 promotes its nuclear translocation and activation of stress response genes, including Xbp1 (Schroder, 2005). The activation of Xbp1 and ATF6 results in transcriptional induction of ER chaperones, of genes encoding ER components, and of factors required to degrade un/misfolded proteins through ER-associated degradation (ERAD), thus enhancing ER folding capacity and proteostatic tolerance (Wang, 2015 and references therein).

Studies in worms have shown that, in addition to these cell-autonomous responses to ER stress, local activation of the UPRER can trigger UPRER responses in distant tissues, indicating that endocrine processes exist that coordinate such stress responses across cells and tissues. The mechanism(s) regulating and mediating these non-autonomous responses remain elusive (Wang, 2015).

By regulating eIF2α, ATF4 and Nrf2, PERK activation integrates the response to both protein misfolding in the ER and to misfolding-associated oxidative stress. Accumulation of un/misfolded proteins in the ER results in the production of reactive oxygen species (ROS), most likely due to the generation of hydrogen peroxide as a byproduct of protein disulfide bond formation by protein disulfide isomerase (PDI) and ER oxidoreductin 1 (Ero1) (Wang, 2015).

The coordinated control of cellular protein and redox homeostasis by the UPRER and other stress signaling pathways is likely critical to maintain SC function, as the intracellular redox state significantly impacts SC pluripotency, proliferative activity, and differentiation. Recent studies shown that this coordination is achieved in Drosophila ISCs by integration of Nrf2/CncC-mediated responses and Xbp1-mediated ER stress responses (Wang, 2014). The fly orthologue of Nrf2, CncC (Cap 'n' collar isoform-C), counteracts intracellular oxidants and limits proliferative activity of ISCs (Hochmuth, 2011). In ISCs, CncC is inhibited in response to high ER stress (as in Xbp1 loss-of-function conditions), resulting in increased oxidative stress and activation of ISC proliferation (Wang, 2015).

The Drosophila ISC lineage exhibits a high degree of functional and morphological similarities with the ISC lineage in the mammalian small intestine. ISCs self-renew and give rise to transient, non-dividing progenitor cells called EnteroBlasts (EBs) that are lineage-restricted (by Robo/Slit signaling and differential Notch signaling) to differentiate into either absorptive EnteroCytes (ECs) or secretory EnteroEndocrine (EEs) cells. ISCs are the only dividing cells in the posterior midgut of Drosophila and their entry into a highly proliferative state is regulated by multiple stress and mitogenic signaling pathways, including Jun-N-terminal Kinase (JNK), Jak/Stat, Insulin, Wnt, and EGFR signaling (Wang, 2015).

During aging, flies develop epithelial dysplasia in the intestine, caused by excessive ISC proliferation and deficient differentiation of EBs (Biteau, 2008; Choi, 2008). This phenotype is a consequence of an inflammatory condition initiated by immune senescence and dysbiosis of the commensal bacteria, and causes metabolic decline, loss of epithelial barrier function, and increased mortality (Rera, 2012; Biteau, 2010; Guo, 2014), and is associated with a strong tissue-wide increase in ER stress (Wang, 2014). Increasing ER proteostasis in ISCs (by over-expressing Xbp1 or the ERAD-associated factor Hrd1) prevents the age-related over-proliferation of ISCs, suggesting that limiting ER stress-associated signaling in ISCs may be beneficial for tissue homeostasis (Wang, 2015).

This study has tested this hypothesis. The regulation of ISC proliferation by cell-autonomous and non-autonomous UPRER responses was explored in detail, and the consequences of limiting ER stress responses in ISCs for longevity were explored. By analyzing loss of function conditions for Ero1L this study finds that the induction of ISC proliferation by ER stress can be uncoupled from the production of ROS, but that ISC-specific activation of PERK is critical for the proliferative response. Interestingly, PERK activation in ISCs is triggered both by ER stress within ISCs and non-autonomously by ER stress in other cells of the intestinal epithelium, which activate PERK in ISCs through the secretion of Unpaired ligands and activation of JAK/Stat signaling in ISCs. PERK thus integrates epithelial stress responses to control ISC proliferation under challenging proteostatic conditions. Strikingly, PERK is also essential for normal cell proliferation in the ISC lineage, and excessive or chronic PERK activity in ISCs is a cause for the development of epithelial dysplasia in aging flies. Accordingly, this study demonstrates that limiting PERK expression in ISCs is sufficient to extend lifespan (Wang, 2015).

This study identifies the PERK branch of the UPRER as a central node in the control of proliferative homeostasis in the intestinal epithelium, and establishes a previously unrecognized role for PERK in promoting regenerative responses to both tissue-wide and cell-autonomous ER stress. This critical function of PERK in tissue regeneration, however, also results in the aging-associated loss of proliferative homeostasis in the intestinal epithelium, limiting organismal lifespan. The unique and specific increase in eIF2α phosphorylation in ISCs in stressed and aging conditions suggests a differential activation of the PERK-eIF2α branch of the UPRER between ISCs and their daughter cells. It remains unclear whether this differential regulation reflects different strategies in combating ER stress between these cell populations, and additional studies are necessary to address this interesting question (Wang, 2015).

Drosophila ISCs, as many other stem cell types, are controlled extensively by redox signals. Previous work, as well as the results shown in this study, suggests that ER-induced oxidative stress plays a central role in the control of ISC proliferation after a proteostatic challenge. The results support the notion that ER-induced ROS is a consequence of the PDI/Ero1L system, as has been proposed in mammalian cells (Harding, 2003). However, Ero1L, as a thiol oxidase, may also affect the proper folding and maturation of Notch directly (as described previously), inhibiting ISC differentiation, and resulting in stem cell tumors. The phenotype of Ero1L-deficient ISC lineages supports a role for Ero1L in Notch signaling (tumors with elevated numbers of Dl+ cells). At the same time, this study's results also support a role for Ero1L in limiting ISC proliferation directly through the UPRER (and independently of Notch signaling or oxidative signals), as loss of Ero1L induces PERK activity without promoting ROS production in these cells. PERK itself is required for the induction of cell cycle and DNA replication genes in ISCs responding to TM treatment, yet it also induces antioxidant genes under these conditions, suggesting complex crosstalk between PERK-mediated control of mitotic activity of ISCs and the control of redox homeostasis in these cells (Wang, 2015).

The fact that loss of Ero1L activates PERK while not inducing Xbp1 in ISCs suggests selective activation mechanisms for these two branches of the UPRER. The study proposes that this selectivity is associated with the production of ROS and that ER protein stress activates the Xbp1 branch when associated with a ROS signal, while PERK can be activated by unfolded proteins independently of ROS production. Further studies are needed to dissect the relative contribution of ROS production, PERK activation and Notch perturbation in the control of ISC proliferation in Ero1L loss of function conditions (Wang, 2015).

This study highlights the interaction between cell-autonomous and non-autonomous events in the ER stress response of ISCs and support the notion that improving proteostasis by boosting ER folding capacity in stem cells improves long-term tissue homeostasis and can impact lifespan. The regulation of PERK activity in ISCs by the JAK/Stat signaling pathway provides a tentative mechanism for the interaction between IECs experiencing ER stress and ISCs: the study proposes that JNK-mediated release of JAK/Stat ligands from stressed IECs results in JAK/Stat mediated activation of PERK in ISCs, and that this activation is required for the proliferative response of ISCs to epithelial dysfunction. The activation of JAK/Stat signaling in the intestinal epithelium of animals in which Xbp1 is knocked down in ECs, the requirement for JNK activation and Upd expression in ECs for ISC proliferation in response to stress, and the requirement for Stat (and Hop and Dome) in ISCs for the activation of eIF2α phosphorylation and stress-induced ISC proliferation, support this model. The mechanisms by which Stat mediates activation of PERK remain unclear, and will be interesting topics of further study (Wang, 2015).

Studies in worms have established the UPRER as a critical determinant of longevity, and Xbp1 extends lifespan by improving ER stress resistance. This study's data further support the notion that regulating ER stress response pathways is critical to increase health- and lifespan. Here, chronic PERK activation can be considered a downstream readout of the buildup of proteotoxic stress in the intestinal epithelium during aging, which then perturbs proliferative homeostasis by continuously providing pro-mitotic signals to ISCs. Knocking down PERK in ISCs limits these pro-mitotic signals, improving homeostasis and barrier function, and extending lifespan. Lifespan is generally extended when ISC proliferation is limited in older flies, but not when it is completely inhibited. Accordingly, they observe lifespan extension when PERK is knocked down using an RNAi approach that does not completely ablate PERK function.

ER stress has been documented as tightly associated with intestinal inflammation and the development of IBDs in mice and humans. Genetic variants in Xbp1 are associated with higher susceptibility to IBD and a recent study indicates that Xbp1 can act as a tumor suppressor in the intestinal epithelium, by limiting intestinal proliferative responses and tumor development through the control of local inflammation. In this context, the specific role of PERK in the control of ISC proliferation in the fly gut is consistent with the function of PERK in the intestinal epithelium of mice, where activation of PERK can promote transition of ISCs into the transient amplifying cell population. While the Drosophila midgut epithelium does not contain a transit amplifying cell population, this study's data suggest that a role for PERK in the proliferative response of the ISC lineage to ER stress is conserved (Wang, 2015).

Due to the importance of the UPRER in the maintenance of tissue homeostasis in aging organisms, therapies targeting the UPRER are promising strategies to delay the aging process. Accordingly, pharmaceuticals that can limit ER stress (such as Tauroursodeoxycholic acid, TUDCA and 4-phenylbutyrate, PBA) have had therapeutic success in various human disorders. Interestingly, flies fed PBA show increased lifespan, yet the effects of PBA on intestinal homeostasis have not yet been explored. Based on this work, it is likely that further characterization of the effects of UPRER-targeting drugs on ISC function and intestinal homeostasis will help develop clinically relevant strategies to limit human aging and extend healthspan (Wang, 2015).

Oogenesis

Regulation of epithelial stem cell replacement and follicle formation in the Drosophila ovary

Though much has been learned about the process of ovarian follicle maturation through studies of oogenesis in both vertebrate and invertebrate systems, less is known about how follicles form initially. In Drosophila, two somatic follicle stem cells (FSCs) in each ovariole give rise to all polar cells, stalk cells, and main body cells needed to form each follicle. One daughter from each FSC founds most follicles but that cell type specification is independent of cell lineage, in contrast to previous claims of an early polar/stalk lineage restriction. Instead, key intercellular signals begin early and guide cell behavior. An initial Notch signal from germ cells is required for FSC daughters to migrate across the ovariole and on occasion to replace the opposite stem cell. Both anterior and posterior polar cells arise in region 2b at a time when approximately 16 cells surround the cyst. Later, during budding, stalk cells and additional polar cells are specified in a process that frequently transfers posterior follicle cells onto the anterior surface of the next older follicle. These studies provide new insight into the mechanisms that underlie stem cell replacement and follicle formation during Drosophila oogenesis (Nystul, 2010).

The Drosophila ovary is a highly favorable system for studying epithelial cell differentiation downstream from a stem cell. New follicles consisting of 16 interconnected germ cells surrounded by an epithelial (follicle cell) monolayer are continuously produced during adult life and develop sequentially within ovarioles (see Prefollicle cells associate with cysts in an ordered fashion downstream from follicle stem cells). Follicle formation begins in the germarium, a structure at the tip of each ovariole that houses 2-3 germline stem cells (GSCs) and 2 follicle stem cells (FSCs) within stable niches. Successive GSC daughters known as cystoblasts are enclosed by a thin covering of squamous escort cells and divide asymmetrically four times in sucession to produce 16-cell germline cysts, comprising 15 presumptive nurse cells and a presumptive oocyte. At the junction between region 2a and region 2b, cysts are forced into single file as they encounter the FSCs, lose their escort cell covering, and begin to acquire a follicular layer. Follicle cells derived from both FSCs soon mold them into a 'lens shape' characteristic of region 2b. Under the influence of continued somatic cell growth, cysts and their surrounding cells round up, enter region 3 (also known as stage 1), and bud from the germarium as new follicles that remain connected to their neighbors by short cellular stalks (Nystul, 2010).

A complex sequence of signaling and adhesive interactions between follicular and germline cells is required for follicle budding, oocyte development, and patterning. However, the mechanisms orchestrating the initial association between follicle cells and cysts within the germarium are less well understood. While lineage analysis indicates the presence of two FSCs, low fasciclin III (FasIII) expression has been claimed to specifically mark FSCs, leading to the conclusion that more FSCs are present under some conditions (Nystul, 2010).

The differentiation of polar cells at both their anterior and posterior ends is required for normal follicle production, and depends on Notch signals received from the germline. Subsequently, anterior polar cells send JAK-STAT and Notch signals that specify stalk cells. While the source of these signals and their effects are clear, the timing of polar cell specification and its dependence on cell lineage are not. Some anterior and posterior polar cells (but not stalk cells) were inferred by lineage analysis to arise and cease division within region 2b. In contrast, on the basis of marker gene expression it was concluded that anterior polar cells are specified later, in stage 1, and posterior polar cells in stage 2. Up to four polar cells may eventually form, but apoptosis reduces their number to a single pair at each end by stage 5. Moreover, polar and stalk are believed to arise exclusively from 'polar/stalk' precursors that separate from the rest of the FSC lineage and these cells were proposed to invade between the last region 2b cyst to affect follicle budding (Nystul, 2010).

This study analyzes the detailed behavior of FSCs and their daughters in the germarium. No evidence of polar/stalk precursors was observed, and it was shown that the first anterior and posterior polar cells are specified in region 2b, prior to the previously accepted time of follicle cell specialization. Additional polar cells are also formed later during stages 1 and 2. Follicle cell differentiation appears to be independent of cell lineage, but is orchestrated by sequential cell interactions, and in particular by Notch signaling. These results reveal the sophisticated, self-correcting behavior of an epithelial stem cell lineage at close to single-cell resolution (Nystul, 2010).

The data provide a much clearer picture of the follicle stem cell lineage than was previously available. They suggest that key aspects of FSC regulation depend on mechanisms that move cysts into a single file and program the loss of their escort cells precisely as they encounter FSCs and enter region 2b. Contact with incoming region 2a cysts likely induces FSC divisions, ensuring that cysts acquire a daughter cell from each stem cell as they stretch out to span the width of the germarium at the region 2a/2b junction. The asymmetry in cyst organization exposes FSC daughter cells to different cyst faces and, therefore, potentially to different signals. The FSC and daughter located on the same side as the entering cyst are exposed to the posterior face of the cyst while it is still in region 2a, covered by escort cells. In contrast, the opposite FSC and daughter contact the anterior face of the cyst as it migrates into 2b, at a time when the cyst is shedding its escort cell layer and exposing the Delta signal on the germ cell surface. Since region 2a cysts tend to interdigitate in forming a single file, cyst entry will usually alternate sides as successive cysts pass, causing FSC daughters arising from the same side to alternate migration paths. An advantage to this system may be its flexibility, allowing follicles to form normally even if multiple cysts enter from the same side in succession (Nystul, 2010).

Notch signaling in early FSC daughters promotes a 'prefollicle' state by blocking follicle cell differentiation. Consistent with this, it was observed that FSCs and their early daughters have much lower levels of differentiation markers such as FasIII and IMP-GFP. This developmental delay may prevent prefollicle cells from immediately incorporating into the differentiated follicular epithelium, allowing them to instead retain a more mesenchymal character conducive to cross-migration, and may also contribute to their ability to compete with the resident FSC for niche occupancy. Notch mutant daughters did not replace wild-type FSCs, most likely because they were unable to migrate into proximity. A role for Notch in suppressing differentiation downstream from the FSC might also explain why cells expressing activated Notch failed to migrate posteriorly (Nystul, 2010).

Follicle cell fates are specified by intercellular signals rather than lineage: The two FSC daughters and their descendants, with few exceptions, continue to associate with the cyst they first contact at the 2a/2b boundary throughout subsequent development. Their division rate increases briefly, because daughters divide four times in the time it takes to generate three new cysts. Despite their growing number, however, all the cells retain the ability to produce main body, stalk, and polar cells for at least the first two to three divisions (8- to 16-cell stage). In contrast to previous reports, no evidence was found that polar and stalk cells derive from a lineage-restricted polar/stalk precursor population. Claims of a polar/stalk fate were based on experiments using higher rates of clone induction than in the experiments reported in this study. While many clones were also observed in these studies that contained both polar and follicle cells or both stalk and follicle cells, they were discounted as double clones (Nystul, 2010).

By examining clones induced at low frequency (more than threefold lower than in previous studies) it was possible to minimize the need for statistical correction for double clones. Furthermore, by studying clones induced at multiple times downstream from the FSC, overweighting small clones induced just as the polar and stalk cell fates are being determined by signaling within small cell groups was avoided. This has the effect of increasing the proportion of clones containing only one or two cell types even in the absence of any lineage restriction. At early, intermediate, and late times in somatic cell development in the germarium, clones that included all combinations of cell fates were always observed, indicating that follicle cells are multipotent prior to polar or stalk specification. This fits well with recent studies showing that many additional cells in the germarium can be induced to take on a polar cell fate by strong Notch signaling, while high levels of JAK-STAT signaling can induce more stalk cells. In contrast, no mechanism, time, or location where putative polar/stalk precursor cells are specified has ever been documented. Previous models also did not explain how these cells would preferentially arrive in the zone of cells separating regions 2b and 3 or what would become of the many extra cells that can sometimes be found in this region beyond the number needed for these fates (Nystul, 2010).

The finding that polar cells are initially specified in region 2b suggests that more spatial information is available within region 2b follicles than has been detected in earlier experiments. It was found that the first anterior and posterior polar cells are specified when cysts are associated with 8- to 16-cell follicle cells, in mid-to-late region 2b. This agrees closely with previous studies, which found that polar cells were first specified at the 14-cell stage. The early polar cells are detected by lineage because they cease dividing; however, no gene expression markers specific for these cells have been identified. Consequently, it remains uncertain where they are located at the time of induction or whether they function while remaining in region 2b. Since evidence was observed of Notch signal reception within individual follicle cells located at the anterior and posterior regions of stage 2b cysts, the simplest model is that these polar cells are induced by Delta signaling from the germline in a normal anterior/posterior (A/P) orientation. Although no Upd expression was detected at this time, these cells may nonetheless signal to the surrounding somatic cells to establish the graded levels of cadherin that define the initial anterior/posterior axis of the cyst (Nystul, 2010).

Where does the information come from that allows a small number of polar cells to be specified at this time? One possibility is a 'signal relay' from more posterior follicles. Highly accurate timing of polar cell formation relative to the signaling events during follicle budding might help to further test this model. However, the observation of localized Notch signal reception and polar cell specification in region 2b follicles suggests that the germline at this stage is already sufficiently polarized to signal in a limited manner along the future A/P axis. Some of this information may come from the inherent asymmetry within the germline cyst whose cells differ systematically in their fusome content, organelle content, and microtubule organization. The future oocyte and its sister four-ring canal cell are always located in the center of the region 2b cysts and hence might be one source for this inductive signal. Alternatively, there may be additional differences within this region of the germarium that have yet to be detected and that may contribute (Nystul, 2010).

These studies confirm previous conclusions that additional polar cells are formed during the process of budding and provide new insight into the budding process itself. Anterior-biased clones were almost always confined to a single follicle, but a significant fraction of posterior-biased clones (~33%) extended onto the next older follicle where they encompassed both an anterior polar cell and 2-30 anterior follicle cells. This suggests that cells at the posterior of the nascent follicle outgrow their cyst as it rounds up and are forced into the space between the posterior 2b cyst and the budding cyst. The origin of these cells has long been a mystery. A fraction of the interstitial cells likely contact and move onto the anterior of the downstream cyst where those that happen to lie adjacent to the existing polar cells are induced as new polar cells and stalk cells. Any remaining interstitial cells likely rejoin the main body of follicle cells as budding is completed or are eliminated by apoptosis as the stalk resolves to its final size (Nystul, 2010).

This study of early follicle cell development provides a rare opportunity to analyze how epithelial cells behave downstream from a stem cell. Most characterized Drosophila stem cell daughters receive information asymmetrically from their mother stem cell and differentiate rapidly. Germline stem cells and their niches ensure that cystoblasts receive an asymmetric fusome segment as well as differential environmental signals that program exactly four stereotyped divisions prior to entering meiosis. Under nonstressed conditions, intestinal stem cells utilize Notch signals to specify their daughters as either enterocytes or enteroendocrine cells and to terminate subsequent division. Neuroblasts program a stereotyped sequence of daughter cell fates by differential division and signaling. In contrast, FSC daughters undergo eight to nine divisions and differentiate independently of lineage over the course of several divisions and are capable of producing normal follicles even when the usual pattern of cellular interactions is altered. The increased resolution of follicle cell behavior afforded by these studies provides a valuable opportunity to study how epithelial cells are able to robustly bring about defined outcomes in the absence of the precise early programming (Nystul, 2010).

Several mechanisms are likely to contribute to successful follicle formation. First, genes characteristic of a polarized epithelium turn on slowly downstream of the FSC. The cross-migrating cell and several other cells frequently lacked such gene expression, but instead expressed genes characteristic of escort cells, suggesting that follicles are able to maintain some germline-soma interactions while completely replacing their somatic coverings. The early differentiation of polar cells may help guide subsequent cell behavior. In conjunction with the intrinsic asymmetric structure of germline cysts, differential adhesive interactions between germ and somatic cells across the follicle, differential pressures resulting from cell growth, and the resistive forces of the ovariolar wall, signals from these cells may be sufficient to ensure that the oocyte moves to the posterior and that cysts begin to round (Nystul, 2010).

These characteristics of the FSC lineage, although unique among well-studied stem cells in Drosophila, may be closer to those governing the epithelial lineages within many mammalian tissues. Thus, the mechanisms that give FSCs and their daughters their developmental flexibility and robustness are likely to be both widespread and medically relevant (Nystul, 2010).

Dynamics of the basement membrane in invasive epithelial clusters in Drosophila: JAK/STAT signalling and recruitment of outer border cells are required for correct shedding and migration

The basement membrane (BM) represents a barrier to cell migration that has to be degraded to promote invasion. However, the role and behaviour of the BM during the development of pre-invasive cells is only poorly understood. Drosophila border cells (BCs) provide an attractive genetic model in which to study the cellular mechanisms underlying the migration of mixed cohorts of epithelial cells. BCs are made of two different epithelial cell types appearing sequentially during oogenesis: the polar cells and the outer BCs. The pre-invasive polar cells undergo an unusual and asymmetrical apical capping with major basement membrane proteins, including the two Drosophila Collagen IV alpha chains, Laminin A and Perlecan. Capping of polar cells proceeds through a novel, basal-to-apical transcytosis mechanism that involves the small GTPase Rab5. Apical capping is transient and is followed by rapid shedding prior to the initiation of BC migration, suggesting that the apical cap blocks migration. Consistently, non-migratory polar cells remain capped. JAK/STAT signalling and recruitment of outer BCs are required for correct shedding and migration. The dynamics of the BM represent a marker of migratory BC, revealing a novel developmentally regulated behaviour of BM coupled to epithelial cell invasiveness (Medioni, 2005).

The migration of cohorts of cells is an alternative to single-cell migration, which is used by normal and cancer cells to invade tissues. One advantage for mixed clusters is to transport tumorigenic (for example, apoptotic resistant) cells with no migratory abilities to a distant destination that they could not reach on their own. In this case, migration is executed by migratory capable cells within the cluster. Clusters illustrate how separate functions (tumorigenesis and migration) can be merged through collaboration between two cell populations. It is thus important to understand how migrating cell clusters are assembled and organized. The BC cluster is made of two distinct populations of cells, i.e. the polar cells and the outer BCs, making it a good model with which to determine the cellular mechanisms underlying the recruitment and migration of mixed cohorts of cells. Three novel steps in the formation of BCs have been identified. (1) It was shown that a developmentally regulated basal to apical transport of BM material takes place in the polar cells, the first population of cells to form in the cluster. The apical cap is the earliest known marker of anterior polar cells. (2) The asymmetrical positioning of the apical cap suggests that despite an apparent identity, the two polar cells are different and might play distinct roles. (3) The data indicate that a two-way interaction takes place between the two differentiated subpopulations of invasive cells before they migrate. A first signal, activating the JAK/STAT pathway is sent by the polar cells to recruit the outer BCs. In a second step, the outer BCs are essential for shedding the apical cap of polar cells (Medioni, 2005).

Outer BCs are not required for apical cap formation. Similarly, outer BCs form normally in the absence of a cap, indicating that apical capping is not a pre-requisite for outer BCs to be recruited and the cluster to be assembled. Interestingly, it was found that immotile polar cells remain capped. Thus, a possible role for apical capping is to block the migration of immature clusters, a finding that could explain the long standing observation that isolated polar cells cannot migrate on their own. Indeed, the coordination between apical cap degradation and the recruitment of outer BCs indicates that degradation of the apical cap could serve as a check point or quality control ensuring that only finalized clusters can start migration. It is important to note that degradation of the ECM at the leading edge of migrating clusters is essential for tumour progression, and examples of cancer cells showing a reduction or absence of some basement membrane markers, including Collagen IV, have been reported. In particular, human alpha3/alpha4 type IV Collagen is found at the apical surface in normal colon tissue, but is absent in colorectal neoplastic cells, making the differential distribution of type IV collagens potential diagnostic markers for the invasiveness of cancer cells. The BC model will be central for future studies aimed at understanding BM dynamics and function in invasive clusters (Medioni, 2005).

Requirement for JAK/STAT signaling throughout border cell migration in Drosophila

The evolutionarily conserved JAK/STAT signaling pathway is essential for the proliferation, survival and differentiation of many cells, including cancer cells. Recent studies have implicated this transcriptional pathway in the process of cell migration in humans, mice, Drosophila and Dictyostelium. In the Drosophila ovary, JAK/STAT signaling is necessary and sufficient for the specification and migration of a group of cells called the border cells; however, it is not clear to what extent the requirement for cell fate is distinct from that for cell migration. It was found that STAT protein is enriched in the migrating border cells throughout their migration and is an indicator of cells with highest JAK/STAT activity. In addition, statts mutants exhibit border cell migration defects after just 30 minutes at the non-permissive temperature, prior to any detectable change in the expression of cell fate markers. At later times, cell fate changes became evident, indicating that border cell fate is labile. JAK/STAT signaling was also required for organization of the border cell cluster. Finally, it is shown that both the accumulation of STAT protein and nuclear accumulation are positively regulated by JAK/STAT activity. The activity of the pathway is negatively regulated by overexpression of a suppressor of cytokine signaling (SOCS) protein and by blocking endocytosis. Together, these findings suggest that the requirement for STAT in border cells extends beyond the initial specification and delamination of cells from the epithelium (Silver, 2005).

Extra and ectopic border cells can be induced in at least two different ways. When ectopic polar cells form, for example in eyes absent or costal 2 (costa) mutant clones, or following mis-expresssion of activated Notch, they recruit surrounding cells into a cluster and these are capable of migration. Alternatively, ectopic expression of UPD, HOP or HOPTum is sufficient to induce large numbers of ectopic migrating border cells. These cells migrate in a variety of sizes of clusters, which lack polar cells, and can even migrate as individual cells. These findings indicate that UPD might be the only factor produced by polar cells that functions to recruit border cells and sustain their motility. To test this hypothesis, ectopic expression of UPD was induced in single anterior follicle cells, or in pairs of cells, to see whether UPD expression alone is as effective as polar cells in recruiting border cells. For comparison, the same method was also used to express a form of UPD that contains a transmembrane domain (UPDTM). Expression of the wild-type form of UPD results in the recruitment of neighboring cells into a cluster, and these cells express high levels of STAT protein, like normal border cells. However, UPD alone is not as effective as a normal polar cell because, normally, two polar cells recruit four to eight cells to surround them, whereas UPD alone results in the recruitment of an average of only 1.1 border cell per UPD-expressing cell. Cells expressing the UAS-UPDTM are actually more effective at recruiting border cells than cells expressing wild-type UPD. A single cell expressing the membrane-tethered form of UPD recruits an average of 3.25 cells to surround it, similar to normal or ectopic polar cells. In the case of the membrane-tethered form of UPD, ectopic migratory cells are observed only adjacent to the UPD-expressing cells. These findings suggested that JAK/STAT signaling contributes to the organization of the migrating border cell cluster. This was investigated further by analyzing the organization of migrating border cells following the reduction of JAK/STAT function (Silver, 2005).

Suppressors of cytokine signaling are thought to inhibit the JAK/STAT pathway, either by blocking JAK or STAT function via a SOCS SH2 domain, or by causing the destruction of these proteins by ubiquitination. There are three SOCS genes in Drosophila but, to date, no loss-of-function mutants have been reported. Using both slbo-GAL4,1310 and c306-GAL4 drivers, it was found that overexpression of wild-type SOCS36E inhibits border cell migration and recruitment, and overexpression of SOCS36E lacking the SOCS domain weakly inhibits migration. By contrast, overexpression of a SOCS36E protein that lacks the SH2 domain, or of slbo-GAL4,1310 or c306-GAL4 alone failed to disrupt border cell migration. Compared with an average border cell number of six for wild-type egg chambers, slbo-GAL4,1310;UAS-SOCS and c306-GAL4;UAS-SOCS egg chambers have an average number of border cells of 3.3 and 4.9, respectively. This is consistent with findings that reduction of JAK/STAT activity inhibits border cell recruitment. Those border cells that do form and migrate, frequently do so as single cells rather than as a cluster. In addition, the border cells had reduced levels of STAT (Silver, 2005).

Consistent with a requirement in cluster organization, when JAK/STAT signaling is reduced by overexpression of a dominant-negative form of dome lacking the cytoplasmic domain, border cells migrate slower than wild-type cells, and frequently as single cells. This is consistent with the finding that in dome mosaic egg chambers, border cells often fail to migrate in a cluster. Taken together, these results support the idea that signaling through the JAK/STAT pathway is responsible for the organization of the border cell cluster (Silver, 2005).

Normally border cells migrate as a cohesive cluster with the non-migratory, UPD-expressing cells in the center and the migratory cells surrounding them. These two cell types are dependent upon each other, since the central cells cannot migrate and are carried by the surrounding cells, and the migratory cells cannot move in the absence of the UPD signal from the central cells. Thus, the organization of the border cell cluster is crucial for normal migration. In addition to its function in border cell specification and motility, several lines of evidence demonstrate the role of UPD/JAK/STAT in organizing the border cell cluster. Ectopic expression of UPD in single anterior follicle cells, for example, is sufficient to recruit adjacent cells to form a cluster capable of migration. In addition, a variety of treatments that reduce STAT activity (dome mosaic clones, overexpression of dominant-negative Dome, and overexpression of SOCS) leads to disruptions of cluster formation. Disruption of the cluster is likely to affect migration through the egg chamber. For example, PAR6, an epithelial protein required for polarity and the migration of border cells, is disrupted in border cells in which dominant-negative Dome is overexpressed, lending support to the idea that JAK/STAT signaling helps to regulate the organization of cells within the cluster. Once the cluster is disrupted, the migratory cells become separated from the polar cells, presumably reducing STAT activity further and aggravating the migration defect. Thus, STAT activity promotes cluster organization, which feeds back to promote efficient UPD/DOME/JAK/STAT signaling (Silver, 2005).

Taken together, the results presented here demonstrate several inter-related properties of JAK/STAT signaling in the control of border cell migration and function. Both anatomical and biochemical mechanisms feed back upon each other to regulate the level of STAT activity precisely throughout the six hours of border cell migration. Positive-feedback mechanisms include maintaining close contact between UPD-expressing cells and the migratory cells, as well as stabilization and nuclear enrichment of STAT protein in response to signaling. One negative regulatory mechanism is the expression of SOCS36E (Silver, 2005).

The findings described here may also have relevance for understanding the requirement of STAT signaling in the progression of cancer. Constitutively activated STAT3 is associated with the aggressive clinical behavior of a number of cancers, including ovarian and renal cancers. Blocking STAT3 in pancreatic cancer cells inhibits tumor growth and metastases in mice, whereas expression of activated STAT3 promotes metastasis. Inhibiting STAT3 expression or activation in ovarian carcinoma cells impedes their motility in vitro. Thus, cancer cells too appear to require sustained activation of this pathway to survive, proliferate and migrate. The finding that JAK/STAT signaling appears to be tightly regulated by its own activity, by that of SOCS inhibitors and by endocytic processes suggests that these may provide points of clinical intervention in the treatment of STAT-dependent cancers (Silver, 2005).

Location and strength of JAK/STAT signaling within the somatic cells of the developing male and female gonad help program gonad morphology and niche structure

The stem cell niches at the apex of Drosophila ovaries and testes have been viewed as distinct in two major respects. While both contain germline stem cells, the testis niche also contains 'cyst progenitor' stem cells, which divide to produce somatic cells that encase developing germ cells. Moreover, while both niches utilize BMP signaling, the testis niche requires a key JAK/STAT signal. This study shows, by lineage marking, that the ovarian niche also contains a second type of stem cell. These 'escort stem cells' morphologically resemble testis cyst progenitor cells and their daughters encase developing cysts before undergoing apoptosis at the time of follicle formation. In addition, JAK/STAT signaling also plays a critical role in ovarian niche function, and acts within escort cells. These observations reveal striking similarities in the stem cell niches of male and female gonads, and suggest that they are largely governed by common mechanisms (Decotto, 2005).

Stem cells are controlled within local tissue microenvironments known as niches that are generated by nearby stromal cells. One of the best-characterized niches supports germline stem cells (GSCs) within the Drosophila ovary. A GSC niche is located at the tip of each ovariole within the germarium, a generative region that is divided into regions 1, 2a, 2b, and 3. The niche itself contains five to seven nondividing somatic cap cells that anchor two or three GSCs via adherens junctions and stimulate reception of an essential BMP signal. Following each GSC division, the posterior daughter cell leaves the niche, differentiates into a 'cystoblast', undergoes four synchronous, incomplete divisions to form a 16-cell germline cyst, and steadily moves in a posterior direction through the germarium. The niche signal directly regulates stem cell fate by repressing transcription of the cystoblast determinant gene bag-of-marbles (bam) in GSC proximal, but not GSC distal, daughters. Less is known about the anatomy and regulation of a second niche that controls the somatic stem cells (SSCs) located midway along the germarium near the start of region 2b. SSCs divide in response to somatic Hedgehog signals to produce follicle cells that encapsulate passing cysts (Decotto, 2005).

Inner germarium sheath (IGS) cells, which line the surface of regions 1 and 2a, support germ cell differentiation and somatic cell production. Thin cytoplasmic processes from IGS cells envelop cystoblasts and cysts for several days prior to follicle formation. In addition, IGS cells located halfway down the germarium anchor SSCs via adherens junctions and are postulated to play a critical role in defining the SSC niche. Both anterior IGS cells and those near the SSCs have been reported to be differentiated and immobile but capable of maintaining parity with cyst number by undergoing sporadic division or death. Following GSC loss, IGS cells gradually disappear by apoptosis as preexisting cysts leave the anterior germarium and acquire follicle cells. This destroys the SSC niches, but the released SSCs can often associate with cap cells in the vacated GSC niche and continue to divide (Decotto, 2005 and references therein) .

The stem cell niche located at the apical tip of the Drosophila testis shows both similarities to and differences from this model of the ovarian niche. As in the ovary, critical signals are sent by a small cluster of somatic cells, the hub, that directly contact GSCs, but not their gonialblast (cystoblast) daughters. In both males and females, newly formed cysts can revert to the stem cell state under certain circumstances. However, testis GSCs require JAK/STAT signals from the hub, while this signaling pathway in the ovary has been reported to act later, during follicle formation. Recently, BMP signals that repress bam have also been shown to contribute to testis stem cell maintenance, but do not control cyst initiation. A further accepted difference between the two gonadal tips concerns the origin of the thin somatic cells surrounding the developing cysts. Contrary to the reported quiescence of female IGS cells, the somatic cells surrounding male cysts descend from stem cells known as 'cyst progenitor cells' that are interspersed between the GSCs and that maintain their own attachments to the hub. Thus, the male GSC niche contains two types of stem cells, while the female niche is thought to contain only one (Decotto, 2005).

The idea that ovarian cystoblasts interact with stationary IGS cells rather than newly generated, mobile somatic cyst cells as in the testis presents several difficulties. Permanent IGS cells would have to stretch and periodically break their cytoplasmic processes to allow cysts to move and contact more posterior IGS cells. How would permanent IGS cells keep pace with changes in the size and number of cysts within the anterior germarium that occur as a function of age? Finally, how can this model be reconciled with the fact that IGS cells are labeled quite frequently in lineage tracing experiments under conditions where cyst numbers are not changing (Decotto, 2005)?

To address these questions, IGS cell behavior was investigated using lineage labeling. It was found that a distinct subset of 12-8 IGS cells, which have been termed 'escort cells', are maintained by 4-6 terminally located 'escort stem cells' (ESCs) that extensively contact GSCs within the niche. Escort stem cells strongly resemble the cyst progenitor cells of the testis in morphology, location, and behavior. Following a single division, escort stem cell daughters move with cysts through the germarium until they are lost by apoptosis and replaced by follicle cells. Moreover, as in the testis, JAK/STAT signaling is essential for the maintenance and division of both the GSCs and ESCs. These findings reveal striking similarities in the cellular organization at the apex of both male and female gonads, and show that the anatomy and regulation of male and female germline stem cell niches are more similar than previously believed (Decotto, 2005).

These studies reveal that a previously unknown type of stem cell, escort stem cells, closely contacts the GSCs within the niche at the tip of each Drosophila ovariole. ESC daughters encase newly produced cystoblasts and remain tightly associated as they grow into 16-cell cysts and enter meiosis. The existence of thin somatic cells that interact with early female germ cells was known previously. However, these inner germarium sheath cells were believed to divide only rarely, and to remain attached in place along the wall of the germarium. The discovery that escort cells are a dynamic cell population supported by distinctive stem cells provides essential information for the further study of early germ cell differentiation, including the processes of GSC regulation, cyst formation, oocyte determination, meiosis, and germline sex determination. All these events take place between the stem cell stage and early region 2b, where germline cysts are found to lose their escort cells by apoptosis and acquire a follicle cell covering (Decotto, 2005).

These findings indicate that the cellular architecture within which early germ cell development occurs is more similar between males and females than previously appreciated. ESCs resemble testis cyst progenitor cells, which are precursors of the two thin somatic cyst cells that surround each developing male 16-cell cyst. There are already indications that escort and somatic cyst cells are involved in common processes and express similar genes. Early male and female germ cells may send common signals to these overlying somatic cells, because both require the activity of stem cell tumor (stet), a rhomboid-like membrane protease that activates Egfr ligands in the signaling cell. Reciprocal signals between germline and escort or somatic cyst cells may terminate cyst growth, prevent reversion toward the stem cell state, and mediate germline sex determination. Thin somatic cell processes also surround mouse ovarian germ cells as they form interconnected cysts and enter meiosis, suggesting that some functions mediated by escort-like cells may occur during vertebrate as well as invertebrate gametogenesis (Decotto, 2005).

JAK/STAT signaling is required during early female gametogenesis, as it is in the male. Moreover, clonal analysis shows that the escort lineage is a major mediator of this requirement. When escort cells (but not germ cells or follicle cells) lack STAT activity, the shape of the germarium tip, and the organization of its epithelial and muscle sheath, is greatly altered. The sheath swells and fills with material whose nature remains unknown. Moreover, large polyploid nuclei arise in this region, either by division of normal sheath cells or possibly by migration from another location within the ovariole. Escort cells line the outer surface of the anterior germarium and may exert these influences through changes in their cytoskeletal organization, by altering the structure of the basement membrane, or through relay signals to the affected cell types (Decotto, 2005).

GSCs are also subject to a STAT-dependent influence from escort cells. When their associated escort cells lacked STAT activity, one or both GSCs lose their attachment to cap cells, fail to maintain their spectrosomes in an anterior orientation, and cease division. GSCs and ESCs contact each other over a large portion of their surfaces, but it is not known what direct signals and adhesive interactions occur. It was found that ESCs divide in a manner that is at least generally coordinated with the rate of cyst production. Even when later stages of cyst development are disrupted, as in Sxlf4 and bamΔ86 germaria, the number and morphology of ESCs is little changed. The stem cell-stem cell interactions likely act in both directions, because ESCs are absent in agametic germaria, and existing ESCs are lost within a few days after forced GSC differentiation (Decotto, 2005).

Strong similarities have long been recognized between niches that support GSCs in ovarioles and at the tip of the testis. In both sexes, GSCs form tight, cadherin-based junctions with cap or hub cells, and undergo mostly asymmetric divisions to yield cyst-forming cells that embark on a program of rapid, incomplete divisions. The structure and behavior of fusomes and ring canals during GSC divisions are highly similar. However, it has previously been thought that differences in anatomy, such as the presence of cyst progenitor cells only in the testis, and in distinct regulatory signals, BMP versus JAK/STAT, indicate that these niches display fundamental differences. Recently, however, BMP signaling was shown to be required in male as well as female GSCs, and to act by repressing bam expression. Now, by showing that ovariole tips contain ESCs analogous to cyst progenitor cells, and require JAK/STAT signaling, these studies strongly suggest that male and female GSC niches develop and operate using fundamentally similar cells and regulatory mechanisms. Remaining differences include the fact that in male GSCs, STAT activity is required cell autonomously. Moreover, bam expression is required to initiate cyst development in females, but not males (Decotto, 2005).

Sex-specific gonadal morphology ultimately arises due to autonomous aspects of germline sex determination that first manifest during embryogenesis, and to differences in the structure of the embryonic male and female somatic gonad. Recently, a major difference between the male and female gonads during early larval development was shown to be the male-specific expression of JAK/STAT ligand(s) in anterior somatic cells and the consequent activation of JAK/STAT signaling within male but not female germ cells. Induced activation of JAK/STAT signaling in female germ cells at this time is sufficient to elicit male-specific germ cell responses such as cell division (Decotto, 2005).

Differing levels of JAK/STAT and BMP signaling during the development of male and female gonads may program and pattern the corresponding GSC niches, prior to the emergence of significant autonomous germ cell sexual differences. The level of JAK/STAT signaling affects the overall shape of the distal end of the gonad, possibly through interactions with muscle and sheath cells. Elevated levels of upd expression produce a more testis-like ovariole, with a rounded tip containing cysts that move away in a less-ordered manner. Reduction of STAT activity generates a hyper thin ovariole within an abnormal organization of its sheath. The number of ESCs near the cap cells increases when JAK/STAT signaling is elevated, and decreases when it is reduced. All these observations suggest that the location and strength of JAK/STAT signaling within the somatic cells of the developing gonad help program its morphology and niche structure. These findings open the way for a detailed analysis of how male- and female-specific niche development is programmed (Decotto, 2005).

Regulation of stem cells by intersecting gradients of long-range niche signals

Drosophila ovarian follicle stem cells (FSCs) were used to study how stem cells are regulated by external signals. and three main conclusions were drawn. First, the spatial definition of supportive niche positions for FSCs depends on gradients of Hh and JAK-STAT pathway ligands, which emanate from opposite, distant sites. FSC position may be further refined by a preference for low-level Wnt signaling. Second, hyperactivity of supportive signaling pathways can compensate for the absence of the otherwise essential adhesion molecule, DE-cadherin, suggesting a close regulatory connection between niche adhesion and niche signals. Third, FSC behavior is determined largely by summing the inputs of multiple signaling pathways of unequal potencies. Altogether, these findings indicate that a stem cell niche need not be defined by short-range signals and invariant cell contacts; rather, for FSCs, the intersection of gradients of long-range niche signals regulates the longevity, position, number, and competitive behavior of stem cells (Vied, 2012).

Stem cells are generally maintained in appropriate numbers at defined locations. It is therefore expected that a specific extracellular environment defines a supportive niche and regulates stem cell numbers. However, the mechanisms for supporting and regulating stem cells may vary widely. In the Drosophila germarium, GSCs are principally regulated directly by a single (BMP) pathway that is activated by signals from immediately adjacent Cap cells and acts within GSCs to prevent differentiation. This study shows that in the same tissue, FSCs are regulated by the activity of at least four major signaling pathways, that the ligands for at least three of these pathways (Wnt, Hh, and JAK-STAT) derive from distant cells and that these pathways appear to collaborate in order to define supportive niche positions for FSCs and the number of FSCs that are supported. Most crucially, FSCs provide a particularly interesting paradigm where the intersection of gradients of long-range niche signals regulates stem cell maintenance, position, number and competitive behavior (Vied, 2012).

How the strength of a signaling pathway specifies FSC numbers and supportive niche positions was examined by manipulating the Hh pathway. Normally, Hh pathway activity is marginally higher in FSCs than in their daughters and is progressively lower in more posterior cells, consistent with Hh emanating from Cap and Terminal Filament cells at the anterior tip of the germarium. Small reductions in Hh pathway activity led to FSC loss while small increases caused FSCs to outcompete their neighbors. FSCs must therefore reside reasonably close to the anterior of the germarium in order to receive sufficient stimulation by Hh, but what prevents FSCs from moving progressively further anterior and enjoying even stronger Hh stimulation? Wg is expressed in anterior Cap cells along with Hh. Here it was found that excess Wnt pathway activity strongly impairs FSC maintenance and that loss of Wnt pathway activity during FSC establishment can lead to enhanced FSC function and to a modest accumulation of Wg-insensitive FSC derivatives in ectopically anterior positions. These observations suggest that anterior Wg expression contributes to limiting the anterior spread of FSCs. However, Wg-insensitive cells do not spread to the extreme anterior of germaria, suggesting that additional factors control the position of FSCs along the anterior-posterior axis of the germarium (Vied, 2012).

In fact, apparent FSC duplication and anterior movement of FSC derivatives, including Fas3-negative FSC-like cells, was seen very dramatically in response to elevated JAK-STAT pathway activity. Furthermore, the pattern of expression of a reporter of JAK-STAT pathway activity and its response to localized inhibition of ligand production showed that the JAK-STAT pathway in FSCs is activated primarily by ligand emanating from more posterior, polar cells. Hence, it is suggested that normal FSCs are unable to function in significantly more anterior positions because they would receive inadequate stimulation of the JAK-STAT pathway (Vied, 2012).

Thus, the combination of graded distributions of Hh, Wnt, and JAK-STAT pathway ligands appears to be instrumental in setting the anterior-posterior position of FSCs and how many FSCs may be supported in each germarium. Neither the Hh nor the JAK-STAT pathway activity gradients appear to be classical smooth gradients but both are high in the central 2a/b region of the germarium (where FSCs are located) and considerably lower in either the anterior (JAK-STAT) or posterior (Hh) third of the germarium. Although FSCs are normally supported by both Hh and JAK-STAT pathways, JAK-STAT pathway hyperactivity could substantially compensate for loss of Hh pathway activity to support FSCs that are neither rapidly lost nor displace wildtype FSCs. It is therefore concluded that the sum of quantitative inputs of these two pathways is a key parameter for supporting normal FSC function (Vied, 2012).

It was first considered that Hh, Wnt, and JAK-STAT pathways might have a major effect on the migratory or adhesive properties of FSCs partly because favorable pathway manipulations led to ectopically positioned FSC-like cells in the germarium and displacement of wild-type FSCs. It is possible that enhanced proliferation could also contribute significantly to these phenotypes. Indeed, FSC proliferation is likely modulated by several signaling pathways and has been shown to be important for FSC retention in the niche. However, to date, manipulation of cell proliferation alone in an FSC has not produced the displacement of wild-type FSCs that has been observed in response to altered Hh, JAK-STAT, and Wnt pathways. Further evidence for FSC signals regulating adhesion comes from the observation that favorable mutations in all four signaling pathways that were investigated in this study obviated, to a remarkable degree for Hh and JAK-STAT pathways, the normal requirement of FSCs for DE-cadherin function. Again, it is possible that enhanced FSC proliferation may compensate for defective niche adhesion. In fact, partial restoration of FSC maintenance has previously been seen in response to excess Cyclin E or E2F activity for FSCs lacking a regulator of the actin cytoskeleton likely to contribute to adhesion. Nevertheless, the continued retention of FSCs in the germarium despite the absence of DE-cadherin is most simply explained if Hh and JAK-STAT pathways alter FSC adhesive properties to favor FSC retention (Vied, 2012).

The cellular interactions guiding the location of FSCs are likely quite complex, involving prefollicle cells, Escort Cells, germline cysts and the basement membrane. Some of the observations made in this study suggest that JAK-STAT signaling might act, in part, by promoting integrin interactions with the basement membrane. Normally, laminin A ligand and strong integrin staining along the basement membrane do not extend further anterior than the FSCs (O'Reilly, 2008). Perhaps excess JAK-STAT signaling facilitates increasingly anterior deposition of laminin A and organization of adhesive integrin complexes, promoting simultaneous anterior migration and basement membrane adhesion of cells of the FSC lineage. In support of this hypothesis, anterior extension of integrin staining and apparent anterior migration of Hop-expressing cells were seen, principally along germarial walls. However, the requirement or sufficiency of these changes in integrin organization remains to be tested (Vied, 2012).

For excessive Hh signaling, ectopic cells also often associated with germarial walls but these cells did not accumulate in far anterior positions or change the pattern of integrin staining, so enhanced integrin-mediated associations seem unlikely to explain the phenotype. The Hh hyperactivity phenotype is very strong in the absence of DE-cadherin function in FSCs, so what other adhesive function might be altered by Hh signaling? Partial restoration of smo mutant FSC maintenance by increased DE-cadherin expression provides some further support that adhesive changes are an important component of the FSC response to Hh. It has been noted that ptc mutant follicle cells rarely contact germline cells in mosaic egg chambers, preferentially occupying positions between egg chambers or surrounding the follicle cell epithelium, suggesting that excess Hh pathway activity in cells of the FSC lineage may reduce their affinity for germline cells or their propensity to integrate into an epithelium. Adhesion to posteriorly moving germline cysts and a nascent follicular epithelium would seem a priori to be the major influences tending to pull FSCs and their daughters away from a stable germarial association. A reduction in FSC or FSC daughter interactions with germline cysts or with prefollicle cells might therefore lead to increased retention of FSCs in the neighborhood of the normal FSC niche, facilitating accumulation of extra FSCs or allowing FSC retention even in the absence of DE-cadherin (Vied, 2012).

Most cancers involve signaling pathway mutations and several such mutations likely originate in stem cells, where selective pressures may eliminate or amplify mutant cell lineages. It is therefore important to understand how signaling pathways regulate stem cells. The current studies on FSCs highlight some significant principles that may be widely relevant to human epithelial cell cancers. First, activating mutations in signaling pathways normally required for maintenance of the stem cell in question can amplify the number of stem or stemlike cells in a local environment. This produces an increased number of identical but independent genetic lineages, greatly facilitating the acquisition and selection of secondary mutations that push a mutant stem cell lineage toward a cancerous phenotype. Second, signaling pathway mutations can enhance a stem cell’s ability to compete for niche positions, promoting occupation of all available niches in an insulated developmental compartment. These stem cells are now no longer vulnerable to competition from wild-type stem cells and are effectively immortalized if, as for FSCs, daughter cells readily replace lost stem cells. Third, signaling pathway alterations can compensate for deficits in other pathways or other contributors to normal stem cell function. Hence, stem cell self-renewal can now tolerate significant further mutations and changes in their environment that accompany cancer progression. Loss of epithelial cadherin function provides a specific example of a significant mutation that would be expected often to contribute to cancer development by spurring an epithelial to mesenchymal cell transition, but which can (in FSCs) only be propagated in stem cells after mutational hyperactivation of a key signaling pathway. Finally, these studies emphasize that it is possible for many pathways to exert strong influences on a single stem cell type; in FSCs, Hh, JAK-STAT, and PI3K pathway hyperactivity phenotypes are extremely strong, while Wnt and BMP pathways can also play significant roles (Vied, 2012).

The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary

The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator Yorkie (Yki). YAP (a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. This study used border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. This study found that hpo and wts are required for migration of the border cell cluster. In outer border cells, over-expression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo, wts, or over-expression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced-expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, these data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: 1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; 2) in polar cells, it regulates upd expression to control border cell induction and migration (Lin, 2014).

Without children is required for Stat-mediated zfh1 transcription and for germline stem cell differentiation

Tissue homeostasis is maintained by balancing stem cell self-renewal and differentiation. How surrounding cells support this process has not been entirely resolved. This study shows that the chromatin and telomere-binding factor Without children (Woc) is required for maintaining the association of escort cells (ECs) with germ cells in adult ovaries. This tight association is essential for germline stem cell (GSC) differentiation into cysts. Woc is also required in larval ovaries for the association of intermingled cells (ICs) with primordial germ cells. Reduction in the levels of two other proteins, Stat92E and its target Zfh1, produce phenotypes similar to woc in both larval and adult ovaries, suggesting a molecular connection between these three proteins. Antibody staining and RT-qPCR demonstrate that Zfh1 levels are increased in somatic cells that contact germ cells, and that Woc is required for a Stat92E-mediated upregulation of zfh1 transcription. These results further demonstrate that overexpression of Zfh1 in ECs can rescue GSC differentiation in woc-deficient ovaries. Thus, Zfh1 is a major Woc target in ECs. Stat signalling in niche cells has been previously shown to maintain GSCs non-autonomously. This study now shows that Stat92E also promotes GSC differentiation. The results highlight the Woc-Stat-Zfh1 module as promoting somatic encapsulation of germ cells throughout their development. Each somatic cell type can then provide the germline with the support it requires at that particular stage. Stat is thus a permissive factor, which explains its apparently opposite roles in GSC maintenance and differentiation (Maimon, 2014).

Spatially distinct downregulation of Capicua repression and Tailless activation by the Torso RTK pathway in the Drosophila embryo: Stat92E transcription factor plays a role as a mediator of Tor signalling

Specification of the terminal regions of the Drosophila embryo depends on the Torso RTK pathway, which triggers expression of the zygotic genes tailless and huckebein at the embryonic poles. However, it has been shown that the Torso signalling pathway does not directly activate expression of these zygotic genes; rather, it induces their expression by inactivating, at the embryonic poles, a uniformly distributed repressor activity. In particular, it has been shown that Torso signalling regulates accumulation of the Capicua transcriptional repressor: as a consequence of Torso signalling Capicua is downregulated specifically at the poles of blastoderm stage embryos. Extending the current model, it is shown that activation of the Torso pathway can trigger tailless expression without eliminating Capicua. In addition, analysis of gene activation by the Torso pathway and downregulation of Capicua unveil differences between the terminal and the central embryonic regions that are independent of Torso signalling, hitherto thought to be the only system responsible for confering terminal specificities. These data provide new insights into the mode of action of the Torso signalling pathway and on the events patterning the early Drosophila embryo (de las Heras, 2006).

While the Tor pathway is normally activated only at the embryonic poles, tor constitutive mutations trigger its activation over the entire embryo in a ligand-independent manner. In these cases, expression of the tor target genes is expanded too much broader domains and embryos develop head and tail structures lacking most of the segmented trunk. According to the current model one would expect that tll domain expansion in these mutations would be accompanied by an expansion of the Cic downregulation domain (de las Heras, 2006).

Embryos from mutant females bearing the torD4021 constitutive mutation (a strong gain-of-function mutation that acts as a dominant female sterile) have been analyzed and instead it was found that Cic protein is still downregulated only at the poles, as in the wild-type embryos. Therefore, while in the wild-type the posterior tll domain is complementary to the domain of Cic accumulation, in embryos from torD4021/+females these domains overlap and tll is expressed in spite of the presence of nuclear Cic. This behaviour is not allele-specific since embryos from homozygous females for another tor constitutive mutation (torRL3) display the same kind of Cic distribution and tll expression (de las Heras, 2006).

It has been postulated that wild-type Tor receptors and Tor receptors activated by ligand-independent constitutive mutations could signal through distinct downstream effectors. Therefore, whether the persistent accumulation of Cic in embryos from tor constitutive mutant females could be due to a distinct property of these mutations was analyzed. Alternatively, the persistent Cic accumulation could reflect a difference in response between Tor activation in the middle versus the terminal embryonic regions. To test these possibilities, ligand-dependent activation of the Tor receptor was triggered over the entire embryo by general expression of the torso-like (tsl) gene. tsl is the only known gene in the Tor pathway whose expression is locally restricted. Indeed its restricted expression in a group of cells at each end of the developing oocyte is the determinant for the local activation of the Tor pathway, since its ectopic expression is sufficient to induce widespread activation of the Tor receptor. Accordingly, it was found that driving tsl expression with a tubGAL4 driver in the oocyte gives rise to an expansion of the tll expression domain and to the generation of embryos with a tor-gain-of-function phenotype, in that they develop head and tail structures and lack most of the segmented trunk. However, and similarly to what is described above for tor constitutive mutations, in these embryos Cic downregulation is not expanded to a broader domain, indicating that even ligand-induced activation of the Tor pathway is unable to inhibit Cic protein accumulation in the embryonic middle regions (de las Heras, 2006).

In the experiments described above, activation of the Tor pathway over the whole embryo did not result in an expansion of Cic downregulation. Paradoxically, activated Tor could trigger downstream targets in the middle region even though Cic was still present. These observations raise the question of whether under these circumstances Cic is still able to act as a transcriptional repressor. Alternatively, Tor signalling could impair cic activity without removing Cic protein from the nuclei. To address this issue, the contribution of cic function was analyzed in embryos from tor constitutive mutants (de las Heras, 2006).

The strong transformations associated with the ectopic activation of the Tor pathway due to torD4021 mutations and tubGAL4 driven expression of tsl make it difficult to assess the operational state of the Cic repressor under these circumstances. To overcome this difficulty use was made of the weaker torRL3 constitutive mutation and cuticular transformations, which are more sensitive to small changes in the expression of tor targets genes than what can be visualized by whole mount in situs, were scored. Besides, in the following experiments the torRL3 genotype was examined in a trunk (trk) background to eliminate ligand-induced activation. On its own, a single copy of torRL3 gives rise to a very mild phenotype, in which occasionally one abdominal segment is deleted. In contrast, removing just one copy of the cic gene does not affect the embryonic pattern. However, a single copy of the torRL3 mutation combined with the removal of just one copy of the cic gene gives rise to prominent transformations; embryos from such females display variable phenotypes but in every case they show major deletions of the embryonic segments. Accordingly, there is an expansion of the domain of tll expression, which also in that case overlaps with the domain where Cic accumulates. In this situation, whether nuclear Cic protein is still functional can be assessed by removing the remaining copy of the cic gene and comparing the two phenotypes. Indeed, embryos from trk torRL3/+; cic/cic have a much stronger phenotype that those from trk torRL3/+; cic/+. Therefore, the Cic protein present in trk torRL3/+; cic/+ embryos is still at least in part functional implying that the torRL3 mutation is able to trigger tll activation without eliminating all cic repression activity (de las Heras, 2006).

What mechanisms are activated by Tor signalling that could bypass the need for Cic downregulation to activate terminal target genes? It has been suggested that the Stat92E transcription factor plays a role as a mediator of Tor signalling elicited by a Tor constitutive mutant receptor, but not in Tor signalling promoted by ligand-dependent activation of the receptor at the poles. The role of Stat92E was assessed in the tor constitutive mutant background. A reduction was found in the transformations associated with the trk torRL3/+; cic/+ genotype by removing a single copy of the stat92E gene. Whether this could also apply in the case of ectopic activation of the Tor pathway through ligand binding was analyzed; also in this case it was found that there is a reduction of the strength of the phenotype. In this case, however, the reduction is smaller, which could be due to the fact that the original transformation generated by the tubGAL4/UAStsl combination is much stronger and/or to a weaker involvement of stat92E in ligand-induced Tor signalling. Regardless, the results suggest that there is no fundamental difference in the role of stat92E between ligand-induced or constitutive activation of the Tor receptor. In support of this conclusion there is the recent observation that Stat92E is specifically phosphorylated at the poles by ligand-induced Tor signalling. Therefore, similarly to what was observed in the embryonic middle regions, it is proposed that Tor could also induce tll activation in the poles, and this occurs by a Cic downregulation-independent mechanism via stat92E. Altogether these results suggest that Tor signalling could normally trigger tll expression at the poles of wild-type embryos by two kinds of regulatory mechanisms, relief of cic repression and positive activation of tll expression. The positive effect of Tor signalling on tll expression could have been obscured by the fact that there is also a still unidentified Tor-independent activator, since terminal fate is specified in embryos lacking both Tor signalling and Cic repression. Accordingly, it has to be noted that stat92E mutants suppress ectopic activation of tll in the middle embryonic regions but not tll activation at the poles, which suggests that the role of stat92E on Tor signalling could be somehow redundant at the poles but absolutely required when Tor signalling is triggered in the embryonic middle regions (de las Heras, 2006).

The following conclusions can be drawn from these results. First, while activation of the Tor pathway at the embryonic poles downregulates Cic, Tor signalling appears to be necessary but not sufficient to eliminate Cic protein, as it can do so only at the embryonic poles. In this regard, it has to be noted that recent results indicate that the posterior maternal system can also affect Cic downregulation. Second, impairment of Cic repressor function is not an absolute requirement for tll expression, since tll can be expressed in situations where Cic repressor is still functional. In this regard, tll expression appears to be the result of a balance between repressor and activator factors and Cic repression might be overcome provided that activation is enhanced. And finally, there are differences between the terminal and the central embryonic regions that are independent of Tor signalling, as judged by the spatially restricted capacity of the Tor pathway to inhibit Cic accumulation and by the apparently distinct regional redundancy of stat92E function in Tor-dependent patterning. These results suggest that the Tor signalling pathway is not the only system that establishes a difference between the terminal and the central regions of the Drosophila embryo (de las Heras, 2006).

Drosophila follicle cells are patterned by multiple levels of Notch signaling and antagonism between the Notch and JAK/STAT pathways

The specification of polar, main-body and stalk follicle cells in the germarium of the Drosophila ovary plays a key role in the formation of the egg chamber and polarisation of its anterior-posterior axis. High levels of Notch pathway activation, resulting from a germline Delta ligand signal, induce polar cells. This study shows that low Notch activation levels, originating from Delta expressed in the polar follicle cells, are required for stalk formation. The metalloprotease Kuzbanian-like, which cleaves and inactivates Delta, reduces the level of Delta signaling between follicle cells, thereby limiting the size of the stalk. Notch activation is required in a continuous fashion to maintain the polar and stalk cell fates. Mutual antagonism between the Notch and JAK/STAT signaling pathways provides a crucial facet of follicle cell patterning. Notch signaling in polar and main-body follicle cells inhibits JAK/STAT signaling by preventing STAT nuclear translocation, thereby restricting the influence of this pathway to stalk cells. Conversely, signaling by JAK/STAT reduces Notch signaling in the stalk. Thus, variations in the levels of Notch pathway activation, coupled with a continuous balance between the Notch and JAK/STAT pathways, specify the identity of the different follicle cell types and help establish the polarity of the egg chamber (Assa-Kunik, 2007).

Stalk formation between adjacent egg chambers is induced by directional signaling from the anterior polar cells of the older (posterior) egg chamber. Signaling via the JAK/STAT pathway provides an essential component of this process, but various indications have suggested a role for the Notch pathway as well. To verify the requirement for Notch signaling in the induction of stalk cells, follicle cell clones were generated that are mutant for Dl, the primary Notch ligand during oogenesis. Despite the proper specification of polar cells, egg chambers containing Dl follicle cell clones often failed to form a stalk on their anterior side, and as a result fused to the neighboring egg chamber. Such clones always encompassed follicle cells at the anterior portion of the egg chamber, indicating that Dl produced by anterior follicle cells is necessary to form an anterior stalk. However, the stalk positioned on the posterior side of these egg chambers was normal, even when the Dl clone surrounded the entire germline cyst. This is in keeping with the observation that posterior follicle cells do not contribute to stalk formation (Assa-Kunik, 2007).

In order to determine which cells of the anterior follicle cell population provide the signal for stalk formation, small anterior Dl-mutant follicle cell clones were analyzed. In all cases where Dl-mutant clones led to loss of the stalk, the anterior polar cells were included in the mutant clone, suggesting that these cells are the source of Dl signaling. A few instances were observed in which an anterior stalk formed even though both polar cells were mutant for Dl. Since the polar cell population defined by expression of Fng is initially larger, and is reduced to two cells by programmed cell death, this most probably resulted from the presence of wild-type Dl-expressing polar cells that provided the signal prior to their apoptosis. No phenotype was observed when the stalk cells themselves were mutant for Dl, indicating that Dl production by the stalk cells is not required for stalk specification (Assa-Kunik, 2007).

These results indicate that Notch signaling is required for at least two processes of follicle cell patterning during early oogenesis: specification of polar cells induced by Dl from the germ line and induction of stalk by Dl provided by anterior polar cells. How are these two signals distinguished, and what is the temporal relationship between them (Assa-Kunik, 2007)?

The universal Notch transcriptional reporter Gbe+Su(H)m8-lacZ was to follow the activation profile of Notch signaling throughout oogenesis. During stages 2-3 of oogenesis, variations were observed in the strength of Notch pathway activation within different anterior follicle cell types. Activation of Notch was observed in the polar cells, but no activation could be detected at this resolution in the stalk cells. These observations indicate that the level of Notch activation in the stalk cells is significantly lower than in the polar cells. Utilization of a second Notch reporter (m7-lacZ) identified essentially the same pattern. However, as this reporter appears to be more sensitive than Gbe+Su(H)m8-lacZ, low levels of Notch activation in the stalk cells at early stages could also be observed (Assa-Kunik, 2007).

Expression of Fng specifically in the future polar cells, provides a possible basis for the enhanced magnitude of Notch signaling in these cells. Polar cells are also part of the follicle cell population adjacent to the germline nurse cell complex, in which overall levels of Dl protein appear relatively high. However, the fraction of Dl localized to the nurse-cell membranes is difficult to quantify, preventing attribution with confidence the differences in signaling levels during early oogenesis to this parameter (Assa-Kunik, 2007).

To define the temporal sequence of polar and stalk cell induction, the expression of specific markers was followed for each cell type. Polar and stalk cell markers are first detected in stage 1 egg chambers (region 3 of the germarium). Markers of both cell types could be detected simultaneously in some egg chambers, where they were aligned as broad adjacent bands, with the polar cell marker always positioned towards the posterior. All other egg chambers at this stage displayed expression of the polar cell marker alone. These observations imply that polar cells are induced first, and, in agreement with the genetic evidence, are properly positioned to signal and induce stalk cell formation at the anterior end of the egg chamber (Assa-Kunik, 2007).

Taken together, these data suggest that distinctions in both the strength of signaling via the Notch pathway and the temporal sequence of pathway activation contribute to distinct cell-fate outcomes within the population of anterior follicle cells during early Drosophila oogenesis (Assa-Kunik, 2007).

It has been shown that the metalloprotease Kuzbanian-like (Kul) cleaves Dl in a cell-autonomous manner, leading to its downregulation. Modulation of Kul levels therefore provides a sensitive tool for manipulating Dl signaling activity in vivo. Attempts were made to determine whether Kul functions within follicle cells during early oogenesis. The expression pattern of Kul during oogenesis was monitored by fluorescent RNA in situ hybridization. Whereas Kul RNA was not detected in the germ line, prominent expression was observed in follicle cells, up to stage 3 (Assa-Kunik, 2007).

Kul levels can be effectively reduced by expression of a specific UAS-dsRNA construct. Since expression of Kul dsRNA by various GAL4 drivers resulted in lethality, expression of this construct was restricted to adult stages through the use of a temperature-sensitive GAL80 inhibitor system. This approach was used throughout the study to enable expression of various UAS-based transgenes during oogenesis. The GAL80ts system was used in conjunction with the neur-GAL4 driver (A101-GAL4) to specifically express Kul dsRNA in polar cells, and assess the effect of Kul on Notch signaling in early follicle cells. Notch transcriptional reporter activity was examined in these egg chambers, and the position and intensity of staining compared with wild-type egg chambers that were processed under identical conditions. Following expression of dskul in polar cells, Notch reporter levels were significantly elevated, both in the germarium and in stage 1-3 egg chambers. These observations indicate that Kul acts as an attenuator of Dl signaling in early-stage follicle cells. Interference with Kul function in this fashion thus provides a means to address the significance of follicle cell Dl levels for proper stalk cell induction. Indeed, expression of dskul in the polar cells led to a significant increase in stalk-cell number, from an average of 7.0 to 10.3 cells per stalk (Assa-Kunik, 2007).

These results indicate that the size of the stalk is highly sensitive to the amount of Dl signaling between follicle cells. This is in agreement with previous experiments, in which the size of the stalk was dramatically increased following a mild hyperactivation of Notch. Consistent with these data, ovaries from heterozygous Dl females have a reduced number of stalk cells, underscoring the sensitivity of the system to levels of Dl signaling (Assa-Kunik, 2007).

To determine whether stalk cells remain sensitive to Notch pathway signaling following their differentiation, dskul was expressed in the stalk cells themselves, using the 24B-GAL4 stalk cell-specific driver, and an increase was observed in the number of stalk cells to an average of 9.0. Kul thus attenuates Dl levels even after the stalk is formed, implying that stalk-cell number is regulated by Dl signaling from both polar cells and the stalk cells themselves. In a converse experiment, Notch signaling was reduced or eliminated from the stalk cells. Expression of dsNotch, or of a dominant-negative Notch construct, by the 24B-GAL4 stalk cell-specific driver led to the disappearance of the stalk marker Big brain (Bib). Thus, persistent, low level activation of Notch is required to maintain stalk cell fate. The low levels of Dl employed for this purpose are presented initially at the polar cell-stalk cell boundary, but as the stalk becomes elongated they might be displayed by neighboring stalk cells (Assa-Kunik, 2007).

Dl is required for establishment and maintenance of the stalk cell fate. The sensitivity of stalk size to the levels of Dl provided by the stalk cells themselves suggests that Dl also affects stalk cell proliferation or survival. To examine this possibility, the anti-apoptotic protein p35 was expressed in both polar and stalk cells using the 109-53-GAL4 driver. A greater abundance was observed of cells not properly arranged into a one-cell-wide stalk. This suggests that excess stalk cells are normally eliminated by apoptosis, and would support a model in which Dl is required for stalk cell survival, as well as stalk differentiation (Assa-Kunik, 2007).

The above observations suggest that different levels of Notch signaling determine the final fate of cells from within the polar/stalk precursor population - a strong germline signal induces the polar cell fate, whereas a weaker follicle cell signal induces the stalk. As an additional test of this model, the effects were examined of strongly elevating the Notch follicle cell signal, by overexpression of Dl specifically in polar cells. Overexpression of Dl using polar cell-specific GAL4 drivers had dramatic effects on anterior follicle cell fate and tissue morphology. Significantly, this alteration in Notch signaling resulted in an excess of polar cells. Supernumerary polar cells formed primarily at the expense of stalk cells, as evidenced by their expression of both polar and stalk cell markers, and as fusions between adjacent egg chambers. Some of the excess polar cells expressed the main-body follicle cell marker Eya, suggesting that the elevated Dl signal was capable of recruiting polar cells from this neighboring population as well. Furthermore, overexpression of Dl within the stalk cells themselves, using the 24B-GAL4 driver, induced the expression of a polar cell marker within the stalk (Assa-Kunik, 2007).

The JAK/STAT ligand Upd is expressed in polar cells, and like Dl is required for induction of the stalk. The binding of Upd to its receptor, Domeless, activates the JAK kinase Hopscotch, which then phosphorylates STAT (Stat92E) to induce its translocation into the nucleus, where it regulates transcription. The observed shift from stalk to polar cell fate upon overexpression of Dl implies that Notch activation has the capacity to antagonize JAK/STAT signaling. To explore this issue further, the Notch m7-lacZ and the STAT92E-GFP transcriptional reporters were used to simultaneously monitor Notch and JAK/STAT signaling in the ovary. Two distinct distributions of transcriptional activation wee observed. During early stages of oogenesis, Upd signaling from the polar cells is capable of inducing strong STAT activation in stalk cells, but fails to elicit activation in either the polar cells themselves, or in the neighboring main-body follicle cells. At later stages, however, follicle cell populations, including main-body and border cells, exhibited concomitant Notch and STAT activation. This analysis highlights a continuous requirement for both the Notch and JAK/STAT signaling pathways during follicle cell differentiation, throughout oogenesis. As predicted, Notch signaling can antagonize STAT activation in follicle cells, but this capacity is spatially and temporally restricted (Assa-Kunik, 2007).

The antagonistic effect of Notch signaling in early egg chambers was further pursued by following nuclear localization of STAT as an assay for JAK/STAT pathway activity. Nuclear STAT staining was pronounced throughout the stalk separating the germarium from the polar cells of the adjacent, posterior egg chamber in wild-type ovaries. Consistent with the STAT92E-GFP reporter pattern, the anterior polar cells did not exhibit nuclear localization of STAT, indicating that although they produce the Upd ligand, they themselves are refractory to this signal. STAT also remained cytoplasmic in the main-body follicle cells adjacent to the polar cells (Assa-Kunik, 2007).

When Upd was overexpressed using a polar cell-specific driver, the anterior range of nuclear STAT localization was significantly increased. Consistent with this enhanced activation of JAK/STAT signaling, longer stalk-like structures were observed. In spite of the higher levels of Upd, nuclear STAT was still only seen in cells anterior to the source, including the future stalk and posterior polar cells of the adjacent younger egg chamber. By contrast, JAK/STAT signaling in the anterior polar cells themselves, and in the neighboring main-body follicle cells, was not activated (Assa-Kunik, 2007).

In light of the suggestion of an antagonistic relationship between Notch and JAK/STAT signaling, one possible explanation for failure of the polar and main-body follicle cells to respond to Upd is the higher level of Notch activation in these cells. To test this hypothesis, Notch-mutant clones were generated in the main-body follicle cells, and the nuclear localization of STAT was monitored. Elimination of Notch in these cells led to nuclear accumulation of STAT in mutant cells situated within four cell-diameters of the polar cells. No nuclear localization was detected in Notch-mutant cells situated further away, presumably owing to restricted diffusion of Upd from the polar cells (Assa-Kunik, 2007).

These results indicate that moderate to high levels of Notch activation inhibit JAK/STAT signaling, and that this inhibition acts before the nuclear translocation of activated STAT. Furthermore, the results demonstrate that correct specification of the polar, main-body and stalk follicle cells depends on crosstalk between distinct levels of Notch activity and the JAK/STAT pathway. High Notch activation induces polar cell fate, including expression of Upd, and antagonizes JAK/STAT signaling. Intermediate levels of Notch activation in the main-body follicle cells antagonize JAK/STAT signaling, without inducing expression of Upd. Finally, low levels of Notch activation synergize with Upd signaling to induce stalk cell fate and to regulate the size of the stalk (Assa-Kunik, 2007).

Maintaining the moderate level of Notch signaling that is induced by Dl expressed in the follicle cells, is essential for producing a stalk with the correct cell number, and this is achieved at least in part by the activity of Kul in the signal-sending cells. The possibility that Notch signaling is also attenuated in the signal-receiving cells by the activity of JAK/STAT was examined by monitoring oogenesis in hopscotch (hop) hypomorphs, in which JAK/STAT signaling is compromised. Stalks formed at early stages of oogenesis in hopmv1/GA32 females, and the oocyte moved to the posterior of the egg chamber as in wild type. However, stalk cells failed to intercalate, and the stalk consisted of two rows of cells linked by adherens junctions. At later stages, the stalk collapsed and, as was observed for strong hop alleles, the stalk cells reverted to the polar cell fate. These cells now clustered at the anterior corners of the older cyst, whilst remaining in contact with the oocyte of the younger egg chamber (Assa-Kunik, 2007).

The conversion of stalk cells to polar cells when the level of JAK/STAT signaling was compromised suggests that Notch signaling in the stalk cells is normally attenuated by the JAK/STAT pathway. When this inhibition is relieved in hop hypomorphs, the increase in the level of Notch signaling leads to their conversion to polar cells. Since the entire polar/stalk precursor cell population expresses Fng, even activation by the lower levels of Dl produced by these cells may be sufficient to give rise to polar cells, in the absence of repression by JAK/STAT (Assa-Kunik, 2007).

Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut

Cells in intestinal epithelia turn over rapidly due to damage from digestion and toxins produced by the enteric microbiota. Gut homeostasis is maintained by intestinal stem cells (ISCs) that divide to replenish the intestinal epithelium, but little is known about how ISC division and differentiation are coordinated with epithelial cell loss. This study shows that when enterocytes (ECs) in the Drosophila midgut are subjected to apoptosis, enteric infection, or JNK-mediated stress signaling, they produce cytokines (Upd, Upd2, and Upd3) that activate Jak/Stat signaling in ISCs, promoting their rapid division. Upd/Jak/Stat activity also promotes progenitor cell differentiation, in part by stimulating Delta/Notch signaling, and is required for differentiation in both normal and regenerating midguts. Hence, cytokine-mediated feedback enables stem cells to replace spent progeny as they are lost, thereby establishing gut homeostasis (Jiang, 2009).

Rates of cell turnover in the intestine are likely to be in constant flux in response to varying stress from digestive acids and enzymes, chemical and mechanical damage, and toxins produced by both commensal and infectious enteric microbiota. This study shows feedback from differentiated cells in the gut epithelium to stem and progenitor cells is a key feature of this system. Genetically directed enterocyte ablation, JNK-mediated stress signaling, or enteric infection with P. entomophila all disrupt the Drosophila midgut epithelium and induce compensatory ISC division and differentiation, allowing a compromised intestine to rapidly regenerate. Other recent reports note a similar regenerative response following three additional types of stress: detergent (DSS)-induced damage (Amcheslavsky, 2009), oxidative stress by paraquat (Biteau, 2008), and enteric infection with another less pathogenic bacterium, Erwinia carotovora (Buchon, 2009a). Remarkably, the fly midgut can recover not only from damage, but also from severe induced hyperplasia, such as that caused by ectopic cytokine (Upd) production. Thus, this system is robustly homeostatic (Jiang, 2009).

Each of the three stress conditions that were studied induced all three Upd cytokines, and genetic tests showed that Upd/Jak/Stat signaling was both required and sufficient for compensatory ISC division and gut renewal. Although JNK signaling was also activated in each instance, it was not required for the stem cell response to either EC apoptosis or infection, implying that other mechanisms can sense EC loss and trigger the cytokine and proliferative responses. JNK signaling may be important in specific contexts that were not tested, such as following oxidative stress, which occurs during some infections, activates JNK, and stimulates midgut DNA replication (Biteau, 2008; Jiang, 2009).

Following P. entomophila infection, virtually the entire midgut epithelium could be renewed in just 2-3 days, whereas comparable renewal took more than 3 weeks in healthy flies. Despite this radical acceleration of cell turnover, the relative proportions of the different gut cell types generated (ISC, EB, EE, and EC remained similar to those in midguts undergoing slow, basal turnover. These data suggested that de-differentiation did not occur, and little evidence was obtained of symmetric stem divisions (stem cell duplication) induced by enteric infection. Hence, it is suggested that asymmetric stem cell divisions as described for healthy animals, together with normal Delta/Notch-mediated differentiation, remain the rule during infection-induced regeneration. The results obtained using Reaper to ablate ECs are also consistent with this conclusion, as are those from detergent-induced midgut regeneration (Jiang, 2009).

Unlike infection, direct genetic activation of JNK or Jak/Stat signaling promoted large increases not only in midgut mitoses, but also in the pool of cells expressing the stem cell marker Delta. Cell type marker analysis discounted de-differentiation of EEs or ECs as the source of the new stem cells, but the reactivation of EBs as stem cells seems possible. For technical reasons, no tests were performed to whether stem cell duplications occur in response to Jak/Stat or JNK signaling, and this also remains possible. The ability of hyperplastic midguts to recover to normal following the silencing of cytokine expression suggests that excess stem cells are just as readily eliminated as they are generated. Further studies are required to understand how midgut stem cell pools can be expanded and contracted according to need (Jiang, 2009).

How the Upds are induced in the midgut by JNK, apoptosis, or infection remains an open question. Paradoxically, ISC divisions triggered by Reaper required EC apoptosis but not JNK activity, whereas ISC divisions triggered by JNK did not require apoptosis, and ISC divisions triggered by infection required neither apoptosis nor JNK activity. These incongruent results suggest that different varieties of gut epithelial stress may induce Upd cytokine expression via distinct mechanisms. In the case of EC ablation, physical loss of cells from the epithelium might drive the cytokine response. In the case of infection, it is expected the critical inputs to be the Toll and/or IMD innate immunity pathways, which signal via NF-kappaB transcription factors. Functional tests, however, indicated that the Toll and IMD pathways are required for neither Upd/Jak/Stat induction nor compensatory ISC mitoses following enteric infection by gram-negative bacteria. Hence, other unknown inputs likely trigger the Upd cytokine response to infection (Jiang, 2009).

Is the cytokine response to infection relevant to normal midgut homeostasis? This seems likely. Low levels of Upd3 expression and Stat signaling are observed in healthy animals, and midgut homeostasis required the IL-6R-like receptor Dome and Stat92E even without infection. Wild Drosophila subsist on a diet of rotting fruit, which is a good source of protein because it is teeming with bacteria and fungi. Given such a diet it seems likely that midgut cytokine signaling is constantly modulated by ever-present factors that impose dietary stress -- food composition and commensal microbiota -- even in healthy animals (Jiang, 2009).

Although studies in mammals have yet to unravel the details of a feedback mechanism underlying gut homeostasis, experimental evidence implies that such a mechanism exists and involves Cytokine/Jak/Stat signaling. As in Drosophila, damage to the mouse intestinal epithelium caused by detergents or infection can stimulate cell proliferation in the crypts, where stem and transient amplifying cells reside. In a mouse model of detergent (DSS)-induced colitis, colon epithelial damage caused by DSS allows exposure to commensal microbes, activating NF-kappaB signaling in resident macrophage-like Dentritic cells. These cells respond by expressing inflammation-associated cytokines, one of which (IL-6) activates Stat3 and is believed to promote cell proliferation and regeneration. Consistent with a functional role for Jak/Stat, disruption of the Stat inhibitor SOCS3 in the mouse gut increased the proliferative response to DSS and also increased DSS-associated colon tumorigenesis. Also pertinent is the presence of high levels of phospho-Stat3 in a majority of colon cancers, where it correlates with adverse outcome, and the observation that IL-6 can promote the growth of colon cancer cells, which are thought to derive from ISCs or transient amplifying cells. Increased colon cancer incidence is associated with gut inflammatory syndromes, such as inflammatory bowel disease (IBD) and Crohn's disease, which are likely to involve enhanced cytokine signaling. Whether cytokines mediate gut epithelial turnover in healthy people or only during inflammation is presently unclear, but it nevertheless seems likely that the mitogenic role of IL-6-like cytokines and Jak/Stat signaling in the intestine is conserved from insects to humans (Jiang, 2009).

The connection to inflammation suggests that these findings may also be relevant to the activity of nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, and celecoxib, as suppressors of colorectal carcinogenesis. These drugs target the cyclooxygenase activity of prostaglandin H synthases (PGHS, COX), which are rate-limiting for production of prostaglandin E2, a short-range lipid signal that promotes inflammation, wound healing, cell invasion, angiogenesis, and proliferation. Notably, COX-2 has been characterized as an immediate early gene that can be induced by signals associated with infection and inflammation, including the proinflammatory cytokines IL-1beta and IL-6, which activate NF-kappaB and STAT3, respectively. Whether prostaglandins mediate the effects of Jak/Stat signaling in the fly midgut remains to be tested, but insects do produce prostaglandins, and Drosophila has a functional COX homolog, pxt, whose activity can be suppressed by NSAIDs (Jiang, 2009).

Effects of Mutation or Deletion

STAT mutants show a reduced viability and exhibit patterning defects that include formation of ectopic wings and abnormalities in segmentation [Images]. There is also a maternal-effect segmentation phenotype. Paternally rescued embryos show a consistant deletion of the fifth abdominal segment and additional defects in thoracic segments, and the fourth abdominal segment (Hou, 1996 and Yan, 1996a).

A dominant negative mutation, which results in a truncated Marelle protein, exhibits patterning defects similar to those seen in mutants of the epidermal growth factor pathway. Specifically, adults exhibit partial ectopic wing vein formation in the posterior wing compartment. Abormalities in embryonic and adult segmentation and in tracheal development are also observed. hopscotch and dominant negative marelle mutations can partially compensate for each other genetically, and hop overexpression can increase marelle transcriptional activity in vitro, indicating that the gene products act in a common regulatory pathway (Yan, 1996b).

The Jak (Janus) family of nonreceptor tyrosine kinases plays a critical role in cytokinesignal transduction pathways. In Drosophila, the dominant hopTum-l mutation in the Hop Jak kinase causes leukemia-like and other developmental defects. The HopTum-l protein might be a hyperactive kinase. The new dominant mutation hopT42, causes abnormalities that are similar to but more extreme than those caused by hopTum-l. HopT42 contains a glutamic acid-to-lysine substitution at amino acid residue 695 (E695K). This residue occurs in the JH2 (kinase-like) domain and is conserved among all Jak family members. HopTum-l and HopT42 both hyperphosphorylate and hyperactivate D-Stat when overexpressed in Drosophila cells. The hopT42 phenotype is partially rescued by a reduction of wild-type D-stat activity. Generation of the corresponding E695K mutation in murine Jak2 results in increased autophosphorylation and increased activation of Stat5 in COS cells. These results demonstrate that the mutant Hop proteins do indeed have increased tyrosine kinase activity, that the mutations hyperactivate the Hop-D-Stat pathway, and that Drosophila is a relevant system for the functional dissection of mammalian Jak-Stat pathways. A model is presented for the role of the Hop-D-Stat pathway in Drosophila hematopoiesis (Luo, 1997).

Overactivation of receptor tyrosine kinases (RTKs) has been linked to tumorigenesis. To understand how a hyperactivated RTK functions differently from wild-type RTK, a genome-wide systematic survey was conducted for genes that are required for signaling by a gain-of-function mutant Drosophila RTK Torso (Tor). Chromosomal deficiencies were screened for suppression of a gain-of-function mutation tor (torGOF); this screen led to the identification of 26 genomic regions that, when in half dosage, suppress the defects caused by torGOF. Testing of candidate genes in these regions revealed many genes known to be involved in Tor signaling (such as those encoding the Ras-MAPK cassette, adaptor and structural molecules of RTK signaling, and downstream target genes of Tor), confirming the specificity of this genetic screen. Importantly, this screen also identified components of the TGFß (Dpp) and JAK/STAT pathways as being required for TorGOF signaling. Specifically, it was found that reducing the dosage of thickveins (tkv), Mothers against dpp (Mad), or STAT92E (aka marelle), respectively, suppress torGOF phenotypes. Furthermore, it has been demonstrated that in torGOF embryos, dpp is ectopically expressed and thus may contribute to the patterning defects. These results demonstrate an essential requirement of noncanonical signaling pathways for a persistently activated RTK to cause pathological defects in an organism (Lia, 2003).

The JAK/STAT pathway and oogenesis

The JAK/STAT signaling pathway, renowned for its effects on cell proliferation and survival, is constitutively active in various human cancers, including ovarian. JAK and STAT are required to convert the border cells in the Drosophila ovary from stationary, epithelial cells to migratory, invasive cells. The ligand for this pathway, Unpaired (Upd), is expressed by two central cells within the migratory cell cluster. Mutations in upd or jak cause defects in migration and a reduction in the number of cells recruited to the cluster. Ectopic expression of either Upd or JAK is sufficient to induce extra epithelial cells to migrate. Thus, a localized signal activates the JAK/STAT pathway in neighboring epithelial cells, causing them to become invasive (Silver, 2001).

Polar cells emit a short-range signal that causes adjacent follicle cells to surround them and acquire the ability to migrate through the nurse cells. The results reported here suggest that Upd is the major signal secreted by the polar cells that both recruits adjacent follicle cells into the cluster and causes them to become migratory. Both of these functions are carried out by activation of JAK and STAT in the neighboring follicle cells. Signaling through this pathway is necessary, both for recruitment of border cells to the cluster and for motility once the cells are recruited. This is based on the observations that in the majority of mutant egg chambers, border cell clusters contain fewer than the normal number of cells, and that even clusters with normal numbers of cells fail to migrate normally (Silver, 2001).

It is worth noting that while some migration is observed in JAK and STAT border cell mutants, the loss of Upd in the polar cells completely prevents migration. This may reflect greater perdurance of JAK and STAT proteins in the mosaic clones, compared to Upd, if Upd is normally present at lower levels and/or is more labile. Alternatively, these differences may imply that in addition to its activation of JAK and STAT, Upd can activate other signaling pathways (Silver, 2001).

Activation of the JAK/STAT pathway is not only necessary but is also sufficient to convert epithelial follicle cells to become migratory. Numerous extra border cells were observed following overexpression of upd, hop, or hopTum, many of which invaded the nurse cell cluster. These extra cells did not result from excess proliferation because follicle cells cease dividing at stage 6, at least 12 hr prior to border cell differentiation. Furthermore, no difference in phospho-histone H3 antibody labeling was observed in cells overexpressing upd or in cells lacking stat, ehrn compared to wild-type. Moreover, it was possible to obtain large clones lacking upd, hop, or stat activity, indicating that homozygous mutant cells retain the ability to divide numerous times. Thus, activation of the JAK/STAT pathway leads to border cell specification and migration, without effects on proliferation. In addition, while extra follicle cells could become migratory as a secondary consequence of ectopic polar cell formation, activation of the JAK/STAT pathway results in the appearance of additional migratory cells in the absence of extra polar cells (Silver, 2001).

The question of whether signaling through this pathway might be sufficient to cause epithelial cells to become invasive was addressed ectopically expressing Upd, Hopscotch (Hop), or the constitutively active form of Hop, HopTum1, using the GAL4/UAS expression system. In this method, the yeast transcriptional activator GAL4 is expressed under the control of a cell type-specific enhancer, in this case slbo-GAL4 and c306-GAL4. In stage 9 egg chambers, slbo-GAL4 induces expression of genes that are under the control of the yeast upstream activating sequence (UAS) in approximately 20 anterior follicle cells, a subset of which normally become the border cells. This is nearly identical to the ß-gal expression from an enhancer trap insertion into the slow border cells (slbo) locus, even though Slbo protein expression is normally restricted to the border cells at stage 9. C306-GAL4 drives expression in a larger number of anterior, as well as posterior, follicle cells, compared to slbo-GAL4. C306-GAL4 also begins expressing earlier in oogenesis than slbo-GAL4 (Silver, 2001).

Egg chambers from c306-GAL4; UAS-hop females exhibit a dramatic increase in the number of border cells compared to wild-type. Up to 90 slbo expressing cells are produced, about 60 of which invade the nurse cell cluster and 20 of which have completed migration by early stage 10. Similar, though less dramatic, phenotypes are observed when the constitutively activated kinase is expressed with either slbo-GAL4 or c306-GAL4. Likewise, slbo-GAL4;UAS-upd and c306-GAL4;UAS-upd females contain numerous extra slbo-expressing cells compared to wild-type, in the absence of extra polar cells. This is in marked contrast to the effect of excessive Hedgehog pathway signaling, which causes ectopic border cells to form as a secondary consequence of ectopic polar cell specification. Overexpression of upd does not appear to cause excess cell proliferation, sinces no difference was detected in phospho-histone H3 antibody labeling, which marks mitotic cells, as compared to wild-type (Silver, 2001).

Some of the extra border cells migrate as single cells, whereas others form multiple small clusters, and still others form one large cluster. The ability of the cells to migrate varies according to which protein is being expressed as well as with the timing and level of expression. High levels of ectopic Upd result in egg chambers in which both normal and extra border cells frequently fail to migrate, whereas high levels of wild-type Hop produce the most migratory cells. Thus, ectopic activation of the JAK/STAT pathway is sufficient to cause extra epithelial follicle cells to invade the nurse cell cluster (Silver, 2001).

In order to gain further insight into the mechanism by which STAT regulates border cell migration, the expression of a number of proteins that are highly expressed in border cells was examined, some of which are also required for migration. The first gene identified as playing a critical role in border cell migration was slow border cells (slbo). slbo encodes Drosophila C/EBP, a basic region/leucine zipper transcription factor. Slbo protein expression is undetectable in stat mutant border cells, which were identified using a positive clone marking system known as MARCM. This result was confirmed by examining several additional proteins, the expression of which is reduced in slbo mutant border cells. In wild-type stage 8 and 9 egg chambers, FAK expression is upregulated in migratory border cells. Border cells that lack stat exhibit reduced levels of FAK. In wild-type stage 9 egg chambers, DE-cadherin (Shotgun) is enriched throughout the border cell cluster and is expressed to the highest level in the polar cells. Stat mutant border cells exhibit reduced DE-cadherin expression compared to wild-type border cells of the same cluster. The polar cells, though mutant, do not show reduced DE-cadherin staining, which is also true of slbo mutants. Additional downstream targets of slbo, including PZ6356 and zinc finger transcription factor jing, are also reduced in stat mosaic clones. Thus, even the few mutant cells that are recruited to the cluster fail to express many border cell proteins required for migration. The effect is specific because expression of Taiman, a protein that is required for border cell migration but is independent of the slbo pathway, was not altered. Mosaic clusters containing a mixture of wild-type and mutant cells show variable migration defects. On average, the extent of migration is proportional to the number of wild-type cells in the cluster (Silver, 2001).

Egg chambers from females heterozygous for any of the stat alleles have a semi-dominant border cell migration phenotype. Advantage was taken of this slight haploinsufficiency to test for dominant genetic interactions with other genes required for border cell migration. Dominant genetic interactions were observed with slbo, hop, and upd alleles. A mutation in the gene coding for DE-cadherin, shotgun, also exhibited a dominant interaction with stat. These interactions appeared to be specific, since stat does not interact with other known border cell migration genes, such as tai, jing, or PZ6356 (Silver, 2001).

Recently, a candidate transmembrane receptor for Upd has been identified. Mutation of this gene, which is named domeless, causes embryonic phenotypes that are indistinguishable from those of upd, hop, and stat mutants. In addition, the gene encodes a protein with sequence homology to mammalian cytokine receptors that mediate JAK/STAT signaling. A dominant negative form of Domeless has been generated, which mimics the loss-of-function phenotype (Brown, 2001). Upon expression of the dominant negative receptor specifically in the outer border cells, using slbo-GAL4, dramatic recruitment and migration defects are observed. The average number of outer border cells in these egg chambers was 0.5 and the migration index was 2.6. These results provide further support for the proposal that Upd from the polar cells activates signaling in the surrounding epithelial cells for their recruitment to the cluster and migration (Silver, 2001).

Recently, a putative guidance factor for the border cells was reported. A protein with homology to VEGF and PDGF, known as PVF-1, accumulates in the oocyte beginning at stage 7. It is interesting to note that the receptor for this factor is expressed uniformly on the surfaces of all of the follicle cells. Thus, all of the follicle cells are exposed to the ligand, and all of the follicle cells express the receptor. However, only the border cells detach from the epithelium, invade the nurse cell cluster, and migrate. These observations raise the obvious question as to why it is that such a small subset of cells migrates toward the putative chemoattractant. The results reported here indicate that it is activation of the JAK/STAT pathway that defines the invasive population of follicle cells. Thus, in vivo exposure of epithelial cells to a chemoattractant does not appear to be sufficient to cause them to become migratory. Rather, the cells require an additional signal, which results in substantial changes in gene expression, in order to migrate in response to this factor (Silver, 2001).

STAT proteins have many biological functions, but the key downstream targets that carry out these functions are largely unknown. The mechanism by which STAT activation leads to border cell migration appears to involve activation of slbo expression and its target genes. The evidence for this is that ectopic activation of the JAK/STAT pathway results in ectopic slbo expression, and loss of STAT leads to reduction in the Slbo protein level (Silver, 2001).

The slbo locus encodes Drosophila C/EBP, a basic region-leucine zipper transcriptional regulator. It is intriguing to note that mammalian C/EBPß is expressed in ovarian carcinomas, and its expression increases dramatically with malignancy. STAT3 is constitutively active in ovarian carcinomas, and while it is not known whether STAT3 activates expression of C/EBPß in these cells, it could be that this relationship has been conserved in evolution (Silver, 2001).

In addition to Slbo, expression of each of its known target genes was reduced in stat mutant cells. The contributions of several of these target genes to cell migration is known. For example, dynamic regulation of DE-Cadherin plays a critical role in promoting migration by providing optimal adhesion with the nurse cells. Furthermore, FAK is essential for the migration of numerous mammalian cell types, while jing encodes a zinc finger transcription factor that cooperates with Slbo in regulating border cell migration (Silver, 2001).

Both loss-of-function and gain-of-function of JAK/STAT pathway activity are detrimental to border cell migration. Interestingly, this is also true for Slbo, since slbo mutants show border cell migration defects and overexpression of slbo also impedes migration. This similarity lends further support to the proposal that STAT exerts at least part of its effect on migration by regulating Slbo (Silver, 2001).

Two transcriptional regulatory pathways have been identified that control the invasive behavior of the border cells in vivo. In addition to expression of slbo and its targets, border cell migration requires a global hormonal signal in the form of ecdysone. This global hormonal signal appears to function in a slbo-independent manner, since the expression of neither slbo nor its targets is reduced when ecdysone signaling is compromised, and no genetic interaction has been observed between mutations affecting the ecdysone response and slbo or stat. Taken together with the putative guidance signal PVF-1 and the data presented here, these results indicate that border cell migration requires the integration of at least three signals. The global hormonal signal coordinates multiple events that occur at stage 9, including border cell migration, and PVF-1 contributes to the directional cue for the border cells. Finally, the local paracrine signal through JAK/STAT is necessary to define the population of cells capable of responding to the other signals by detaching from the epithelium and invading the nurse cell cluster. Of these three signals, only the signal through the JAK/STAT pathway is spatially restricted to the migratory population (Silver, 2001).

STAT proteins may regulate epithelial to mesenchymal transitions and cell migration not only in the border cells but also in mammalian cells. Consistent with this proposal, embryos homozygous for deletion of the STAT3 gene fail to gastrulate. Gastrulation in the mammal requires that some of the epithelial cells within the epiblast layer become mesenchymal and migrate through the primitive streak to form the mesoderm (Silver, 2001).

More direct evidence for a role for STAT3 in an epithelial to mesenchymal transition has been reported in a study of a tissue-specific knockout of STAT3. Loss of STAT3 in epidermal keratinocytes results in defects in re-epithelialization following wounding, where STAT3 appears to be required specifically for the migratory component of the response. JAK2 has also been found to be required for hematopoietic progenitor cell migration in response to stromal cell derived factor 1. Thus, there is evidence that JAK and STAT promote cell migration, though the mechanisms by which they do so have not been elucidated. It may be that the constitutive activation of the JAK/STAT pathway that is observed in a variety of human cancers contributes to invasiveness and mestastasis, in addition to the well established effects on survival and proliferation (Silver, 2001).

Tests were performed to see whether domeless and STAT92E interact genetically. Zygotic STAT92E homozygotes have a very mild spiracle phenotype due to the persistence of maternally expressed RNA. Despite this, the weak dome367 phenotype is strongly enhanced by STAT92E mutants, suggesting that both genes are in the same genetic pathway (Brown, 2001).

To determine the phenotype caused by eliminating domeless maternal and zygotic products, germ line clones were induced. Maternal and zygotic dome embryos have segmentation defects identical to those reported for mutations in the STAT92E and hop (JAK) mutants. Defects include the deletion of the A5 and most of the A4 denticle belts, partial or total fusion of A6 to A7, and a variable reduction of the thoracic and the A8 segments. These phenotypes are also observed in Df(1)osUE69, which deletes the ligand, upd. The segmentation defects in STAT92E, upd, and hop have been shown to be due to the abnormal expression of pair rule genes. In dome germ line clones, the expression of even-skipped is affected in stripes 3 and 5, as described for the other members of this pathway (Brown, 2001).

JAK signaling is somatically required for follicle cell differentiation in Drosophila

Janus kinase (JAK) plays several signaling roles in Drosophila oogenesis. The gene for a JAK pathway ligand, unpaired, is expressed specifically in the polar follicle cells, two pairs of somatic cells at the anterior and posterior poles of the developing egg chamber. Consistent with unpaired expression, reduced JAK pathway activity results in the fusion of developing egg chambers. A primary defect of these chambers is the expansion of the polar cell population and concomitant loss of interfollicular stalk cells. These phenotypes are enhanced by reduction of unpaired activity, suggesting that Unpaired is a necessary ligand for the JAK pathway in oogenesis. Mosaic analysis of both JAK pathway transducers, hopscotch and Stat92E, reveals that JAK signaling is specifically required in the somatic follicle cells. Moreover, JAK activity is also necessary for the initial commitment of epithelial follicle cells. Many of these roles are in common with, but distinct from, the known functions of Notch signaling in oogenesis. Consistent with these data is a model in which Notch signaling determines a pool of cells to be competent to adopt stalk or polar fate, while JAK signaling assigns specific identity within that competent pool (McGregor, 2002).

The somatic cells of the ovary consist of multiple subpopulations, each with its own function(s) in the developing egg. While the germline cyst is dividing and developing within the germarium, a monolayer of somatic cells surrounds the cyst as it moves posteriorly through the germarium. As the cyst becomes enveloped by the somatic cells, the egg chamber pinches off from the germarium, entering the vitellarium. At that time, approximately 5-8 somatic cells differentiate into stalk. These flattened, disc-shaped cells are stacked together to form the spacer between successive cysts. Stalk cells connect the anterior end of a more mature egg chamber to the posterior end of the next younger chamber. Also at that time, molecular markers can distinguish the stalk cells from the polar cells, which arise from the same precursors. The polar cells are arranged as two pairs of follicle cells, one pair at either end of each chamber near the stalk cells. While the stalk cells and polar cells cease proliferation at the end of the germarium, the remaining follicle cells, which are referred to as epithelial follicle cells, divide approximately five times to expand the pool of follicle cells. Those epithelial cells later differentiate into various subpopulations with specific functions in the vitellarium. Those subpopulations are pre-patterned with mirror image symmetry along the anterior-posterior axis of the egg. Imposed on that pre-pattern, signaling from the oocyte by the TGFalpha molecule Gurken stimulates the induction of posterior polarity on the somatic cells at the posterior end. The result is an egg with coordinated polarities of the somatic and germline cells. This coordination is essential for the proper localization of maternal determinants that pattern the resulting embryo (McGregor, 2002).

Strikingly, unpaired is expressed very specifically within the ovary. After egg chambers pinch off from the germarium, upd is restricted to the two pairs of polar cells found at the anterior and posterior tips of the egg. In the germarium, upd is expressed in a cluster of somatic cells at the posterior of region 3. Presumably these are the cells that give rise to the stalk and polar cells. Expression in the polar and border cells persists until egg maturation. In situ hybridization to Stat92E RNA reveals that Drosophila STAT is expressed in both the germarium and the vitellarium. Expression in the germarium occurs in all follicle cells in region 2a and 2b; it then begins to be restricted to terminal follicle cells in region 3. In the vitellarium, Stat92E is expressed weakly at the termini of the egg chamber, but in a broader domain than only the two polar cells. After stage 9, Stat92E is strongly expressed in the nurse cells, consistent with the maternal role of Stat92E in the segmentation of the early embryo. Moreover, weak ubiquitous expression of hop is detectable in the follicular epithelium. These data are consistent with a potential role for JAK signaling in oogenesis (McGregor, 2002).

The loss of JAK pathway function in the somatic cells of the Drosophila ovary results in the fusion of adjacent cysts and/or the mislocalization of the oocyte within a cyst. Based on molecular markers for cell identity, mutations in hop or Stat92E cause the loss of stalk cells and an increase in the number of polar cells. This shift in cell fates correlates with the fusion of adjacent cysts. An allelic series of hop mutant combinations shows a range of phenotypic severity, from occasional fusion of two adjacent chambers to complete fusion of all cysts with no morphological distinction between germarium and vitellarium. The severity of the visible phenotypes is reflective of the severity of the follicle cell fate transformations. Effects on fate range from frequent appearance of one extra polar cell in the weakest mutation to consistent appearance of a dozen or more extra polar cells in more severe alleles. Phenotypes seen in mutant clones of hop and Stat92E ovaries are similar to those seen in the heteroallelic combinations of hop mutations. By using the directed mosaic technique, clone production is limited specifically to the somatic cells, thereby demonstrating that the activity of the JAK pathway is required in the follicle cells. Mosaic analysis also demonstrates that the adoption of proper epithelial cell fates requires JAK activity (McGregor, 2002).

All follicle cell subpopulations in an egg are derived from approximately three stem cells in the germarium of each ovariole. While still in the germarium, a common pool of distinct stalk and polar cell precursors is set aside from the epithelial follicle cells. Those precursors then differentiate into either stalk or polar cells. The remaining epithelial cells are pre-patterned with mirror image symmetry along the anteroposterior axis, with three distinct subpopulations at each end. The symmetry is broken at stage 6 when Gurken in the oocyte stimulates EGF receptor in the posterior terminal cells to determine posterior polarity of the egg. The three anterior terminal cell populations then become border cells, stretched (nurse cell-associated) cells, and centripetal cells. The posterior terminal cells are essential for the reorganization of the cytoskeleton in the oocyte. Those cells send an unknown signal to the germline that stimulates the reversal of microtubular polarity in the egg which is necessary for the migration of the oocyte nucleus to the anterior and for the correct localization of polarity determinants in the egg (McGregor, 2002).

Loss of JAK pathway signaling clearly influences the terminal fate of the stalk/polar cell precursors. In heteroallelic mutant combinations of hop, the number of polar cells increases while the number of stalk cells decreases. However, the sum of stalk cells plus polar cells remains approximately the same as in wild type, indicating that loss of JAK signaling is not influencing proliferation of the precursor pool, nor is it causing recruitment of epithelial follicle cells to a polar fate. This suggests a model in which the normal function of the JAK pathway is to promote the adoption of stalk cell fate in a subset of the stalk/polar cell precursor pool. JAK pathway activation may either instruct the adoption of stalk cell fates or prevent the adoption of polar cell fate. Current data do not distinguish between these alternatives (McGregor, 2002).

A second role for JAK signaling in the follicle cells was highlighted by analysis of mosaics. In chambers of the vitellarium, the immature cell marker Fas III is rapidly downregulated in all but the polar cells. However, the epithelial follicle cells do not begin to express markers of terminal differentiation until stage 7. Indeed, these cells continue to proliferate through stage 6. Nonetheless, the loss of Fas III in the epithelial cells beginning around stage 2 suggests that the identity of these cells has already begun to change. Presumably they become preliminarily committed to an epithelial follicle cell fate. In hop or Stat92E mutant clones, younger chambers retain high levels of Fas III in all the mutant cells. In more mature egg chambers (stage 7 or later) there is a consistent lack of Fas III expansion in mutant cells. The transient nature of the increase in Fas III expression suggests that the mutant cells remain in an immature state until later stages. In this model, JAK pathway activity would be necessary for the preliminary commitment step in epithelial cell differentiation that occurs after the egg chamber pinches off from the germarium. At approximately stage 7, the normal stage for terminal differentiation, the Fas III-positive JAK mutant cells lose Fas III expression, presumably because they are cued to differentiate by another signal. The consequence of loss of JAK signaling on terminal epithelial cell fates remains to be investigated (McGregor, 2002).

Several signaling pathways have been implicated in the patterning of the follicular epithelium. The best characterized are the Notch, EGFR and Hedgehog pathways. In the earliest of these activities, strong expression of hh in the terminal filament and cap cells at the anterior tip of the germarium stimulates the proliferation of the somatic stem cells. Loss of Hh signaling results in reduced follicle cell number and consequent failure to properly encapsulate the germline cyst. Recent work has demonstrated that the normal role of Hh in the ovaries is as a somatic stem cell factor and that it is necessary for the proliferation of somatic stem cells (McGregor, 2002).

After Hh activity promotes the production of a pool of follicular precursors, the stalk/polar cell precursor pool is set aside from the epithelial cell pool. The stalk/polar cell precursor pool is distinct from the epithelial pool because it ceases to proliferate as the cyst reaches the posterior end of the germarium. The method by which the stalk/polar cell precursors are determined is not known, but it has been suggested that Notch signaling, enhanced by localized Fringe activity, may be involved in the process. Similar to JAK mutants, the loss of Notch activity causes chamber fusions that are apparently the result of a failure to produce stalk cells. But unlike JAK mutants, N pathway mutants also fail to produce polar cells. Therefore, N signaling is required for the differentiation of both polar and stalk cell fates (McGregor, 2002).

So what distinguishes stalk and polar cells from one another? JAK signaling induces the adoption of stalk cell fates in a subset of the stalk/polar cell precursors. Loss of JAK pathway activity expands polar cells at the expense of stalk cells, while ectopic activation of the pathway causes a reduction of polar cells. Therefore, it is proposed that JAK pathway activity determines the terminal fate of stalk and polar cells. However, JAK activity is limited in assigning stalk cell fates to only competent cells, that is, the stalk/polar cell precursor pool. Thus, another activity, perhaps N signaling, is necessary to induce competence for stalk and polar fates. Alternatively, N signaling may be primarily responsible for the assignment of polar cell fates. One could imagine a mechanism of lateral inhibition, already linked to N signaling in various tissues, in which all the cells of the precursor pool have N activity, but that the signal becomes limited to and maintained only in the polar cells. It may be the activity of the N pathway that then drives stable expression of upd and allows the induction of stalk cell fates in neighboring cells (McGregor, 2002).

While polar and stalk cell fates are adopted as chambers exit the germarium, differentiation of the epithelial follicle cell fates is not obvious until later. At approximately stage 7, epithelial follicle cells express markers for each of the terminal identities with a clear anterior-posterior orientation. But in the absence of Grk/EGFR signaling at the posterior, a symmetrical mirror image pattern of three terminal populations of epithelial fates at each end is revealed. In wild-type ovaries, up to approximately stage 6, the oocyte signals to the overlying posterior follicle cells through Gurken. The terminal follicle cells that receive the Grk signal are induced to become posterior follicle cells. The resulting posterior follicle cells then signal to the oocyte to stimulate a cytoskeletal rearrangement. The resulting microtubular polarity drives the migration of the oocyte nucleus from the posterior to the anterior and establishes the AP axis that allows the sequestration of anterior and posterior maternal products to their respective poles. The signal from the soma for polarization of the oocyte microtubules is not yet known (McGregor, 2002).

When the developing cyst exits the germarium, there is a distinct change in the epithelial cell precursors. The level of Fas III, a marker for immature follicle cells, is rapidly reduced in all epithelial cell precursors. However, these cells do not begin to express markers for new cell identities until around stage 7. Therefore, it seems that the epithelial cells become committed to a fate early in the vitellarium, but do not terminally differentiate until later. This is consistent with the fact that the epithelial follicle cells continue to divide until stage 6. Furthermore, Grk/EGFR signaling does not impose posterior identity on epithelial cells until stage 6. So the loss of Fas III in epithelial cell precursors in the early vitellarium marks an intermediate step in specific epithelial identities. JAK signaling is involved in this step, because clones of JAK pathway mutations cause the persistence of Fas III in epithelial cell precursors in the early vitellarium. The normal loss of Fas III expression in epithelial precursors of the early vitellarium may indicate the establishment of a pre-pattern of epithelial identities determined by JAK signaling. It is attractive to speculate such a role because the secreted JAK pathway ligand Upd is expressed symmetrically at the termini of the chamber. It is easy to envision a scheme in which the strength of the Upd signal received by the epithelial cell precursors determines the ultimate epithelial identity. However, these epithelial cells would remain in a proliferative, undifferentiated program until stage 7. The event that allows terminal differentiation is unclear, but could also be a N signal, as suggested above for competence of stalk and polar cells. This is consistent with the report of a pulse of Delta protein, a N ligand, that occurs at stages 5-7. Additional work will determine whether JAK signaling is instructive for specific epithelial fates (McGregor, 2002).

The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis

During Drosophila oogenesis, border cells perform a stereotypic migration. Slbo, a C/EBP transcription factor, is required for this migration. Drosophila Stat92E has been identified in a screen for gain-of-function suppressors of the slbo mutant phenotype. By clonal analysis for Stat92E and hop mutants it has been found that the JAK/STAT pathway is required in border cells for their migration. The activating ligand for the pathway, Unpaired, is expressed in polar cells. Polar cells are specialized cells that can induce border cell fate in anterior follicle cells. On its own, ectopic expression of Unpaired can induce ectopic expression of border cell markers, including Slbo. However, Stat92E mutant cells still express normal levels of Slbo protein, thus Stat92E must regulate other targets critical for border cell migration (Beccari, 2002).

Egg chambers are initially formed in the germarium, which also contains the germ line and somatic stem cells. When an egg chamber buds off from the germarium, few somatic follicle cells show any specialization. The specialized cells are the two polar cells present at each end of an egg chamber (follicle) and the interfollicular stalk cells. Polar cells can be identified by expression of specific molecular markers in early as well as late egg chambers. Polar and stalk cells stop dividing before exit from the germarium. The remaining follicle cells continue to proliferate, and differentiate after stage 6 when the egg chamber has its final complement of 700-1000 follicle cells (Beccari, 2002).

From stage 9 of oogenesis, both marker gene expression and cell shape can be used to distinguish different follicle cell populations at the anterior end of the egg chamber. Most anterior are the border cells, consisting of the polar cells themselves plus about six immediately adjacent cells ('outer' border cells). The border cells delaminate from the follicular epithelium as a tight cluster at the beginning of stage 9 and migrate between the nurse cells to the oocyte. Adjacent to the border cells are the stretch cells, which stretch very thin to cover the nurse cells. Finally the centripetal cells, which later will cover the anterior end of the egg, are located between the stretch cells and the columnar 'main body' follicle cells. Anterior follicle cell fates are repressed in the corresponding follicle cells at the posterior end of the egg chamber by prior Gurken-EGFR signaling from the oocyte (Beccari, 2002).

Production of ectopic polar cells by exposing early egg chambers to increased Hedgehog expression appears sufficient to induce ectopic migrating border cells at stage 9. A slbo-lacZ enhancer trap is induced in extra migrating clusters at stage 9. Similar ectopic border cell clusters have been observed in egg chambers with clones of follicle cells mutant for costal2, a negative regulator of the Hedgehog signal transduction pathway. Thus the presence of polar cells, and absence of posteriorizing signal from the oocyte, may be sufficient for the induction of border cells at the appropriate developmental stage. What signals from polar cells may be responsible for induction of border cell fate in adjacent follicle cells? There is good evidence that Upd is a key signal from polar cells: Upd is specifically expressed in polar cells and acts non cell autonomously; ectopic expression of Upd induces two border cell markers; and the JAK/STAT pathway is required in border cells. Previous studies of the JAK/ STAT pathway in Drosophila have indicated that Upd expression induces Stat92E activation through the JAK kinase Hop and that the effects of Upd can be explained in this manner. Ectopic expression of Upd induces ectopic expression of Slbo. Since the JAK/STAT pathway is required in border cells and thus must be active there, Upd regulated Stat92E may normally contribute to Slbo up-regulation in border cells (Beccari, 2002).

However, given that the Stat92E mutant affected border cell migration without affecting Slbo expression, the JAK/STAT pathway may not be required for Slbo expression. One reasonable explanation is that another signal from polar cells contributes to activation of Slbo in border cells. This upstream signal may by itself be required for Slbo expression or may act redundantly with Stat92E. The additional signal may be a novel effect of Upd, not mediated by the JAK/STAT pathway. However, given the inability of Upd to convert stretch cells to border cells, it is thought a different signal is more likely. Irrespective of its potential effect on Slbo, the effect of Stat92E mutant clones shows that other targets of Stat92E must be critical for border cell migration (Beccari, 2002).

Just as ectopic Slbo expression is not sufficient to convert other cells into border cells, Upd misexpression and ectopic activation of Stat92E is also not sufficient to convert stretch cells into migrating border cells. In the latter situation, the stretch cells experience both Stat92E activation and Slbo expression. The stretch cells nevertheless do not assume border cell fate. This has several possible explanations. The signal invoked above as upstream regulator of Slbo may, in addition to Slbo, have other target genes required for migration which are not being induced by Upd. Alternatively, there may be yet another signal from polar cells that is required for border cell differentiation. While one of these two explanations is favored, there are other possibilities. The stretch cells may already have been specified at the time of ectopic Upd expression, and thus be refractive to additional inductive signals. Also, when functional ectopic border cells are induced by extra polar cells, the timing and levels of signals to adjacent cells are likely to be relatively normal. This may not be the case when Upd is ectopically expressed (Beccari, 2002).

In addition to the spatial signal described above, a temporal signal must turn on expression of Slbo and other markers at the right stage. Upd and other polar cell markers are expressed in polar cells from earlier stages. Yet normal polar cells, or Hedgehog-induced ectopic polar cells, only induce border cells and border cell markers at stage 9. The temporal signal(s) may either modify polar cell signals to make them functional at the right stage, or act directly on border, stretch and centripetal cells to influence expression of target genes. Given the early expression of Upd and given that marker genes are induced in follicle cells with somewhat different temporal profiles, the latter scenario is favored. Two known candidates for supplying temporal signals are late Delta/Notch signaling and hormonal regulation (ecdysone). Analysis of a temperature-sensitive Notch allele has shown that Notch was required for Slbo expression. It has recently been shown that signaling by germ line Delta to Notch in follicle cells is required for proper differentiation of all follicle cells after stage 6. Although required for differentiation, the direct effect of Delta/Notch signaling at stage 6 is unlikely to explain the onset of Slbo expression at stage 8/9. But a cascade of events initiated at stage 6 might indirectly lead to expression of differentiation markers 16-24 h later. There is also evidence that some stage specific gene expression in egg chambers is regulated by the hormone ecdysone. In addition, the ecdysone receptor, EcR and its partner, Usp, appear to be required for border cell migration. One experiment in this study has suggested that ecdysone regulates timing of border cell migration, but apparently not timing of Slbo expression. Hormone application requires additional ectopic expression of Slbo to induce premature border cell migration. Thus the temporal regulation of anterior follicle cell differentiation may also have multiple components. Given that the stages of oogenesis are well-studied, this will be an interesting system in which to determine how temporal and spatial regulation of differentiation is coordinated (Beccari, 2002).

The Drosophila STAT protein, Stat92E, regulates follicle cell differentiation during oogenesis

Signal transducer and activator of transcription (STAT) proteins are transcription factors that play a critical role in the response of a variety of eukaryotic cells to cytokine and growth factor signaling. In Drosophila, the STAT homolog encoded by the stat92E gene is required for the normal development of multiple tissues, including embryonic segmentation, imaginal discs, blood cells, male germ cells, and sex determination. Stat92E RNA expression is strongest in the differentiating follicle cells in the germarium, as determined by in situ hybridization. An ethylmethane sulfonate-induced, temperature-sensitive allele, stat92EF, in which the mutant protein contains a P506S substitution, located in the DNA binding domain, was used to study the role of stat92E in oogenesis. At the restrictive temperature, mutant females are sterile. Mutant ovaries have multiple defects, including fused egg chambers and an absence of interfollicular stalks cells and functional polar follicle cells. An analysis of mosaic clones, using an apparent null stat92E allele, indicates that Stat92E is required in the polar/stalk follicle cell lineage. It is concluded that stat92E is necessary for the early differentiation of follicle cells and for proper germ line cell encapsulation during Drosophila oogenesis (Baksa, 2002).

The somatic follicle precursor cells are present in stat92E mutants, but these cells appear to be defective in their ability to differentiate into mature polar cells and stalk cells. This defect leads to egg chamber fusions and in some cases oocyte mislocalization. Stat92E activity is required in the somatic follicle precursor cells. Further, the partial loss of Hop activity gives a phenotype similar to that of stat92E mutants, supporting the idea that the Hop-Stat92E (Jak-Stat) pathway is involved in this regulation (Baksa, 2002).

These results suggest two possible models for the regulation of follicle precursor cell fate by stat92E and the Hop-Stat pathway. One model postulates that stat92E can act as the switch to commit follicle precursor cells specifically to a stalk cell fate. In this model, the lack of stat92E activity leads to an excess number of differentiated polar follicle cells, at the expense of stalk cells. An alternative model postulates that stat92E activity is required for the commitment of follicle precursor cells to either a polar cell or stalk cell fate. In this model, the lack of stat92E activity causes the precursor follicle cells to remain in an undifferentiated state. These experiments do not conclusively distinguish between these two possibilities -- however, the latter model is favored. In the enhancer trap line, the coexpression of FasIII and alpha-spectrin in mutant cells expressing ß-gal suggests that daughter cells of this lineage have failed to commit to either the pole or stalk cell lineage. This model is further supported by the observation that stat92E mutant ovarioles frequently contain mislocalized oocytes. The mislocalized oocytes are not adjacent to the LacZ-expressing cells, indicating that the attachment of the germ line to the follicle cells does not occur properly (Baksa, 2002).

Additionally, in stat92EF/stat92E6346 mutants grown at 25°C, the number of cells that coexpress FasIII, alpha-spectrin, and ß-gal (driven by the stat92E6346 enhancer-trap line) equals the number of polar cells and stalk cells expected in normal egg chambers. Strikingly, larger clusters of 20-30 coexpressing cells appear ectopically in the egg chambers of females that had been upshifted to the more restrictive temperature, 29°C. There are several interpretations to explain these abnormalities. The size of the ectopic clusters suggests that cells of the pole cell/stalk cell lineage, which normally exit cell division in the germarium, may have overproliferated. Alternatively, multiple pole cell/stalk cell clusters may have fused as an indirect result of multiple egg chamber fusions. It is also possible that lateral follicle cells, adjacent to the polar/stalk precursor cells, have adopted the polar/stalk cell fate (Baksa, 2002).

The fused egg chambers, loss of stalk cells, and gaps in the follicle cell layer observed in stat92E mutants bear a strong similarity to those described for mutants in other Drosophila genes, particularly components of the Notch signaling pathway. While Notch activity is required at multiple steps of oogenesis, either the loss of the Notch cell surface receptor, or of its ligand Delta, leads to the phenotype of egg chamber fusions and lack of stalk cells. Mutations in daughterless enhance the Notch and Delta phenotypes (Baksa, 2002 and references therein).

It is tempting to postulate a direct interaction between the Hop-Stat92E pathway and that of Notch. However, the precise mechanism by which the Notch pathway regulates stalk cell and polar cell formation is not fully understood. For example, an increase in the number of FasIII-positive cells in Notch ts egg chambers led to the hypothesis that loss of the Notch function causes an increase in polar follicle cells at the expense of stalk cells. More recently, a clonal analysis utilizing a null Notch allele and additional cell markers led to the alternative model, that Notch activity is required for differentiation of the follicle precursor cells into either polar cells or stalk cells. In spite of similarities in the mutant phenotypes, it is equally possible that the Hop-Stat92E and Notch pathways regulate different aspects of stalk cell/polar cell formation. In support of the latter view, there are common aspects to the adult eye defects caused by mutations in the Hop and Notch pathways, although the interaction is complicated and probably not direct (Baksa, 2002).

The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis

Among the diverse cellular processes taking place during oogenesis, the delamination and migration of border cells (BCs), a group of anterior follicle cells, represent a powerful model to study cell invasion in a normal tissue. During stage 9 of oogenesis, BCs detach from the outer epithelium to invade the germline cyst compartment. The BC cluster contains two centrally located polar cells surrounded by approximately six outer border cells and undergoes a nearly 6-hour long posteriorward migration to reach the anterior part of the growing oocyte. Together with centripetal cells, they assemble the micropyle, a specialized structure required for sperm entry. domeless was isolated in a screen to identify genes essential in epithelial morphogenesis during oogenesis. The level of dome activity is critical for proper border cell migration and is controlled in part through a negative feedback loop. In addition to its essential role in border cells, dome is required in the germarium for the polarization of follicle cells during encapsulation of germline cells. In this process, dome controls the expression of the apical determinant Crumbs. In contrast to the ligand Upd, whose expression is limited to a pair of polar cells at both ends of the egg chamber, dome is expressed in all germline and follicle cells. However, Dome protein is specifically localized at apicolateral membranes and undergoes ligand-dependent internalization in the follicle cells. dome mutations interact genetically with JAK/STAT pathway genes in border cell migration and abolish the nuclear translocation of Stat92E in vivo. dome functions downstream of upd and both the extracellular and intracellular domains of Dome are required for JAK/STAT signaling. Altogether, the data indicate that Dome is an essential receptor molecule for Upd and JAK/STAT signaling during oogenesis (Ghiglione, 2002).

dome interacts genetically with the Stat92E and dpias ([a.k.a. Su(var)2-10 gene, Betz, 2001] a negative regulator of the JAK/STAT pathway) during BC migration, and dome phenotypes in ovaries are similar to those found in Stat92E and hop mutants. Furthermore, Stat92E nuclear localization is lost in dome mutant follicle cells, indicating that the mechanisms leading to Stat92E activation and subsequent nuclear translocation require dome. Since dome is epistatic to upd, the data indicate that dome is required downstream of upd and upstream of Stat92E for JAK/STAT signaling in egg chambers. Altogether, these results provide strong evidence that Dome is a receptor molecule for Upd during oogenesis (Ghiglione, 2002).

Previous studies have shown that the migration of BCs is sensitive to gene dosage, making this migration a useful marker for genetic screens. The reduction or elevation of slbo, a gene encoding a C/EBP homolog, is sufficient to produce BC migration defects. Consistently, recent work has shown that Slbo protein levels are tightly regulated by the ubiquitination pathway. BCs are also sensitive to changes in Dome protein levels. Indeed, either a decrease or an increase of Dome causes BC migration defects. There are several mechanisms by which gene activity can be regulated, including post-translational regulation, as with the Slbo protein, or transcriptional regulation. The data suggest that dome expression is regulated in part by a transcriptional negative feedback loop. Two consensus STAT binding sites present in the promoter region of the dome gene may prove to be important for this regulation. Interestingly, it has been shown that vertebrate STAT proteins can have both positive and negative regulatory functions. Further work will be necessary to determine whether Stat92E is a direct repressor of dome (Ghiglione, 2002).

A Notch/Delta-dependent relay mechanism establishes anterior-posterior polarity in Drosophila

The anterior-posterior axis of Drosophila becomes polarized early in oogenesis, when the oocyte moves to the posterior of the germline cyst because it preferentially adheres to posterior follicle cells. The source of this asymmetry is unclear, however, since anterior and posterior follicle cells are equivalent until midoogenesis, when Gurken signaling from the oocyte induces posterior fate. Asymmetry is shown to arise because each cyst polarizes the next cyst through a series of posterior to anterior inductions. Delta signaling from the older cyst induces the anterior polar follicle cells, the anterior polar cells signal through the JAK/STAT pathway to induce the formation of the stalk between adjacent cysts, and the stalk polarizes the younger anterior cyst by inducing the shape change and preferential adhesion that positions the oocyte at the posterior. The anterior-posterior axis is therefore established by a relay mechanism, which propagates polarity from one cyst to the next (Torres, 2003).

The follicle stem cells reside in region 2b of the germarium and give rise to two distinct lineages: the epithelial follicle cell precursors, which proliferate until stage 6 to generate most of the cells that surround each cyst, and the polar/stalk precursors. The latter exit mitosis at stage 1 to 2 of oogenesis and give rise to the symmetric pairs of polar cells at the anterior and posterior poles of the cyst and to the stalk that separates each cyst from the adjacent one. Delta mutant germline clones and Notch follicle cell clones fail to form polar cells, indicating that Delta signals from the germline to activate the Notch receptor in the polar/stalk precursors to induce them to adopt the polar cell fate. This induction requires fringe, which is upregulated in the polar/stalk precursors and renders these precursors competent to respond to the Delta signal. Once the polar cells are specified, they express Unpaired, the ligand for the JAK/STAT pathway, and the resultant activation of JAK/STAT signaling plays two key roles in patterning the rest of the follicle cells. (1) The polar cells induce uncommitted polar/stalk cell precursors to become stalk cells. Overexpression of Unpaired causes all polar/stalk cell precursors to differentiate as stalk, whereas loss-of-function mutations in hopscotch (JAK) or STAT92E cause a loss of the stalk. (2) Unpaired signaling from the polar cells induces the adjacent epithelial follicle cells at each pole of the egg chamber to adopt a terminal fate. This induction is essential for axis formation because only the terminal cells are competent to respond to Gurken by becoming posterior. Unpaired also acts as a morphogen to specify three distinct terminal cell types at the anterior: the border cells, the stretched follicle cells, and the centripetal cells. In the absence of Gurken signaling, all three cell types also form at the posterior of the egg chamber, indicating that the graded activity of JAK/STAT pathway creates a symmetric prepattern at both poles (Torres, 2003 and references therein).

Coactivation of STAT and Ras by Torso is required for germ cell proliferation and invasive migration in Drosophila

Primordial germ cells (PGCs) undergo proliferation, invasion, guided migration, and aggregation to form the gonad. In Drosophila, the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development, and coactivation of STAT and Ras is required for PGC proliferation and invasive migration. Embryos mutant for stat92E or Ras1 have fewer PGCs, and these cells migrate slowly, errantly, and fail to coalesce. Conversely, overactivation of these molecules causes supernumerary PGCs, their premature transit through the gut epithelium, and ectopic colonization. A requirement for RTK in Drosophila PGC development is analogous to the mouse, in which the RTK c-kit is required, suggesting a conserved molecular mechanism governing PGC behavior in flies and mammals (Li, 2003a).

STAT92E plays an essential role in mediating the phenotypic effects of gain-of-function mutations of Torso, TorGOF, but is only minimally required for wild-type Tor function in patterning the terminal structures of the Drosophila embryo. To investigate whether wild-type Tor nevertheless activates STAT92E, an antibody was used that recognizes the phosphorylated, or active form of STAT92E (pSTAT92E) to examine the activation status of STAT92E in different genetic backgrounds. In early embryos, pSTAT92E is detected in the anterior and posterior terminal regions in a pattern reminiscent of Tor activation. By analyzing embryos mutant for loss- or gain-of-function mutations of tor as well as those lacking JAK, encoded by hopscotch (hop), it was concluded that the early STAT92E activation is dependent on Tor but not Hop, suggesting that Tor may activate STAT92E independent of Hop. Because STAT92E contributes only marginally to the expression of the Tor target gene tailless (tll), it was of interest to find whether the early activation of STAT92E by Tor had any other biological functions. It was evident that Tor activation correlates temporally and spatially with the formation of PGCs, which are localized at the posterior pole of the early embryo. Tor-dependent activation of STAT92E as well as that of the Ras-MAPK signaling cassette, as detected by an antibody against activated ERK/MAPK (diphospho-ERK), persists in pole cells at this stage. STAT92E activation was detected in PGCs during their migration and in the gonads of late embryos, that are formed following the migration of pole cells through a complex route. These observations indicate that STAT92E and Ras1/Draf activation may play a role in PGC development (Li, 2003a).

So far the only known function of Tor has been in pattern formation, since Tor protein is present only transiently in early embryos. Therefore, the finding that Tor is involved in germ cell migration was initially unexpected. However, there is a precedent for the requirement of an RTK in germ cell migration in the mouse. Mutations in the mouse genes dominant white-spotting (W) cause migration and proliferation defects in germ cells as well as a few other cell types. W encodes the protooncoprotein c-kit, an RTK that is expressed on the membrane of mouse PGCs. Sl encodes the c-kit ligand termed stem cell factor (SCF), which is localized on the membrane of somatic cells associated with PGC migratory pathways. Interestingly, c-kit and Tor share structural similarities and both are structurally similar to the platelet derived growth factor (PDGF) receptor, in which an insert region separates the intracellular kinase domain. Moreover, similar to Tor and the PDGF receptor, c-kit is able to activate STAT molecules as well as the Ras-MAPK cascade. Although true molecular homologs of c-kit and SCF are not yet found in the Drosophila genome, the functional and structural similarities between Tor and c-kit suggest that flies and mice share molecular mechanisms for regulating primordial germ cell proliferation and migration (Li, 2003a).

In addition to germ cells, the ovarian border cells of Drosophila are also capable of invasive and guided migration. Border cells of the Drosophila ovary are follicle cells that, during oogenesis, delaminate as a cluster six to ten cells from the anterior follicle epithelium, invade the nurse cells, and migrate toward the oocyte. Interestingly, it has been shown that the detachment and guided migration of these cells require STAT92E activation. Mutations in components of the Hop/STAT92E pathway cause border cell migration defects. In addition, border cell migration also requires RTK signaling. An RTK related to mammalian PDGF and VEGF receptors, PVR, is required in border cells for their guided migration toward the oocyte. PVR appears functionally redundant with another fly RTK, EGFR, in guiding border cells. Taken together, these results indicate that the invasive behavior and guided migration of Drosophila ovarian border cells require both STAT92E and RTK activation. In light of the results from analyzing PGC migration, it is proposed that activation of both STAT and components downstream of RTK signaling may serve as a general mechanism for invasive and guided cell migration (Li, 2003a).

It has been shown that actin-based cytoskeletal reorganization plays a crucial role in cell shape changes and movements. The identification of STAT and Ras coactivation as an essential requirement for germ cell migration raises an interesting question of how activated STAT and Ras coordinate the cytoskeletal reorganization required for germ cell migration. STAT92E has been shown to be involved in the transcriptional activation of many signaling molecules as well as key transcription factors. A recent systematic search for STAT92E target genes has revealed a plethora of genes that might be directly activated by STAT92E, among which are those involved in the regulation of cytoskeletal movements and actin reorganization. Upregulation of such genes in response to spatial cues should facilitate cell movements. In addition, Ras and other small GTP proteins have been implicated in multiple cellular processes that require cytoskeletal reorganization. It remains to be determined how these two signaling pathways coordinate germ cell movements in response to guidance cues from surrounding somatic tissues (Li, 2003a).

Localized JAK/STAT signaling is required for oriented cell rearrangement in the hindgut

Rearrangement of cells constrained within an epithelium is a key process that contributes to tubular morphogenesis. Activation in a gradient of the highly conserved JAK/STAT pathway is essential for orienting the cell rearrangement that drives elongation of a genetically tractable model. Using loss-of-function and gain-of-function experiments, it has been shown that the components of the pathway from ligand to the activated transcriptional regulator STAT are required for cell rearrangement in the Drosophila embryonic hindgut. The difference in effect between localized expression of ligand (Unpaired) and dominant active JAK (Hopscotch) demonstrates that the ligand plays a cell non-autonomous role in hindgut cell rearrangement. Taken together with the appearance of STAT92E in a gradient in the hindgut epithelium, these results support a model in which an anteroposterior gradient of ligand results in a gradient of activated STAT. These results provide the first example in which JAK/STAT signaling plays a required role in orienting cell rearrangement that elongates an epithelium (Johansen, 2003).

upd, encoding the ligand for the Drosophila JAK/STAT pathway, is expressed only in the small intestine and is regulated by genes controlling hindgut cell rearrangement. In drm and bowl mutants, expression of upd is missing from the small intestine, while in lin mutants, upd expression is expanded throughout much of the hindgut. These results raise the possibility that localized Upd might provide an orienting cue for rearranging hindgut cells (Johansen, 2003).

If it plays a role in hindgut cell rearrangement, upd must be expressed before and during the period of major hindgut elongation, i.e. between stages 11 and 16; genes encoding the other known components of the Drosophila JAK/STAT signaling pathway should also be expressed at the same stages, both within and adjacent to upd-expressing cells. In situ hybridization was used to characterize the expression of upd, dome, hop and Stat92E during stages just prior to and during hindgut elongation (Johansen, 2003).

Expression of upd in the hindgut is first detected at stage 9 in a narrow ring of cells that will become the small intestine. Expression in the prospective small intestine is maintained during stages 10 and 11, where it can be seen just posterior to the everting renal tubules (note that in the hindgut at these germband-extended stages, 'posterior' is toward the head). During stages 12-14, when the hindgut undergoes a major part of its elongation, upd expression is seen throughout the now distinct small intestine. Expression of upd is maintained throughout the small intestine during the remainder of embryogenesis (Johansen, 2003).

The Janus kinase hop is expressed uniformly throughout the embryo, including the hindgut as it elongates. Expression of both the receptor-encoding gene dome and Stat92E is detected weakly at the anterior of the hindgut beginning at stage 9; it becomes significantly stronger by stage 11, and is maintained through stage 14. For both the receptor- and STAT-encoding genes, expression domains in the hindgut epithelium overlap with and extend beyond the narrow domain of upd expression. Most significantly, expression of dome and Stat92E extends to a more posterior position in the hindgut epithelium than does expression of upd. Thus, the mRNA expression of the ligand, receptor and STAT components in the hindgut prior to and during its elongation is consistent with a role for JAK/STAT signaling in hindgut cell rearrangement (Johansen, 2003).

Elongation of the Drosophila hindgut by cell rearrangement requires the Upd ligand and the JAK/STAT pathway components Dome (receptor), Hop (JAK) and Stat92E. Since elongation does not occur when expression of ligand or activation of the pathway is uniform, but only when the source of ligand is localized to the hindgut anterior, the requirement for localized JAK/STAT signaling in hindgut elongation can be characterized as instructive, rather than permissive. Since patterning is normal in hindguts both lacking and uniformly expressing upd, the required role of JAK/STAT signaling in hindgut morphogenesis is likely via direct effects on cell movement (Johansen, 2003).

The rescue of the upd phenotype by anteriorly localized expression in the hindgut of upd, but not of activated JAK (Hopscotch), demonstrates that there is a requirement for upd function that is not cell autonomous. In other words, upd is required in cells (those of the large intestine that undergo the greatest rearrangement) that are different from cells that produce it (those of the small intestine). A number of examples have been described in which localized expression of a signaling molecule (including Upd) is required non-autonomously for cell rearrangement, morphogenesis or motility. In the Drosophila eye imaginal disc, expression of Upd at the midline is required to establish a dorsoventral polarity that orients ommatidial rotation. In both Drosophila tracheae and the vertebrate lung, branching morphogenesis of the epithelium depends on localized expression of FGF in adjacent mesenchyme (Johansen, 2003).

Localized activation of JAK/STAT signaling has been shown to play a role in cell motility in a number of contexts. In Drosophila, localized expression of Upd in the anterior polar cells of the egg chamber acts to coordinate the migration of the adjacent border cells. In mammals, cytokines expressed in target tissues act to attract both migrating lymphocytes and tumor. The finding that localized (only in the small intestine) expression of upd is both necessary and sufficient for rearrangement of cells in the large intestine indicates that Upd must have an organizational, action-at-a-distance function in controlling cell rearrangement during tubular morphogenesis (Johansen, 2003).

Rescue experiments establish that there is a cell non-autonomous requirement for upd in hindgut elongation. Consistent with this, there is evidence that Upd is present and required in an anteroposterior gradient in the hindgut. Prior to and during hindgut elongation, both Stat92E mRNA and Stat92E protein are detected not only in the small intestine epithelium (and the visceral mesoderm surrounding the small intestine), but also in the epithelium posterior to the small intestine; this expression of Stat92E appears to be in a gradient. In the Drosophila eye imaginal disc, a gradient of Upd is required to orient the rotation of ommatidial cell clusters; in addition, there is evidence for a gradient of Upd and Stat92E in patterning of the follicular epithelium of the Drosophila egg chamber. Since expression of Stat92E depends on upd, it is likely that Upd protein is present in the hindgut epithelium as an anteroposterior gradient, with its highest level in the upd-expressing cells of the small intestine, and lowest level in posterior, upd non-expressing cells of the large intestine. Expression of SOCS36E (suppressor of cytokine signaling at 36E), which is regulated by upd, overlaps with and extends significantly beyond the domain of upd expression, further supporting the idea that there is a gradient of Upd in the hindgut (Johansen, 2003).

In the Drosophila eye imaginal disc, anti-Upd staining and the behavior of clones of mutant cells that have lost components of the JAK/STAT pathway indicate that Upd is present in a gradient that extends at least 50 µm beyond its midline mRNA expression domain. In the Drosophila hindgut, Stat92E is a reliable reporter for the presence of Upd. Two to four hours after upd is first expressed at the anterior of the hindgut (stage 9), Stat92E can be detected at least 30-40 µm from the site of upd expression (stages 11 and 12). These time and distance parameters are similar to those observed during generation of the Upd gradient in the eye, and the Dpp and Wg gradients in wing imaginal discs, which form over distances of roughly 40-80 µm in 1-8 hours. Thus, it is reasonable to imagine that a gradient of Upd is established in the developing hindgut in a short enough time frame to affect cell rearrangement (Johansen, 2003).

The essential consequence of JAK/STAT signaling is activation of the STAT protein, which leads to altered transcriptional programs. STAT has been shown in a number of contexts to be required for cell motility, and therefore probably regulates expression of genes controlling cytoskeletal assembly and cell adhesion. In these contexts, however, activation of STAT does not appear to be required to orient cell movement, but rather to facilitate or promote it. As Stat92E is required for hindgut elongation, and its protein product appears to be present in a gradient along the anteroposterior axis, this raises the intriguing question of how a gradient of a transcription factor might orient cell rearrangement (Johansen, 2003).

Biological functions of the ISWI chromatin remodeling complex NURF: Interaction with JAK/STAT pathway

The nucleosome remodeling factor (NURF) is one of several ISWI-containing protein complexes that catalyze ATP-dependent nucleosome sliding and facilitate transcription of chromatin in vitro. To establish the physiological requirements of NURF, and to distinguish NURF genetically from other ISWI-containing complexes, mutations were isolated in the gene encoding the large NURF subunit, nurf301. NURF is shown to be required for transcription activation in vivo. In animals lacking NURF301, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, NURF is shown to be required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate the phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that causes melanotic tumors to form (Badenhorst, 2002).

During the course of this analysis it was noticed that nurf301 mutant animals display a high incidence of melanotic tumors. Melanotic tumors have previously been reported in a number of mutant backgrounds and are generally caused by neoplastic transformation of the larval blood cells. The circulating cells (hemocytes) of the larval blood or hemolymph provide one tier of the innate immune system of insects by encapsulating or engulfing pathogens. A number of mutations have been shown to trigger the overproliferation and premature differentiation of hemocytes. Tumors form when these cells aggregate, or invade and encapsulate normal larval tissues Badenhorst, 2002).

Melanotic tumors are observed both in EMS-induced nurf301 mutants that truncate NURF301, the P-element induced mutation that reduces nurf301 transcript levels, and allelic combinations of these mutants. Tumor penetrance is extremely high (100% for nurf3012 at 25°C). Consistent with tumor development, circulating hemocyte cell number was increased dramatically in hemolymph isolated from nurf301 mutant animals. A large percentage of animals lacking ISWI, the catalytic subunit of NURF, also displayed melanotic tumors confirming that disrupted NURF function induces tumor formation. In iswi mutant animals the number of circulating hemocytes is also increased. In both nurf301 and iswi mutant hemolymph, small aggregates of hemocytes are often observed. All hemocyte cell types are present, from small round cells (prohemocytes) to crystal cells and lamellocytes (Badenhorst, 2002).

In Drosophila, larval blood cell transformation and melanotic tumor formation can be induced by inappropriate activation of either of two distinct signaling cascades: the Toll or the JAK/STAT pathway. Inappropriate activation and nuclear-localization of the Drosophila NF-kappaB homolog Dorsal, caused either by constitutive activation of the Toll receptor or removal of the inhibitor, the Drosophila IkappaB Cactus, leads to melanotic tumors in third instar larvae. In the second pathway, gain-of-function mutations in Hopscotch (Hop), the Drosophila Janus Kinase (JAK), induce melanotic tumors. Hop gain-of-function mutants cause tumor development by triggering constitutive activation and DNA-binding by the Drosophila STAT transcription factor, STAT92E (Badenhorst, 2002).

To resolve whether the melanotic tumors seen in the nurf301 mutants were caused by misregulation of either the TOLL or HOP/STAT92E pathways, whether nurf301 mutants enhance tumor phenotypes seen in constitutively active Toll or Hop mutant lines was tested. Tumor incidence in animals carrying one copy of a gain-of-function Hop mutation -- hopTum-1 -- is increased by simultaneous reduction in NURF301 levels. In contrast, removal of one copy of NURF301 fails to enhance the Toll gain-of-function allele Tl10b. The results suggest that NURF acts as a negative regulator within the Drosophila JAK/STAT signaling pathway (Badenhorst, 2002).

Molecular signatures of both JAK and Toll activation have been defined. It is known that Hop gain-of-function mutants induce expression of a complement-like protein TEP1. Overactivation of the Toll pathway also induces TEP1 synthesis but primarily induces expression of antimicrobial peptides, including Drosomycin (Drs) and Diptericin (Dpt). Loss of nurf301 induces tep1 but fails to induce drs or dpt, demonstrating that NURF301 principally affects the Hop/STAT92E pathway. Whether nurf301 interacts genetically with other known components of the Hop/STAT92E pathway was tested. Certain mutations in unpaired (upd, also known as outstretched), which encodes a ligand for the Hop receptor, display a characteristic wings-out phenotype, due to decreased activation of Hop and consequently decreased STAT92E function. When NURF301 levels are simultaneously decreased in these mutant backgrounds, animals are mostly restored to the wild-type. These genetic interactions confirm that NURF301 acts as a negative regulator of the Hop/STAT92E pathway, at a point downstream of Hop. Hence, disruption of NURF could affect either STAT92E or the targets of STAT92E. In nurf301 mutants, levels of the STAT92E transcription factor are not elevated, suggesting that NURF acts to repress the activity of STAT92E or the expression of some STAT92E target genes (Badenhorst, 2002).

A sensitized genetic screen to identify novel regulators and components of the Drosophila janus kinase/signal transducer and activator of transcription pathway

The JAK/STAT pathway exerts pleiotropic effects on a wide range of developmental processes in Drosophila. Four key components have been identified: Unpaired, a secreted ligand; Domeless, a cytokine-like receptor; Hopscotch, a JAK kinase, and Stat92E, a STAT transcription factor. The identification of additional components and regulators of this pathway remains an important issue. To this end, a transgenic line was generated where the upd ligand was misexpressed in the developing Drosophila eye. GMR-upd transgenic animals have dramatically enlarged eye-imaginal discs and compound eyes that are normally patterned. The enlarged-eye phenotype is a result of an increase in cell number, and not cell volume, and arises from additional mitoses in larval eye discs. Thus, the GMR-upd line represents a system in which the proliferation and differentiation of eye precursor cells are separable. Removal of one copy of stat92E substantially reduces the enlarged-eye phenotype. An F1 deficiency screen was performed to identify dominant modifiers of the GMR-upd phenotype. Nine regions have been identified that enhance this eye phenotype and two specific enhancers: C-terminal binding protein and Daughters against dpp. Twenty regions have been identified that suppress GMR-upd and 13 specific suppressors: zeste-white 13, pineapple eye, Dichaete, histone 2A variant, headcase, plexus, kohtalo, crumbs, hedgehog, decapentaplegic, thickveins, saxophone, and Mothers against dpp (Bach, 2003).

These results indicate that Upd and the JAK/STAT pathway control the size of the Drosophila eye. Heteroallelic hypomorphic combinations of upd result in a small adult eye, while ectopic misexpression of upd in the developing fly eye results in a greatly enlarged eye. This phenotype is specific to activation of the JAK/STAT pathway in the developing eye because reduction in the dose of stat92E or the eye-specific transcription factor glass results in suppression of the enlarged eye. The results suggest that ectopic misexpression of upd in the developing eye results in additional mitoses of precursor cells in the region of the eye disc anterior to the furrow. These additional cells are patterned normally by the morphogenetic furrow, resulting in increased numbers of ommatidia in GMR-upd discs (Bach, 2003).

The enlarged-eye phenotype observed by ectopic misexpression of an activated form of ras85D using the ey enhancer, ey-rasV12, is the result of ectopic R7 cells and also appears very rough. The results indicate that the GMR-upd phenotype is distinct from the ey-rasV12 because GMR-upd eyes are patterned normally, are not rough, and are not modified by ras85D mutations. The enlarged eyes observed with misexpression of the Drosophila InR using GMR-Gal4 results primarily from increased cell volume. The results indicate that in the Drosophila eye the JAK/STAT and InR pathways do not interact, at least when ectopically misexpressed. Reduction in doses in InR pathway genes, such as InR, Pten, and chico, do not modify the GMR-upd phenotype. Moreover, the GMR-upd phenotype results from increased cell numbers, not from increased cell volume. In fact, cells in GMR-upd adult eyes actually exhibit decreased cell volumes when compared to wild type. Interestingly, the enlarged-eye phenotype in GMR-upd shares similarities with that produced as a nonautonomous effect of expression of an activated form of Notch (Nintra) in the eye, with prominent dorsal outgrowths. This observation is also interesting in light of the fact that CtBP, which represses N pathway activity, was identified as an enhancer of GMR-upd. It is possible that CtBP represses Stat92E itself or negatively regulates transcriptional coactivation by Stat92E (Bach, 2003).

The GMR-upd line was identified as a sensitized genetic background and an F1 screen for dominant modifiers of the GMR-upd phenotype was performed using a set of overlapping deletions of the Drosophila genome. Twenty loci were identified that suppress and nine that enhance the enlarged-eye phenotype. The gene(s) in these deficiencies, responsible for the modification of the phenotype, may represent new components of or new interactors with the JAK/STAT pathway. Thirteen mutations were identified as Su(GMR-upd): zw13, crb, pie, D, His-2Av, kto, hdc, px, hh, dpp, tkv, sax, and Mad. In addition, two mutations were identified as En(GMR-upd): CtBP and Dad (Bach, 2003).

zw13 interacts genetically with the meiotic kinesin-like genes nod and ncd and encodes a poorly characterized protein with RNA-recognition motifs. Therefore, Zw13 may be important in regulating upd expression. crb was also identified as a suppressor of GMR-upd. Crb is a PDZ-containing protein involved in the establishment and maintenance of apical-basal polarity in epithelia. crb may suppress the GMR-upd phenotype by altering the localization of Dome and/or Upd or the signaling output of the JAK/STAT pathway in the eye (Bach, 2003).

Several transcription factors were identified as suppressors of GMR-upd: pie, D, His2Av, kto, px, and hdc. Pie is a nuclear protein that contains a PHD finger, which is a C4HC3 zinc-finger-like motif thought to facilitate chromatin-mediated transcriptional regulation. Eyes from pie homozygotes show irregular spacing of ommatidia, although the ommatidia have the normal array of photoreceptors. Notably, pie homozygous flies also have held-out wings, a phenotype shared by os flies and flies that overexpress full-length Dome. In embryonic segmentation, D directly regulates the expression of the pair-rule gene, even-skipped (eve), by binding to multiple sites located in downstream regulatory regions that direct formation of eve stripes 1, 4, 5, and 6. This overlaps with the function at Stat92E, which is needed for proper expression of eve stripes 3 and 5. Interestingly, fish and upd share related expression patterns and phenotypes. The early expression pattern of fish is almost identical to that of upd. Like upd, fish is also required in the hindgut, and the D held-out wing phenotype is very similar to that of os. His2Av belongs to the H2AZ variant subclass, which is involved in chromatin stability, chromatin remodeling, and transcriptional control. Given that mammalian STATs have been shown to mediate transcriptional changes within seconds of activation, it is possible that histone modification must be coordinated with transcriptional coactivation. Kto is the homolog of thyroid-hormone receptor associated protein (TRAP230), which was originally identified as part of the trithorax group, a large transcriptional coactivation complex. kto is involved in photoreceptor differentiation because homozygous mutant clones in the eye disc fail to develop into photoreceptors, although mutant cells can respond to Hh by expressing dpp. hdc encodes a nuclear factor involved in tracheal development, where it acts nonautonomously in an inhibitory signaling mechanism to determine the number of cells that will form unicellular sprouts in the trachea. Interestingly, it has been recently noted that stat92E is also required in tracheal development. However, whether hdc and stat92E interact, if at all, in this tissue is not known, nor is it understood whether any interaction exists in the eye disc. Px is a nuclear protein that, like Pie, contains a PHD zinc finger and is involved in venation in the wing. It is not known if px mutants exhibit an eye phenotype. Clearly, future work must focus on the elucidation of any biochemical interaction between Stat92E and these transcription/nuclear factors and also whether they regulate the transcription of a common set of genes required for growth of the eye disc (Bach, 2003).

The other modifiers identified in the modifier screen are genes in the Dpp pathway, specifically dpp, tkv, sax, mad, hh, and Dad. It was initially reasoned that upd may exerts its proliferative effects through hh or dpp. However, hh and dpp are expressed normally in GMR-upd. In addition, ectopic misexpression of hh or dpp in the os/os1A flies does not rescue the small-eye phenotype whereas upd does and ectopic expression of upd in flip-out clones does not induce hh. These results suggest that upd may not directly regulate dpp or hh expression. These data also suggest that Upd and Dpp and/or Hh may coregulate genes involved in the proliferation of eye precursor cells. This hypothesis is supported by observations in mammalian systems. The cytokines leukemic inhibitory factor and bone morphogenic protein 2 activate Stat3 and Smad1, respectively, and act synergistically in fetal neuroepithelial cultures to promote the differentiation of astrocytes from progenitor cells. The synergism requires functional Stat3 and Smad1. However, these proteins do not physically interact; rather, they both bind to p300/CBP to promote transactivation of target genes, such as glial fibrillary acidic protein, a marker of astrocyte differentiation (Bach, 2003).

In both mammals and flies, the JAK/STAT pathway plays an important role in the control of organ/tissue size. Stat5 knock-out mice are runted due to impaired growth-hormone signaling. Similarly, Socs-2 knock-out mice are significantly larger than their wild-type littermates, due to a lack of negative regulation of the growth-hormone pathway in vivo in the absence of the Socs-2 gene. Overexpression of an activated, constitutively dimerized STAT, c-Stat3, results in the formation of tumors in mice. Importantly, the only gain-of-function mutations in any JAK are found in Drosophila hop. hopTum-l and hopT42 are independent point mutations that give rise to hyperactive Hop proteins, overproliferation and premature differentiation of Drosophila larval blood cells (a so-called fly 'leukemia'), melanotic tumors, and lethality. Overexpression of upd or hop in the developing Drosophila eye leads to a greatly enlarged eye due to an increase in the number of cells in the eye disc. In contrast, hypomorphic mutations in upd, for example, os or os/os1A, lead to a small adult eye (Bach, 2003).

Although proliferation is clearly a result of activation of the JAK/STAT pathway in mammals and Drosophila, little is known about how this pathway regulates the increase in cell number or the cell cycle. The data suggest that activation of the JAK/STAT pathway in the eye disc increases the number of cycling cells, possibly by shortening the G1 phase or by regulating the G2/M transition of the cell cycle. As a secreted molecule, Upd presumably acts in a cell-nonautonomous manner and may promote proliferation directly through activation of Hop and Stat92E. However, the observed proliferation in GMR-upd may in fact be due to the ability of Upd to induce another molecule that can also act cell nonautonomously. At the moment it is not possible to differentiate between these two possibilities. Nonetheless, the fact that more cells are observed in GMR-upd indicates that Upd may regulate genes involved in proliferation in the eye disc. In addition to the 15 modifiers of GMR-upd described here, several uncharacterized mutations have been identified that modify GMR-upd and may encode potentially novel molecules and uncover new functions of the JAK/STAT pathway. Given the high conservation between the Drosophila and mammalian JAK/STAT pathways, it is likely that the genes and functions uncovered in this screen will also be relevant to higher organisms (Bach, 2003).

Drosophila C-terminal Src kinase negatively regulates organ growth and cell proliferation through inhibition of the Src, Jun N-terminal kinase, and STAT pathways

Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. The Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk–/– overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. The data provide in vivo evidence that Src activity requires JNK and STAT function (Read, 2004).

Partial reduction of Src64B, Src42A, or Btk29A activity suppresses the dCsk–/– phenotype, providing functional data to support the view that the imaginal disc overgrowth, defective larval and pupal development, and lethality of dCsk–/– mutants results from inappropriate activation of the Src-Btk signal transduction pathways. Mutations in Btk29A more strongly suppress dCsk phenotypes than either Src42A or Src64B mutations, perhaps reflecting that (1) Src paralogs act redundantly to each other in Drosophila as in mammals and (2) Btk29A has been shown to act downstream of Src family kinases (SFK) in flies and in mammals. In vivo evidence is provided that loss of Csk function hyperactivates Btk to drive cell cycle entry in development, demonstrating that Tec-Btk family kinases are critical to SFK-mediated proliferation. The data raise the possibility that partial reduction of Tec-Btk kinase activity could reduce proliferation in other cellular contexts in which overgrowth is driven by hyperactivated SFKs, such as in colon tumors (Read, 2004).

Tissue culture models show that constitutively activated SFK signal transduction modulates the function of numerous downstream effector molecules and pathways. Using a loss-of-function approach to identify effectors that mediate the dCsk overgrowth phenotypes, some of these pathways were not implicated in dCsk function. For example, SFKs up-regulate the SOS-Ras-ERK pathway in multiple tissue culture studies and Drosophila overexpression models. However, although dRas1 signaling is active throughout retinal development, reduced dEGFR, Sos, and Jra (c-jun) gene dosage failed to affect the dCsk phenotype. dCsk mutations also failed to modify a hypermorphic allele of dEGFR. Levels of doubly phosphorylated and activated ERK appeared unaltered in dCsk–/– tissue. Moreover, the dCsk phenotype failed to phenocopy defects caused by Ras pathway hyperactivation. For example, constitutively active dRas1 causes increased cell size and patterning defects in the developing imaginal discs, defects that were not observed in dCsk mutant eye tissues. These data argue that not every signal transduction pathway implicated in SFK tissue culture models necessarily functions as predicted within a developing epithelial tissue (Read, 2004).

These studies emphasize the importance of two signaling pathways in dCsk and SFK function. Since certain defects in dCsk–/– animals, such as a split notum, resembled those of hep (JNKK) mutants, it is suspected that JNK pathway activity is involved in dCsk function. Phenotypic and FACS analysis established that reduced JNK (bsk) function suppresses the phenotypes and cell cycle defects caused by loss of dCsk. These results confirm studies indicating that JNK functions downstream of the Src-Btk pathway in Drosophila and mammalian tissue culture cells. Components of the JNK pathway are required for Src-dependent cellular transformation, but the exact role of JNK in these cells is unknown. Importantly, the data show that the JNK pathway mediates proliferative responses to Src signaling in vivo. Further work will be needed to precisely understand its role in proliferation (Read, 2004).

Genetic studies also highlight the importance of the Jak/Stat signal transduction pathway. dCsk proves a negative regulator of Jak/Stat signaling; for example, dCsk mutant tissues show up-regulation of Stat92E protein, a hallmark of Jak/Stat activation in Drosophila. Stat92E, the sole Drosophila STAT ortholog, is most similar to mammalian STAT3. In mammalian cells, Src directly phosphorylates and activates STAT3 and STAT3 function and activation are required for Src transforming activity. Conversely, overexpression of Csk blocks STAT3 activation in v-Src transformed fibroblasts. Activating mutations in STAT3 can also promote oncogenesis in mice. However, the physiological significance of these interactions within developing epithelia remains unclear (Read, 2004).

dCsk; Stat92E double mutant clones reveal that blockade of STAT function in dCsk mutants severely reduces Src-dependent overgrowth and promoted apoptosis of mutant tissue. dCsk–/–; Stat92E–/– EGUF adult eyes (the EGUF method produces genetically mosaic flies in which only the eye is exclusively composed of cells homozygous for the mutation) are nearly identical to phenotypes caused by overexpression of Dacapo, the fly ortholog of the cdk inhibitor p21, and PTEN, a negative regulator of cell proliferation and growth. Importantly, removing Stat92E function in dCsk mutant tissue led to a synthetic small eye phenotype and did not simply rescue the dCsk–/– proliferative phenotype. This outcome distinguishes Stat92E from mutations in Src64B, Btk29A, or bsk, which rescue dCsk-mediated defects toward a normal phenotype. The loss of tissue in dCsk–/–; Stat92E–/– clones indicates that Src-Btk signaling provokes apoptosis in the absence of Stat92E function. Consistent with this interpretation, reduced Btk29A function rescued the dCsk–/–; Stat92E–/– EGUF phenotype to a more normal phenotype, demonstrating that the reduced growth and increased apoptosis observed in the dCsk–/–; Stat92E–/– tissues is indeed Src-Btk pathway dependent (Read, 2004).

The data suggest the existence of a Src-dependent proapoptotic pathway that is normally suppressed by STAT. One possible component of this pathway is JNK, given that JNK signaling is an important activator of apoptosis in both flies and mammals. Perhaps Src-dependent hyperactivation of Bsk (JNK) in dCsk–/–; Stat92E–/– tissue contributes to cell death in the absence of proliferative and/or survival signals provided by Stat92E. However, a number of other candidate pathways may also mediate this response. The further characterization and identification of these pathways may have important implications for interceding in Src-mediated oncogenesis (Read, 2004).

Together, these observations indicate that, in tissue that contains hyperactive Src or reduced Csk, blocking STAT function is sufficient to trigger apoptosis and decrease proliferation in the absence of any further mutations or interventions. Reduced STAT3 function can promote apoptosis within breast and prostate cancer cells that show elevated SFK activity, but the molecular pathways driving apoptosis in these cells are unknown. These cells may require survival signals provided by STAT3 to counteract apoptosis due to chromosomal abnormalities or other defects. Alternatively, these cells may die because of proapoptotic signals provided by hyperactive SFKs in the absence of STAT3 function. The data argue that the latter may be true, which suggests the intriguing possibility that therapeutic blockade of STAT function in tumors with activated Src may actively provoke Src-dependent apoptosis and growth arrest in tumor tissues (Read, 2004).

Mutational analysis reveals separable DNA binding and trans-activation of Drosophila STAT92E

In the canonical model of JAK/STAT signalling STAT transcription factors are activated by JAK mediated tyrosine phosphorylation following pathway stimulation by external cytokines. Activated STAT molecules then homo- or hetero-dimerise before translocating to the nucleus where they bind to DNA sequences within the promoters of pathway target genes. DNA-bound STAT dimers then activate transcription of their targets via interaction with components of the basal transcription machinery. This study describes a missense mutation in the SH2 domain of the single Drosophila STAT92E homologue that results in an amino-acid substitution conserved in both the canonical SH2 domain and STAT-like molecules previously identified in C. elegans and the mosquito Anopheles gambiae. This mutation leads to nuclear accumulation and constitutive DNA binding of Drosophila STAT92E even in the absence of JAK stimulation. Strikingly, this mutant shows only limited transcriptional activity in tissue culture based assays and functions as a dominant-negative at both the phenotypic and molecular levels in vivo. These features represent aspects of both dominant gain-of-function and dominant-negative activities and imply that the functions of DNA binding can be functionally separated from the role of STAT92E as a transcriptional activator. It is thus possible that an alternative post-translational modification, in addition to tyrosine phosphorylation, may be required to allow STAT to act as a transcriptional activator and suggests the existence of an alternative mechanism by which STAT transcriptional activity may be regulated in vivo (Karsten, 2005).

Drosophila STAT92E containing a mutation of methionine 647 to histidine results in a molecule that constitutively nuclear accumulates, is constitutively DNA-bound and is likely to be constitutively tyrosine phosphorylated. However, STAT92EM647H-GFP is largely incapable of activating transcription and acts as a dominant-negative in vivo such that its expression is sufficient to inhibit endogenous JAK/STAT pathway activity both at the phenotypic and gene expression levels. Given these findings it appears possible that the dominant-negative effect occurs via target gene promoter occupation by transcriptionally incompetent STAT92EM647H-GFP complexes, which block access for endogenous STAT92E (Karsten, 2005).

In the light of these results it is perhaps surprising that the original substitution on which the Drosophila mutation was based has been described as a gain-of-function allele. In this report, human STAT5AN643H was identified on the basis of its ability to rescue cytokine independent growth of the Ba/F3 cell line. While this rescue acts as the initial selection criteria, analysis of known STAT5A targets revealed only relatively modest increases in transcription induced by the activated molecule suggesting that a small increase in the level of endogenous JAK/STAT pathway activity may be sufficient to rescue the Ba/F3 cell line. Given that multiple STATs are expressed in Ba/F3 cells, such a low level increase in activity could conceivably be mediated by hetero-dimers containing transcriptionally competent endogenous STATs and constitutively phosphorylated STAT5AN643H molecules. By contrast, the low redundancy of the Drosophila system reduces the likelihood that similar heterodimeric combinations form in vivo or in S2 cells, especially given the relatively high levels of expression that result from transient transfection and the Gal4/UAS system. As such, a scenario in which STAT92EM647H-GFP is constitutively DNA-bound yet transcriptionally incompetent may represent the more accurate description of this particular mutation in vivo (Karsten, 2005).

Although this study has shown that the DNA binding and transcriptional activation activities of STA92E represent two distinct and separable processes, the mechanism by which STAT92EM647H-GFP is constitutively phosphorylated in the absence of active Hop is not clear (Karsten, 2005).

As shown in this study, the inhibition of endogenous phosphatase activity by Sodium-ortho-vanadate treatment is sufficient to stimulate Y704-dependent DNA binding of wild type STAT92E-GFP. Given the lack of detectable endogenous JAK/STAT pathway activity in S2 cells, it appears that tyrosine kinases other than Hop are able to phosphorylate STAT92E. Furthermore, Sodium-ortho-vanadate treatment shows that the action of these non-specific kinases is normally countered by the activity of endogenous phosphatase activity. In the case of STAT92EM647H-GFP, however, JAK-independent phosphorylation of Y704 does not appear to be countered by endogenous phosphatases. Intriguingly, the possibility of an increased pY/SH2 affinity in STAT92EM647H-GFP is consistent with molecular modeling, based on the known structures of homo-dimerised STAT3. Using such an approach, it appears that vertebrate STAT5AN643H and Drosophila STAT92EM647H mutants both substitute residues with the potential to physically interact with the phospho-tyrosine residue of the dimerised partner. Whether the difference between wild type and STAT92EM647H is a consequence of biophysical factors such as an increased affinity between pY and the SH2 domain and/or decreased accessibility for phosphatases remains to be determined (Karsten, 2005).

A second question raised by the activity of STAT92EM647H-GFP is the nature of the mechanism by which STAT92E functions as a transcriptional activator. Numerous studies have shown that endogenous STAT92E acts as a transcriptional activator both in cell culture and in vivo and a number of target genes have been identified, which require active pathway signalling for their expression. Given that STAT92EM647H-GFP appears to be constitutively phosphorylated and DNA-bound the reason for its failure to activate transcription is less clear. Indeed, the STAT92EM647H-GFP molecule contains all domains present in wild type STAT92E and differs by only a single SH2 domain internal residue. While it is possible that the M647H substitution may result in unfolding or instability of a distinct trans-activation domain, this appears to be unlikely given the physical location of the mutated residue and the presence of histidine residues in analogous positions both within other STAT molecules and other SH2 domains shown be active in vivo. Rather, it seems that the full trans-activation activity of STAT92E is not an inherent characteristic of the molecule itself but is likely to require a second post-translational modification, in addition to tyrosine phosphorylation, that is missing in STAT92EM647H-GFP (Karsten, 2005).

Finally, the presence of endogenous histidine residues in mosquito and C. elegans STATs at the position mutated in Drosophila STAT92EM647H-GFP also raises the possibility that STAT-like molecules present in these organisms may be constitutively DNA-bound. Furthermore, despite the availability of genomic sequence data no JAK-like molecule has been identified in C. elegans. While inherently speculative, it is possible that STAT activity in C. elegans may be controlled by mechanisms independent of tyrosine phosphorylation, conceivably via the modulation of transcriptional activation activity (Karsten, 2005).

Identification of Drosophila genes modulating janus kinase/signal transducer and activator of transcription signal transduction

The JAK/STAT pathway was first identified in mammals as a signaling mechanism central to hematopoiesis and has since been shown to exert a wide range of pleiotropic effects on multiple developmental processes. Its inappropriate activation is also implicated in the development of numerous human malignancies, especially those derived from hematopoietic lineages. The JAK/STAT signaling cascade has been conserved through evolution and although the pathway identified in Drosophila has been closely examined, the full complement of genes required to correctly transduce signaling in vivo remains to be identified. A dosage-sensitive dominant eye overgrowth phenotype caused by ectopic activation of the JAK/STAT pathway was used to screen 2267 independent, newly generated mutagenic P-element insertions. After multiple rounds of retesting, 23 interacting loci that represent genes not previously known to interact with JAK/STAT signaling have been identified. Analysis of these genes has identified three signal transduction pathways, seven potential components of the pathway itself, and six putative downstream pathway target genes. The use of forward genetics to identify loci and reverse genetic approaches to characterize them has allowed assembly of a collection of genes whose products represent novel components and regulators of this important signal transduction cascade (Mukherjee, 2006).

Cell cycle proteins: The screen identified genes responsible for the modification of the overgrown eye phenotype associated with P{w+, GMR-updδ3'}. The eye overgrowth induced by P{w+, GMR-updδ3'} results from additional rounds of mitosis in eye-imaginal disc cells anterior to the morphogenetic furrow. Despite the ectopic JAK/STAT pathway activation caused by the misexpression of upd, these cells are patterned essentially normally and go on to form an increased number of ommatidia in the P{w+, GMR-updδ3'} eye disc. Despite this proliferation-dependent phenotype, core cell cycle regulatory proteins failed to show consistent interactions when assayed as part of a candidate approach. While unexpected, this result suggests that the core cell cycle regulatory proteins do not represent components that become rate limiting in the proliferative environment tested (Mukherjee, 2006).

Despite the lack of interaction with core cell cycle components, alleles of did, trbls, and Mob1 were identified as modifiers of the overgrown eye phenotype. Indeed, homozygous did mutants have been described as having small imaginal discs, and a phenotype similar to that is observed in hopM13 mutant third instar larval discs. While not central to cell cycle progression, these loci appear to be involved in its regulation and may imply that the interaction between JAK/STAT signaling and cellular proliferation is indirect (Mukherjee, 2006).

Of particular interest are the inconsistent interactions observed between Cdk4 alleles. Although cdk4 represents the only Drosophila component of the cell cycle machinery proposed to interact with the JAK/STAT pathway, the assay identified only one of the three alleles tested as a weak suppressor of the eye overgrowth phenotype. Previous studies did not utilize loss-of-function experiments but rather utilized the converse approach. When misexpressed by a P{w+, GMR-Gal4} driver, the coexpression of P{w+, UAS-CycD}, P{w+, UAS-Cdk4}, and P{w+, UAS-upd} dramatically enhanced the eye overgrowth phenotype over that mediated by P{w+, UAS-upd} or P{w+, UAS-CycD} and P{w+, UAS-Cdk4} alone. Although it is possible that loss of a single copy of the cdk4 locus does not reduce protein levels below a rate-limiting threshold, the inconsistency of interactions produced by multiple cdk4 alleles is puzzling and true existence or nature of any potential interaction between JAK/STAT signaling and endogenous Cdk4 remains to be established (Mukherjee, 2006).

Transcription factors and coregulators: A number of transcription factors were identified as interacting loci in the screen. One of these is the Drosophila homolog of the nuclear factor of activated T-cells (NFAT), a locus originally identified as an inducer of cytokine gene expression. Intriguingly, it has been shown that human NFAT, in conjunction with NF-kappaB, AP-1, and STATs, represents factors involved in mediating cytokine and T-cell-receptor-induced interferon-γ signaling. Intriguingly, activation of these transcription factors results in the production of numerous intrinsic antiviral factors in the vertebrate system, a role that has also been shown to depend on JAK/STAT signaling within Drosophila fat-body cells. Although further analysis of this interaction is required, this is the first report that suggests an evolutionarily conserved link between NFAT and JAK/STAT signaling in Drosophila (Mukherjee, 2006).

C-terminal binding protein (CtBP), a transcriptional corepressor previously characterized as an enhancer of the Drosophila JAK/STAT pathway, was also identified in the screen. While not all alleles of CtBP show consistent interaction with P{w+, GMR-updδ3'}, cell culture assays utilizing dsRNA-mediated knockdown imply that CtBP is a component of the JAK/STAT pathway, which acts as a positive regulator of signaling. In addition, an independent genomewide RNAi-based screen for JAK/STAT pathway interactors also identified dsRNAs targeting CtBP as a suppressor of pathway signaling. Finally, an upregulation of CtBP transcript is observed in P{w+, GMR-updδ3'} eye discs compared to wild-type eyes. Given the results from cell-based assays and in situ analysis, it appears most likely that CtBP does indeed represent a positive regulator of JAK/STAT pathway activity. This finding is particularly surprising, given the previously identified role for CtBP as a transcriptional repressor, which, in combination with the Groucho corepressor, is involved in repressing Su(H)-mediating expression of Notch pathway target genes. The significance of this result, however, remains to be determined and it is conceivable that the observed interaction with the eye overgrowth phenotype represents an indirect effect, possibly via interaction with Notch pathway signaling activity (Mukherjee, 2006).

Extracellular proteins: One aspect of the screen undertaken is the paracrine mode of Upd signaling required for cellular overproliferation. In the P{w+, GMR-updδ3'} eye, the region of upd expression is spatially separate from the domain in which increased levels of cellular proliferation are observed and the ligand must therefore be able to move to and activate the pathway in neighboring cells. Although it has been shown that Unpaired represents a secreted extracellular signaling molecule that is both post-translationally glycosylated and able to associate with the extracellular matrix (ECM), very little is known regarding the mechanisms regulating these processes (Mukherjee, 2006).

One class of molecules previously shown to be involved in the extracellular trapping and movement of signaling ligands is the heparan sulfate proteoglycans (HSPGs) Dally, Dally-like, Perlecan, and Syndecan. These molecules, and their extensive post-translational modifications, not only play important roles in providing shape and biomechanical strength to organs and tissues, but also have been shown to be required for the transduction of signaling by the Wingless, Hedgehog, and the FGF-like ligands Heartless and Breathless. Despite the significance of HSPGs for the transduction of these ligands, mutations in the HSPGs themselves, as well as mutations in the HSPG-modifying enzymes sugarless and sulphateless, do not appear to interact with the eye overgrowth phenotypes associated with P{w+, GMR-updδ3'} and suggest that Upd is likely to interact with the ECM via different mechanisms. One potential component of this alternative mechanism identified in the screen is Tenascin-major (Ten-m). Ten-M, also known as odd Oz, encodes an extracellular adhesion molecule that was also classified as a component of the JAK/STAT pathway in the tissue-culture-based paracrine signaling assay. Although the tissue culture results imply a direct function of the molecule in pathway signaling, further analysis of the role of Ten-m in controlling the secretion and/or movement of Upd remains to be determined in vivo (Mukherjee, 2006).

Signaling pathways: The Drosophila eye is dispensable in a laboratory environment and sensitized genetic screens that compromise its function have proven to be powerful tools for the identification of signal transduction pathway components. Drosophila eye development is, however, a complex process involving multiple signal transduction pathways including EGFR, Hh, Notch, Dpp, and Wingless. A number of examples of interactions between these pathways and JAK/STAT signaling have been described. For example, a gradient of four-jointed in the developing eye disc is determined by the coordinated activities of Notch, Wingless, and JAK/STAT pathways. Also, at the posterior dorso/ventral border of the eye, Notch and eye gone (eyg) have been shown to cooperatively induce expression of upd, which then acts to promote cell proliferation. Consistent with these complex interactions, the screen identified Bunched (bun), a member of the Dpp signal transduction pathway, and Bearded (brd), a member of the Notch signaling pathway. bunched is a transcription factor that genetically interacts with dpp. Strikingly, Dpp pathway components have previously been reported as modulators of the P{w+, GMR-updδ3'} eye phenotype, with hypomorphic alleles of dpp and Mothers against dpp (Mad) representing strong suppressors of eye overgrowth. Similar interactions in mammalian systems have identified the synergistic activity of STAT3 and Smad1 in the differentiation of astrocytes from their progenitor cells. These proteins, however, do not physically interact, but bind to p300/CBP to promote the transactivation of target genes (Mukherjee, 2006).

The screen also identified mth-like8, a seven-pass trans-membrane protein with predicted G-protein-coupled receptor activity. Although expression of mth-like8 changes in response to JAK/STAT pathway activation, an in-depth analysis of its interaction remains to be undertaken (Mukherjee, 2006).

Regulation of cell adhesion and collective cell migration by hindsight and its human homolog RREB1: HNT affected border cell cluster cohesion and motility via effects on the JNK and STAT pathways

Cell movements represent a major driving force in embryonic development, tissue repair, and tumor metastasis. The migration of single cells has been well studied, predominantly in cell culture; however, in vivo, a greater variety of modes of cell movement occur, including the movements of cells in clusters, strands, sheets, and tubes, also known as collective cell migrations. In spite of the relevance of these types of movements in both normal and pathological conditions, the molecular mechanisms that control them remain predominantly unknown. Epithelial follicle cells of the Drosophila ovary undergo several dynamic morphological changes, providing a genetically tractable model. This study found that anterior follicle cells, including border cells, mutant for the gene hindsight (hnt) accumulated excess cell-cell adhesion molecules and failed to undergo their normal collective movements. In addition, HNT affected border cell cluster cohesion and motility via effects on the JNK and STAT pathways, respectively. Interestingly, reduction of expression of the mammalian homolog of HNT, RREB1, by siRNA inhibited collective cell migration in a scratch-wound healing assay of MCF10A mammary epithelial cells, suppressed surface activity, retarded cell spreading after plating, and led to the formation of immobile, tightly adherent cell colonies. It is proposed that HNT and RREB1 are essential to reduce cell-cell adhesion when epithelial cells within an interconnected group undergo dynamic changes in cell shape (Melani, 2008).

To explore the mechanisms by which HNT affects cluster cohesion and motility, its effects on known signaling pathways were investigated. In the extraembryonic tissue known as the amnioserosa, hnt is a negative regulator of the JNK signaling cascade. Recently, the JNK pathway was shown to be active in the border cells and to affect border cell migration in clusters with reduced PVR activity. In addition, inhibition of the JNK cascade causes a phenotype that strikingly resembles the cluster dissociation phenotype caused by HNT overexpression, suggesting that HNT could be a negative regulator of the JNK pathway or vice versa. By using phospho-Jun antibody staining as a readout of the JNK signaling cascade, the activity of this pathway was seen to be reduced in border cells overexpressing hnt. In clusters in which JNK was reduced by overexpression of either Puckered (the JNK phosphatase) or a dominant-negative form of Basket (Drosophila JNK), cluster disassembly reminiscent of the hnt gain-of-function phenotype was observed. In addition, HNT was upregulated 1.7- and 1.4-fold, respectively. Together, these results indicate that hnt and JNK repress each other. In the embryo, in which HNT also antagonizes JNK, this pathway is required for the turnover of focal complexes and proper dorsal closure. Therefore, HNT appears to play a general role in remodeling of adhesion complexes to facilitate morphogenesis (Melani, 2008).

Although the cluster-disassembly phenotype of HNT could be attributed to effects on JNK signaling, JNK pathway mutations caused milder border cell motility defects than hnt. To determine whether HNT affected, in addition, one of the known border-cell-motility pathways, the effect of hnt on the activity of STAT and its key target SLBO was examined. STAT activation and nuclear translocation is the most upstream event in the differentiation of the border cells and is also required throughout border cell migration. It was found that, in border cells overexpressing HNT, nuclear accumulation of STAT was reduced though not eliminated. In addition, the levels of slbo were dramatically reduced in border cells overexpressing HNT. Because loss of function of either STAT or SLBO causes a dramatic migration defect, the effects of HNT overexpression on STAT and SLBO can account for the severe effect on motility. However, neither stat nor slbo mutant border cells exhibit a cluster-disassembly phenotype. Therefore, it is concluded that HNT mediates its effect on cluster cohesion via JNK and its effect on border cell motility primarily through STAT and SLBO (Melani, 2008).

Although HNT overexpression affects border cell motility via effects on STAT and SLBO, HNT has general effects on cell adhesion and morphogenesis, whereas SLBO appears to be more specific. For example, the effects of hnt on stretched follicle cells and in embryonic tissues are independent of SLBO because this protein is neither expressed nor required in these other cell types. Therefore, it is proposed that HNT plays a general role in regulating cell adhesion and morphogenesis via JNK signaling and a tissue-specific role in motility through STAT and SLBO. In this way, HNT can cooperate with tissue-specific factors to orchestrate a diverse array of collective cell movements (Melani, 2008).

Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT

Neuroblasts (NBs) generate a variety of neuronal and glial cells in the central nervous system of the Drosophila embryo. These NBs, few in number, are selected from a field of neuroepithelial (NE) cells. In the optic lobe of the third instar larva, all NE cells of the outer optic anlage (OOA) develop into either NBs that generate the medulla neurons or lamina neuron precursors of the adult visual system. The number of lamina and medulla neurons must be precisely regulated because photoreceptor neurons project their axons directly to corresponding lamina or medulla neurons. This study shows that expression of the proneural protein Lethal of scute [L(1)sc] signals the transition of NE cells to NBs in the OOA. L(1)sc expression is transient, progressing in a synchronized and ordered 'proneural wave' that sweeps toward more lateral NEs. l(1)sc expression is sufficient to induce NBs and is necessary for timely onset of NB differentiation. Thus, proneural wave precedes and induces transition of NE cells to NBs. Unpaired (Upd), the ligand for the JAK/STAT signaling pathway, is expressed in the most lateral NE cells. JAK/STAT signaling negatively regulates proneural wave progression and controls the number of NBs in the optic lobe. These findings suggest that NBs might be balanced with the number of lamina neurons by JAK/STAT regulation of proneural wave progression, thereby providing the developmental basis for the formation of a precise topographic map in the visual center (Yasugi, 2008).

NE cells are programmed to differentiate into NBs from the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to lateral optic lobe sweeping across the entire NE sheet; it is preceded by the transient expression of the proneural gene l(1)sc. As the NBs at the medial edge are oldest and the more lateral ones are youngest, developmental process of medulla neurons can be viewed as an array of progressively aged cells across optic lobe mediolaterally. This contrasts with NB formation in the embryonic CNS in which a small number of cells are selected from NE cells to become NBs, leaving the majority of NE cells to develop into non-neural cells. The optic lobe proneural wave is reminiscent of the morphogenetic furrow that moves across the developing eye imaginal disc. The morphogenetic furrow is the site where differentiation from neuroepithelium to photoreceptor neurons is initiated. The progression is driven by the secreted Hh expressed in the differentiated photoreceptor cells. By contrast, the proneural wave still progresses even when NB differentiation is impaired, suggesting that its progression is not driven by a factor emanating from differentiated NBs. No progression-defective phenotypes were observed when Hh or Decapentaplegic (Dpp) signaling was reduced. The model is favored that the proneural wave progression is driven by an intrinsic mechanism such as a segmentation clock and is negatively regulated by JAK/STAT pathway. As the JAK/STAT ligand Upd is expressed only by the most lateral NE cells, proliferation of the NE cells moves the source of ligand laterally and as a consequence releases more medial NE cells from negative regulation and allows the proneural wave to progress laterally. Alternatively, distribution of the Upd ligand and/or the response to Upd changes as the NE cells age as graded 10xSTAT-GFP activities are more prominent in the early stage. Non-autonomous action of JAK/STAT signal indicates that it does not directly regulate L(1)sc expression and there are second signal(s) that regulate the expression of L(1)sc under the control of JAK/STAT signal (Yasugi, 2008).

Three out of the four AS-C genes [sc, l(1)sc and ase] are expressed during medulla neurogenesis. l(1)sc is expressed in NE cells and ase in NBs, while sc is expressed both in NE cells and NBs. Deleting all AS-C genes causes as significant delay as da in NB formation but does not completely eliminate NB formation, suggesting that Da-dependent proneural gene activities are required for timely onset of NB formation. Mutation for sc or ase alone does not affect NB formation, but the simultaneous deletion of sc and l(1)sc causes the delay in NB formation and the additional deletion of ase further delays NB formation. ase expression is not altered in the absence of l(1)sc and l(1)sc is not altered in the absence of ase, indicating that l(1)sc and ase both contribute to the differentiation from NE cells to NBs. Although the contribution of Sc cannot be formally excluded, the highly specific expression pattern led to the inference that L(1)sc plays a major role in the proneural wave (Yasugi, 2008).

JAK/STAT signaling is known to regulate stem cell maintenance in the adult germline of Drosophila. In the male testis, germline stem cells (GSCs) attach to a cluster of somatic support cells at the tip (hub) of the testis. When a GSC divides, the daughter retaining contact with the hub maintains self-renewing GSC identity, while the other daughter differentiates into gonialblast. Upd is specifically expressed in the hub cells and activates JAK/STAT signal in the GSCs to maintain stem cell state. In the female ovary, JAK/STAT signaling is required in the somatic escort stem cells whose daughters encase developing cysts. This study shows that in the optic lobe development, JAK/STAT signaling maintains NE cells in an undifferentiated state. It is suggested that a common mechanism operates in both these developmental systems. Loss of Hop or Stat92E function decreases number of stem cells and ectopic expression of Upd results in over proliferation of undifferentiated cells. The cell fate may be determined by the distance of the cells from the source of ligand; the cells farther from the source commence to differentiate (Yasugi, 2008).

In the vertebrate CNS, NE cells first proliferate by symmetric cell divisions and differentiate into neurons and glia in later developmental stages. JAK/STAT signaling has been implicated in maintenance of neural precursor cells, but there is no clear evidence that those cells are in the same developmental stage as described in this study for Drosophila. Further study of JAK/STAT signaling will reveal whether a common mechanism underlies stem cell development in both Drosophila and vertebrates, and should give new insights into vertebrate CNS neurogenesis (Yasugi, 2008).

Development of a precise topographic map (retinotopic map) in Drosophila is known to involve regulation of lamina neuron development with respect to the incoming R axons. The lateral NE sheet is continuous with a groove called the lamina furrow where NE cells are arrested at G1/S phase. The arriving R axons deliver Hh and liberate the arrested NE cells to proliferate and develop into lamina neuron precursors. And, thus, R axons can induce the development of their synaptic partners in their vicinity to balance the number of R axonal termini and lamina neurons. However, medulla development does not depend on inputs from the R axons in the early phase. This study shows that both lamina and medulla neurons are derived from the continuous NE sheet. Large clones of cells mutant for the JAK/STAT signaling cause immature proliferation of medulla NBs at the expense of lamina neurons, suggesting that the number of NE cells serves as the limiting factor to generate precursors for lamina and medulla neurons. Thus, the number of medulla neurons is roughly regulated at the level of NBs whose generation might be balanced indirectly with the number of lamina neurons through regulating proneural wave progression by JAK/STAT signaling. JAK/STAT signaling therefore plays an important role in the formation of a precise retinotopic map in the visual center (Yasugi, 2008).

Invasive and indigenous microbiota impact intestinal stem cell activity through JAK-STAT and JNK pathways in Drosophila

Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. This study addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. The increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, evidence is provided that the JAK-STAT and JNK pathways are both required for bacteria-induced stem cell proliferation. Similarly, it was demonstrated that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, it was shown that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity (Buchon, 2009b).

The JAK-STAT and JNK signaling pathways are required to maintain gut homeostasis upon exposure to a broad range of bacteria. In normal conditions, low levels of the indigenous gut microbiota and transient environmental microbes maintain a basal level of epithelium renewal. The increase in gut microbes in old or Imd-deficient flies is associated with a chronic activation of the JNK and JAK-STAT pathways, leading to an increase in intestinal stem cells (ISC) proliferation and gut disorganization. The impact of pathogenic bacteria can have different outcomes on gut homeostasis, depending on the degree of damage they inflict on the host. Damage to the gut caused by infection with E. carotovora is compensated for by an increase in epithelium renewal. Infection with a high dose of P. entomophila disrupts the homeostasis normally maintained by epithelium renewal and damage is not repaired, contributing to the death of the fly (Buchon, 2009b).

Previous studies have shown that the NADPH oxidase Duox plays an essential role in Drosophila gut immunity by generating microbicidal effectors such as ROS to eliminate both invasive and dietary microbes. Ecc15 is a potent activator of Duox, which in turn is important in the clearance of this bacterium. This oxidative burst is coordinated with the induction of many genes involved in ROS detoxification upon Ecc15 ingestion. This study provides evidence that the observed increase in epithelium renewal upon Ecc15 infection is a compensatory mechanism that repairs the damage inflicted to the gut by this oxidative burst. This is supported by the observation that reducing ROS levels by either the ingestion of antioxidants or silencing the Duox gene reduces epithelium renewal. Although ISC proliferation could be directly triggered by ROS, it is more likely a consequence of signals produced by stressed enterocytes. A number of data support this hypothesis: (1) Ingestion of corrosive agents can also induce ISC proliferation, and (2) physical injury is sufficient to induce local activation of the cytokine Upd3, which promotes epithelium renewal. Interestingly, a significant increase in epithelium renewal was observed in Duox RNAi flies at late time points following infection, correlating with damage attributed to the proliferation of Ecc15 in the guts of Duox-deficient flies. While the increase in epithelium renewal observed with Ecc15 is clearly linked to the damage induced by the host immune response, it is likely that effects on epithelium renewal by other pathogens could be more direct and mediated by virulence factors, such as the production of cytolytic toxins (Buchon, 2009b).

The data indicate that the JAK-STAT and JNK pathways synergize to promote ISC proliferation and epithelium renewal in response to the damage induced by infection. The JAK-STAT pathway is implicated in the regulation of stem cells in multiple tissues and is proposed to be a common regulator of stem cell proliferation. The data extend this observation by showing that the JAK-STAT pathway is also involved in ISC activation upon bacterial infection. The cytokine Upd3 is produced locally by damaged enterocytes and subsequently stimulates the JAK-STAT pathway in ISCs to promote their proliferation. The results globally agree with a recent study showing that the JAK-STAT pathway is involved in ISC proliferation upon infection with a low dose of P. entomophila (Jiang, 2009). This work and the current study clearly demonstrate that the JAK-STAT pathway adjusts the level of epithelium renewal to ensure proper tissue homeostasis by linking enterocyte damage to ISC proliferation. The study by Jiang also uncovered an additional role of this pathway in the differentiation of enteroblasts during basal gut epithelium turnover. The implication of the JAK-STAT pathway in differentiation could explain the accumulation of the small-nucleated escargot-positive cells observed in the gut of flies with reduced JAK-STAT signaling in ISCs. The JAK-STAT pathway was also shown previously to control the expression of some antimicrobial peptides such as Drosomycin 3 (Dro3). Therefore, the JAK-STAT pathway has a dual role in the gut upon infection, controlling both the immune response and epithelium renewal (Buchon, 2009b).

The data show that the lack of JNK pathway activity in ISCs results in the loss of ISCs in guts infected with Ecc15, thus preventing epithelium renewal. The findings are consistent with the attributed function of JNK at the center of a signal transduction network that coordinates the induction of protective genes in response to oxidative challenge. This cytoprotective role against ROS would protect ISCs from the oxidative burst induced upon Ecc15 infection, explaining why ISCs die by apoptosis when JNK activity is reduced. It is likely that JNK signaling is required not only to protect ISCs from oxidative stress, but also to induce stem cell proliferation to replace damaged differentiated cells. This is supported by the observation that overexpression of the JNKK Hep in ISCs is sufficient to trigger an epithelium renewal in the absence of infection. In addition, increased JNK activity in ISCs of old flies has been linked to hyperproliferative states and age-related deterioration of the intestinal epithelium. This study shows that JNK signaling is also required for epithelium renewal upon Ecc15 infection. Thus, infection with Ecc15 recapitulates in an accelerated time frame the impacts of increased stress observed in guts of aging flies (Buchon, 2009b).

The inhibition of the dJun transcription factor in ISCs leads to a loss of stem cells in the absence of infection, suggesting that this transcription factor plays a critical role in ISC maintenance in the gut. There is no definitive explanation for why the dJun-IR construct behaves differently than the basket and hep-IR constructs. It is speculated that this could be due to (1) differences in the basal activity of the JNK pathway, which would be blocked only with the dJun-IR that targets a terminal component of the pathway; (2) effects of Jun in ISCs independent of the JNK pathway; or (3) side effects of the dJun-IR construct (Buchon, 2009b).

In contrast to the requirement of the JNK pathway upon Ecc15 infection, it has been reported that oral ingestion with a low dose of P. entomophila still induced mitosis in the JNK-defective mutant hep1. In agreement, this study found that inhibiting the JNK pathway in ISCs did not block the induction of epithelium renewal by a low dose of P. entomophila. This difference in the requirement of the JNK pathway may be explained by the nature of these two pathogens. Whereas Ecc15 damages the gut through an oxidative burst that activates the JNK pathway, the stimulation of epithelium renewal by P. entomophila could be due to a more direct effect of this bacterium on the gut. Altogether, this work points to an essential role of the JAK-STAT pathway in modulation of epithelium renewal activity, while the role of JNK may be dependent on the infectious agent and any associated oxidative stress. While it is known that the JNK pathway is activated by a variety of environmental challenges including ROS, the precise mechanism of activation of this pathway has not been elucidated. Similarly, the molecular basis of upd3 induction in damaged enterocytes is not known. Future work should decipher the nature of the signals that activate these pathways in both ISCs and enterocytes, as well as the possible cross-talk between the JNK and JAK-STAT pathways in ISC control (Buchon, 2009b).

The observation that flies unable to renew their gut epithelium eventually succumb to Ecc15 infection highlights the importance of this process in the gut immune response. It is striking that defects in epithelium renewal are more detrimental to host survival than deficiency in the Imd pathway, even though this pathway controls most of the intestinal immune-regulated genes induced by Ecc15. The results are in agreement with a previous study indicating that, in the Drosophila gastrointestinal tract, the Imd-dependent immune response is normally dispensable to most transient bacteria, but is provisionally crucial in the event that the host encounters ROS-resistant microbes. However, this study demonstrates that efficient and rapid clearance of bacteria in the gut by Duox is possible only when coordinated with epithelium renewal to repair damage caused by ROS. This finely tuned balance between bacterial elimination by Duox activity and gut resistance to collateral damage induced by ROS is likely the reason why flies normally survive infection by Ecc15. Yet, this calibration also exposes a vulnerability that could easily be manipulated or subverted by other pathogens. Along this line, this work also exposes the range of impact different bacteria can have on stem cell activation. It was observed that infection with high doses of P. entomophila led to a loss of gut integrity, including the loss of stem cells. Moreover, the ability of P. entomophila to disrupt epithelium renewal correlates with damage to the gut and the death of the host. Since both JNK and JAK-STAT pathways are activated upon infection with P. entomophila, this suggests that this bacterium activates the appropriate pathways necessary to repair the gut, but ISCs are unable to respond accordingly. Interestingly, a completely avirulent P. entomophila mutant (gacA) does not persist in the gut and does not induce epithelium renewal. In contrast, an attenuated mutant (aprA) somewhat restores epithelium renewal. These observations, along with the dose response analysis using P. entomophila and corrosive agents, suggest that the virulence factors of this entomopathogen disrupt epithelium renewal through excessive damage to the gut. Of note, recent studies suggest that both Helicobacter pylori and Shigella flexneri, two bacterial pathogens of the human digestive tract, interfere with epithelium renewal to exert their pathological effects. This suggests that epithelium renewal could be a common target for bacteria that infect through the gut. In this respect, the host defense to oral bacterial infection could be considered as a bimodular response, composed of both immune and homeostatic processes that require strict coordination. Disruption of either process results in the failure to resolve the infection and impedes the return to homeostasis (Buchon, 2009b).

In contrast to the acute invasion by pathogenic bacteria, indigenous gut microbiota are in constant association with the gut epithelium, and thus may impact gut homeostasis. Using axenically raised flies, it was established that indigenous microbiota stimulate a basal level of epithelium renewal that correlates with the level of activation of the JAK-STAT and JNK pathways. This raises the possibility that both indigenous and invasive bacteria, such as Ecc15, are capable of triggering epithelium renewal by the same process. Additionally, the data support a novel homeostatic mechanism in which the density of indigenous bacteria is coupled to the level of epithelium renewal. This is the first report that gut microbiota affect stem cell activation and epithelium renewal, concepts proposed previously in mammalian systems but never fully demonstrated. This also implies that variations in the level of epithelium renewal observed in different laboratory contexts could actually be due to impacts from gut microbes (Buchon, 2009b).

Importantly, in this context, it was shown that lack of indigenous microbiota reverts most age-related deterioration of the gut. Aging of the gut is usually marked by both hyperproliferation of ISCs and differentiation defaults that lead to disorganization of the gut epithelium. These alterations have been shown to be associated with activation of the PDGF- and VEGF-related factor 2 (Pvf2)/Pvr and JNK signaling pathways directly in ISCs. Accordingly, inhibition of the JNK pathway in ISCs fully reverts the epithelium alterations that occur with aging. This raises the possibility that gut microbiota could exert their effect through prolonged activation of the JNK pathway. Interestingly, immune-deficient flies, lacking the Imd pathway, also display hyperproliferative guts and have higher basal levels of activation of the JNK and JAK-STAT pathways. The observation that these flies also harbor higher numbers of indigenous bacteria further supports a model in which failure to control gut microbiota leads to an imbalance in gut epithelium turnover. Future work should analyze the mechanisms by which gut microbiota affect epithelium renewal and whether this is due to a direct impact of bacteria on the gut or is mediated indirectly through changes in fly physiology. Moreover, the correlation between higher numbers of indigenous bacteria and increased disorganization of the gut upon aging in flies lacking the Imd pathway raises the possibility that a main function of this pathway is to control gut microbiota. This is in agreement with concepts emerging in mammals that support an essential role of the gut immune response in maintaining the beneficial nature of the host-microbiota association. This function also parallels the theory of 'controlled inflammation' described in mammals, where a low level of immune activation is proposed to maintain gut barrier integrity (Buchon, 2009b).

In conclusion, this study unravels some of the complex interconnections between the immune response, invasive and indigenous microbiota, and stem cell homeostasis in the gut of Drosophila. Based on the evolutionary conservation of transduction pathways such as JNK and JAK-STAT between Drosophila and mammals, it is likely that similar processes occur in the gut of mammals during infection. Interestingly, stimulation of stem cell activity by invasive bacteria is proposed to favor the development of hyperproliferative states found in precancerous lesions. Thus, Drosophila may provide a more accessible model to elucidate host mechanisms to maintain homeostasis and the impact of bacteria on this process (Buchon, 2009b).

JAK/Stat signaling regulates heart precursor diversification in Drosophila

Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the JAK/Stat pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, this study shows that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. The E(spl)-C gene HLHm5 is shown to be expressed in a pattern complementary to tin during phase 3, and this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain (Johnson, 2011).

tin expression can be divided into four distinct spatial-temporal phases. Phase 1 tin expression initiates after gastrulation during which Twist (Twi) activates pan-mesodermal tin expression via the enhancer tinB. Phase 2 begins after FGF-mediated mesoderm spreading in which Dpp signals produced by the dorsal ectoderm maintain tin expression throughout the dorsal mesoderm via a second enhancer, tinD. It is during phase 2 that Tin specifies the major dorsal mesoderm derivatives. Phase 3 initiates after dorsal mesoderm progenitor specification and is characterized by a pronounced restriction of tin to the cardiac and visceral muscle progenitors. Upd and Upd2 are expressed in the ventral ectoderm during the transition from phase 2 to phase 3 expression. Phase 4 initiates after precursor specification and is characterized by further restriction of tin to the cardiac precursors that give rise to the contractile cardiomyocytes and the noncontractile pericardial nephrocytes. Phase 4 expression further directs heart cell diversification and maturation and is dependent on a third enhancer element, tinC (Johnson, 2011 and references therein).

To test the hypothesis that the JAK/Stat pathway functions in the cardiac-specific gene regulatory network, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. The JAK/Stat pathway regulates final cardiac morphology as well as heart precursor diversification. Stat92E loss-of-function analysis identified a discrete function for the JAK/Stat pathway in restricting tin during the transition from phase 2 to phase 3 expression. In addition, Stat92E embryos have an expanded pericardial cell domain arguing that the JAK/Stat pathway regulates tin to ensure proper heart precursor diversification. Mechanistically, it was found that the E(spl)-C gene HLHm5 is expressed in a complementary pattern to tin during phase 3 expression and that the JAK/Stat pathway activates HLHm5 expression in the dorsal mesoderm. The E(spl)-C genes in turn repress twi expression to preserve cardiac morphology and restrict tin and H15 expression to direct heart precursor diversification. These findings provide the first evidence of a role for the JAK/Stat pathway in cardiogenesis and identify a novel tin autoinhibitory circuit involving Stat92E and E(spl)-C (Johnson, 2011).

Stat92E is a direct Tin target gene during phase 2 expression; however, Stat92E is expressed in segmented stripes at this stage whereas tin is expressed throughout the dorsal mesoderm. In addition, embryos lacking only the maternal contribution of Stat92E have mesoderm patterning defects. Tin-regulated Stat92E zygotic transcription is therefore insufficient to coordinate mesoderm development. These data suggest that maternally contributed Stat92E is activated in response to segmented Upd and Upd2 activity, binds the Stat92E locus and co-activates Stat92E zygotic transcription with Tin. In addition, ChIP-chip experiments identified Stat92E binding activity and a conserved Stat92E consensus binding sites (SCBS) within the Tin-responsive Stat92E mesoderm enhancer. It is concluded that Stat92E and tin co-regulate a brief, spatially restricted JAK/Stat signaling event that patterns the mesoderm (Johnson, 2011).

Phase 3 tin expression promotes cell-type diversification and differentiation within the dorsal mesoderm and is indirectly activated by Wg via the T-box transcription factors in the Dorsocross complex and the GATA factor Pannier. A key finding of this study is that the JAK/Stat pathway activates the transcriptional repressor HLHm5 in the dorsal mesoderm to establish phase 3 tin expression. Because the HLHm5 cis-regulatory region lacks a conserved SCBS, it is predicted that Stat92E regulates HLHm5 expression through a non-consensus binding site. Alternatively, Stat92E acts at long distances to regulate gene expression. The SCBSs in E(spl)-C could be a platform from which Stat92E regulates multiple E(spl)-C genes that, in turn, regulate HLHm5 expression. In either event, Stat92E-mediated activation of E(spl)-C genes restricts tin in the dorsal mesoderm to establish phase 3 expression. Tin, therefore, establishes an autoinhibitory circuit by activating its own repressors in the JAK/Stat pathway and in E(spl)-C (Johnson, 2011).

Both Stat92E and Df(3R)Esplδmδ-m6 embryos show an increased number of Tin+ pericardial cells and an expanded H15 expression domain. Misexpressing mid or H15 in the mesoderm expands the number of Tin+ cells in the dorsal vessel and embryos misexpressing mid show a phenotype strikingly similar to Stat92E and E(spl) embryos. As mid, and presumably H15, are positively regulated by Tin during St11/12, unrestricted tin expression in Stat92E or Df(3R)Esplδmδ-m6 embryos expands the H15 expression domain. Ectopic H15 then specifies supernumerary Tin+ pericardial cells. Because mid expression is not affected in Stat92E embryos, the expression of mid and H15 must be controlled by distinct mechanisms and might have non-redundant functions (Johnson, 2011).

Although the Twi target genes directing mesoderm development and subdivision have been studied in detail, the molecular mechanism that restricts twi expression after gastrulation remains unclear. One regulator of twi is the Notch signaling pathway, which acts through E(spl)-C genes to restrict twi expression. However, Notch signaling appears to be active throughout the mesoderm after gastrulation. This study suggests that segmented JAK/Stat signaling activity differentially upregulates E(spl)-C gene expression in concert with Notch to produce periodic twi expression in the mesoderm. In addition, pan-mesodermal twi expression causes cardiac defects independently of cell fate specification, suggesting that the cardiac morphology defects in Stat92E embryos are due to dysregulated twi expression. These results highlight a previously unrecognized role for the JAK/Stat pathway and Twi in regulating cardiogenesis (Johnson, 2011).

Pericardial cell hyperplasia without a concomitant loss of cardioblasts has been reported for dpp hypomorphic embryos and lame duck (lmd) embryos. A late Dpp signal, which occurs after the Dpp signal that regulates phase 2 tin expression, instructs the Gli-like transcription factor Lmd to repress Tin expression in fusion competent myoblasts (FCMs). Tin expression appears to expand into the FCM domain in Stat92E embryos; however, the closest Stat92E chromatin binding domain is over 120 kb distal to the lmd transcriptional start site. This study highlights the possibility that sequential JAK/Stat and then Dpp signals regulate Lmd function to direct heart precursor diversification (Johnson, 2011).

In vertebrates, skeletal myogenesis initiates with the periodic specification of somites in the presomitic mesoderm. Cyclical expression of hairy1 in the chick, the hairy- and E(spl)-related family (Her) in zebrafish, and the orthologous Hes family in mice are under the control of Notch-Delta signaling. Loss of her1 and her7 alters the periodicity with which somite boundaries are specified in fish, and artificially stabilizing Hes7 causes somites to fuse in the mouse. Thus, mesoderm segmentation is governed by Notch-Delta regulation of the E(spl)-C genes in both insects and vertebrates indicating that the two processes share molecular homology. A cell culture model of somitogenesis shows that oscillating Hes1 expression is dependent on Stat activity. This study supports the exciting possibility that JAK/Stat signaling and E(spl)-C form a conserved developmental cassette directing mesoderm segmentation throughout Metazoa (Johnson, 2011).

Characterization of a dominant-active STAT that promotes tumorigenesis in Drosophila

Little is known about the molecular mechanisms by which STAT proteins promote tumorigenesis. Drosophila is an ideal system for investigating this issue, as there is a single STAT (Stat92E), and its hyperactivation causes overgrowths resembling human tumors. This study reports the first identification of a dominant-active Stat92E protein, Stat92E(δNδC), which lacks both N- and C-termini. Mis-expression of Stat92E(δNδC)in vivo causes melanotic tumors, while in vitro it transactivates a Stat92E-luciferase reporter in the absence of stimulation. These gain-of-function phenotypes require phosphorylation of Y(711) and dimer formation with full-length Stat92E. Furthermore, a single point mutation, an R(442P) substitution in the DNA-binding domain, abolishes Stat92E function. Recombinant Stat92E(R442P) translocates to the nucleus following activation but fails to function in all assays tested. Interestingly, R(442) is conserved in most STATs in higher organisms, suggesting conservation of function. Modeling of Stat92E indicates that R(442) may contact the minor groove of DNA via invariant TC bases in the consensus binding element bound by all STAT proteins. It is concluded that the N- and C-termini function unexpectedly in negatively regulating Stat92E activity, possibly by decreasing dimer dephosphorylation or increasing stability of DNA interaction, and that Stat92E(R442) has a nuclear function by altering dimer:DNA binding (Ekas, 2010).

This study used in vivo and in vitro assays to perform a structure-function analysis of Stat92E. Neither the N- terminus (residues 1-133) nor the C-terminus (residues 725-761) is required for Stat92E function in vivo and in vitro. However, when both N- and C-termini are removed, the resulting protein Stat92EΔNΔC has dominant-active properties, including oncogenesis. Furthermore, Stat92EΔNΔC with a Tyr to Phe substitution at residue 711 (Stat92EΔNΔCY711F) is non-functional. It was also shown that both Stat92EY711F and Stat92ER442P are non-functional and manifest dominant-negative activities in vivo. The lack of function of the Stat92EY711F variant is likely due to an inability to form an activated dimer. The data suggest that Stat92ER442P cannot function because it does not maintain normal interactions between activated Stat92E dimers and cognate DNA (Ekas, 2010).

This study is important for several reasons, perhaps foremost of which is the identification of a dominant-active Stat92E. The simultaneous removal of the N-terminal 133 and the C-terminal 36 amino acids results in a truncated protein that has constitutive activity, which causes melanotic tumors when mis-expressed in wild type larvae and transactivates a Stat92E reporter gene in vitro and in vivo in the absence of stimulation. The activity of Stat92EΔNΔC is dependent on the presence of Y711, as is that of Stat92EFL. These data suggest that Stat92EΔNΔC is activated in a manner similar to full length Stat92E (i.e., by reciprocal interactions between SH2 domains and phosphorylated Y711 residues on adjacent Stat92E proteins). Consistent with this, it was shown that Stat92EΔNΔC forms a dimer with endogenous Stat92E (i.e., a Stat92E: Stat92EΔNΔC dimer) after pervanadate stimulation. Therefore, Stat92EΔNΔC appears to behave like wild type Stat92E that has lost a negative regulatory component. One possibility is that removal of the N- and C-termini prolongs the interaction between the Stat92E:Stat92EΔNΔC dimer and DNA, leading to increased gene activation. Another is that Stat92E:Stat92EΔNΔC dimers cannot be efficiently dephosphorylated and/or return to an inactive dimer conformation, which may be required for normal export of Stat92E dimers to the cytoplasm. Constitutive activity is not observed by the removal of either the N or C-terminus domain individually, and therefore, deletion of the both domains must contribute to this activity. The constitutive activity of Stat92EΔNΔC in vitro is significantly robust that it could be used for RNAi screens to identify enhancers and suppressors of Stat92E activity, which may have conserved functions in restricting tumorigenesis in mammals. In addition, the high rate of melanotic tumor formation observed in clones mis-expressing Stat92EΔNΔC will make this mutation an extremely useful in vivo reagent as well (Ekas, 2010).

One unresolved issue is how the constitutive activity of Stat92EΔNΔC is initiated in the first place. The model described above predicts that in the absence of stimulation (1) Stat92E:Stat92EΔNΔC dimers should be detected, if only at very low levels, and/or (2) Stat92EΔNΔC should be tyrosine phosphorylated, again if only at very low levels. Unexpectedly neither result is observed. To rectify these results, a model involked in which a few activated STAT dimers (in this case Stat92E:Stat92EΔNΔC dimers) are constantly generated within a cell, and their activity is quenched by tyrosine phosphatases. Although it has not been possible to detect these dimers, this is a plausible model because treatment of a cell with vanadate, a pan-phosphatase inhibitor, activates STATs in a ligand-independent manner, presumably by inhibiting the enzymes that keep their low level of activation in check. It is hypothesized that Stat92E:Stat92EΔNΔC dimers may better evade the actions of tyrosine phosphatases, resulting in a steady state of increased STAT signaling in a cell and ultimately leading to sustained, increased expression of STAT targets and, in certain cell types like the blood, oncogenesis (Ekas, 2010).

This study also discovered that Stat92E lacking either the N- or the C- terminus functioned similarly to Stat92EFL. They rescued the eye/antennal/head phenotypes, as well as the hatching rate, and they shifted the lethality in either 85C9/85C9 or 397/397 zygotic mutants. The fact that a transgene encoding a Stat92E lacking the last 36 amino acids could rescue the phenotypes associated with homozygosity for the 397 allele, which is predicted to encode a protein lacking all amino acids after Trp594, suggests that the transactivation domain of Stat92E does not residue at the C-terminus, since a Stat92EΔC: Stat92E397 dimer would lack any residue after 724. Alternatively, there could be more than one transactivation domain in Stat92E, one at the C-terminus and the other located elsewhere in the protein. The proteins encoded by the Stat92E85C9 and Stat92E397 alleles are predicted to have N-terminal domains. Previous work has shown that two N-termini are required for the formation of non-phosphorylated dimers, which are the preferred substrated of receptor/JAK complexes and are required for cytokine-dependent activation of STAT4. As such, the single N-terminus present in any potential dimers between Stat92EΔN and Stat92E85C9 or between Stat92EΔN and Stat92E397 is unlikely to support dimer formation/activation in Stat92E85C9 and Stat92E397 homozygous mutant cells, respectively (Ekas, 2010).

This study is also noteworthy in that it is the first analysis of Stat92E structure-function variants that employs in vivo rescue assays in a Stat92E homozygous mutant background. It was previously reported that 90% of animals carrying large patches of Stat92E homozygous mutant tissue in the eye-antennal disc fail to hatch but that these phenotypes were rescued when these animals expressed a full-length version of Stat92E in the eye-antennal disc. This study now shows that mis-expression of Stat92EFL in 85C9/85C9 zygotic mutants significantly shifted the lethal phase, from embryonic/first larval instar to third larval instar/early pupa. Currently it is not understood why the UASP-3HA-Stat92EFL transgene did not rescue to 85C9/85C9 homozygotes to adulthood, but one possibility is low expression. Transgenes driven by UASP, which are designed for expression in the germ-line, are expressed at significantly lower levels than those driven by UAST, which are expressed in the soma, and the levels of Stat92E required to rescue to 85C9 or 397 zygotic mutants to adulthood simply might not have been achieved (Ekas, 2010).

This is the first study to demonstrate that Tyr711 is required for Stat92E function in vivo, which is consistent with in vitro data that a substitution of Tyr711 to Phe abolished the ability of Stat92E to bind DNA. However, no reduction in eye size was observed when this Stat92E construct was over-expressed in a wild type fly eye. It is thought that this is due to the inability to over-express this construct at high enough levels to inhibit the function of endogenous Stat92E. Finally, it was also shown that an Arg residue is specifically required at position 442 for Stat92E function. A homology model of Stat92E suggests that Arg442 is involved in DNA binding and may be a recognition element for the binucleotide TTCnnnGAA in the Stat92E binding site, suggesting that this interaction may be important for Stat92E recognition of its DNA target site. The Arg442 of Stat92E is conserved in the C. elegans STAT, STA-1, and in mammalian STAT2,3,5a,5b and 6. Therefore, it will be interesting in the future to determine if this residue is also required for the function of STAT proteins in which this Arg residue is conserved (Ekas, 2010).

Cytokine signaling through the JAK/STAT pathway is required for long-term memory in Drosophila

Cytokine signaling through the JAK/STAT pathway regulates multiple cellular responses, including cell survival, differentiation, and motility. Although significant attention has been focused on the role of cytokines during inflammation and immunity, it has become clear that they are also implicated in normal brain function. However, because of the large number of different genes encoding cytokines and their receptors in mammals, the precise role of cytokines in brain physiology has been difficult to decipher. This study took advantage of Drosophila's being a genetically simpler model system to address the function of cytokines in memory formation. Expression analysis showed that the cytokine Upd is enriched in the Drosophila memory center, the mushroom bodies. Using tissue- and adult-specific expression of RNAi and dominant-negative proteins, it was shown that not only is Upd specifically required in the mushroom bodies for olfactory aversive long-term memory but the Upd receptor Dome, as well as the Drosophila JAK and STAT homologs Hop and Stat92E, are also required, while being dispensable for less stable memory forms (Copf, 2011).

Using the Drosophila olfactory aversive learning paradigm in combination with a conditional tissue-specific expression system, this study has shown that cytokine signaling through the JAK/STAT pathway is necessary for protein synthesis-dependent LTM but is dispensable for less stable forms of memory. All four major components of this pathway -- the extracellular cytokine Upd, the cytokine receptor Dome, the tyrosine kinase Hop, and the transcription factor Stat92E -- are required within the MBs, the major olfactory learning and memory center for LTM processing (Copf, 2011).

Although cytokine signaling may be required for normal health and physiology of the MBs, this hypothesis is not favored because neither learning nor ARM formation are affected when this signaling pathway is compromised. Rather, it is suggested that the JAK/STAT pathway is specifically recruited for LTM processing. The requirement for de novo gene expression during LTM formation has been widely observed in a number of different model systems. Much attention has been focused on the role of transcription factor cAMP response element-binding protein (CREB) as an LTM-specific regulator of gene expression in Drosophila and other species. A number of other transcription factors have also been found to play an important role in LTM, including Adf-1 in Drosophila and CCAAT/enhancer-binding protein (C/EBP), Zif-268, AP-1, and NF-κB in mammals. Although the JAK/STAT pathway has been shown to be involved in diverse biological processes in flies, this study identifies a role in Drosophila adult brain physiology and behavioral plasticity. In addition, despite the plethora of studies examining the impact of cytokines in memory formation, the experiments presented in this study demonstrate that JAK/STAT signaling contributes to the transcriptional regulation thought to underlie synaptic plasticity and long-lasting memory (Copf, 2011).

To understand how Stat92E modulates memory, it will be necessary to identify its transcriptional targets in the adult MBs. Identification of such target genes could be approached by using bioinformatics and/or transcription profiling. Recent profiling studies have identified a number of putative Stat92E target genes in the Drosophila eye disk and hematopoietic system, some of which include Notch signaling pathway genes that have already been implicated in LTM. Another mode of action of JAK/STAT signaling in LTM could be through chromatin remodeling. Recent findings have identified a noncanonical mode of JAK/STAT signaling that directly regulates heterochromatin stability and cellular epigenetic status, affecting expression of genes beyond those under direct Stat92E transcriptional control. Finally, given that regulation of the actin cytoskeleton is central to both cell motility and neuronal structural plasticity, it will be interesting to determine whether some of the mechanisms by which JAK/STAT signaling drives border cell migration in the Drosophila germ line are also relevant to the formation of stable memories in the MBs (Copf, 2011).

These experiments demonstrate a clear positive role for signaling by the cytokine Upd in olfactory aversive memory, and, in doing so, they contribute to a lively debate as to the role played by cytokines in memory. Mammalian studies in which levels of proinflammatory cytokines are increased to pathogenic levels, either through direct injection or indirectly via induction of inflammation through injection of lipopolysaccharide or bacteria, tend to suggest that augmented cytokine signaling is detrimental for performance in a variety of learning and memory assays. This negative impact of cytokine signaling on memory is supported by studies that take a loss-of-function approach to address the physiological function of interleukins and their receptors in different cognitive tasks under nonpathological conditions. In contrast, several studies describe learning and memory defects attributed to loss of function of other cytokines or their receptors, using a variety of behavioral assays. Thus, despite significant efforts, understanding of the molecular and cellular basis for interactions between the cytokine network and learning and memory remains limited. The complexity of mammalian cytokine signaling, with its vast array of genes encoding ligands, receptors and downstream regulators, and the substantial degree of crosstalk between pathways, ensures that this task remains an enormous challenge. By using Drosophila, a simplified model system encoding single JAK and STAT genes, this study now shows that signaling through a cytokine-regulated JAK/STAT pathway is critical for LTM. In contrast to the mammalian gene-disruption studies, this study has been able to rule out the possibility that the observed memory impairments are attributable to defects in development because targeting of gene expression in this study was limited to adult flies. The crucial role of JAK/STAT signaling in memory, if conserved in vertebrates, may explain why inappropriate up-regulation of the pathway appears to disrupt memory, thus shedding light on the large number of diseases in which neuroinflammation is thought to drive pathogenesis (Copf, 2011).

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. This finds that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. It was further shown that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth (Wang, 2011).

This study has shown that JAK/STAT signaling plays an important role in the maintenance and expansion of the neuroepithelial stem cells in the Drosophila larval optic lobe. Loss of JAK/STAT function leads to the disruption of the adherens junction and disintegration of the OPC neuroepithelium while ectopic JAK/STAT activation is sufficient to generate ectopic neuroepithelial stem cells. The non-cell autonomous induction of NEs in the medulla by ectopic expression of upd strongly suggests that JAK/STAT plays a direct role in the growth and proliferation of neuroepithelial stem cells, rather than promotes growth indirectly by blocking premature differentiation of the NEs into medulla neuroblasts (Wang, 2011).

The Drosophila JAK/STAT pathway has been shown to be essential for stem cell maintenance in the testis and ovary, and in the intestine. This study presents evidence that this pathway is also required for the maintenance and expansion of another type of stem cell, the optic lobe neuroepithelial stem cells. Interestingly, STAT3 activity has been implicated in the maintenance of the neuroepithelial stem cells in the developing mouse brain. Thus, the JAK/STAT pathway may play a conserved role in the maintenance and proliferation of stem cells in general and neural stem cells in particular (Wang, 2011).

The phenotypes of JAK/STAT pathway mutants in the optic lobe are reminiscent of those of Notch mutants. The similar phenotypes of the two pathways indicate that they might interact or cooperate to control optic lobe development. Several recent studies reported the interactions between these two pathways in the optic lobe. It has been proposed that the JAK/STAT pathway acts upstream of Notch because ectopic expression of hopTum-l in clones caused a delay of Dl upregulation on the medial region of the OPC, which suggests that JAK/STAT represses Dl expression. However, this study observed ectopic induction of Dl expression in hopTum-l clones. Others have instead suggested that the two pathways are interdependent and cooperate with one another during optic lobe development, based on the observation that Dl expression was reduced in stat92Ets brains and JAK signaling activity was compromised in Notch signaling mutants that expressed a dominant-negative Su(H) construct, although, late-third instar larval brains were analyzed which should have lost the neuroepithelial cells due to loss of either Notch or JAK signaling activity thus complicating the interpretation of the results. The current data indicate that JAK/STAT signaling stimulates the Notch pathway which controls the maintenance and expansion of the OPC neuroepithelium (Wang, 2011).

Non-autonomous crosstalk between the Jak/Stat and Egfr pathways mediates Apc1-driven intestinal stem cell hyperplasia in the Drosophila adult midgut

Inactivating mutations within adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, are responsible for most sporadic and hereditary forms of colorectal cancer (CRC). This study used the adult Drosophila midgut as a model system to investigate the molecular events that mediate intestinal hyperplasia following loss of Apc in the intestine. The results indicate that the conserved Wnt target Myc and its binding partner Max are required for the initiation and maintenance of intestinal stem cell (ISC) hyperproliferation following Apc1 loss. Importantly, it was found that loss of Apc1 leads to the production of the interleukin-like ligands Upd2/3 and the EGF-like Spitz in a Myc-dependent manner. Loss of Apc1 or high Wg in ISCs results in non-cell-autonomous upregulation of upd3 in enterocytes and subsequent activation of Jak/Stat signaling in ISCs. Crucially, knocking down Jak/Stat or Spitz/Egfr signaling suppresses Apc1-dependent ISC hyperproliferation. In summary, these results uncover a novel non-cell-autonomous interplay between Wnt/Myc, Egfr and Jak/Stat signaling in the regulation of intestinal hyperproliferation. Furthermore, evidence is presented suggesting potential conservation in mouse models and human CRC. Therefore, the Drosophila adult midgut proves to be a powerful genetic system to identify novel mediators of APC phenotypes in the intestine (Cordero, 2012).

Using the Drosophila adult midgut as a model system this study has uncovered a key set of molecular events that mediate Apc-dependent intestinal hyperproliferation. The results suggest that paracrine crosstalk between Egfr and Jak/Stat signaling is essential for Apc1-dependent ISC hyperproliferation in the Drosophila midgut (Cordero, 2012).

Previous studies have demonstrated that Myc depletion prevents Apc-driven intestinal hyperplasia in the mammalian intestine. This study provides evidence that such a dependency on Myc is conserved between mammals and Drosophila. It was further demonstrated that endogenous Myc or Max depletion causes regression of an established Apc1 phenotype in the intestine. Taken together, these data highlight the importance of developing Myc-targeted therapies to inhibit Apc1-deficient cells. Since not all roles of Myc are Max dependent, present efforts are focused on developing inhibitors that interfere with Myc binding to Max and would therefore be less toxic. These data provide the first in vivo evidence in support of the Myc/Max interface as a valid therapeutic target for CRC (Cordero, 2012).

Recent work showed that loss of the tuberous sclerosis complex (TSC) in the Drosophila midgut leads to an increase in cell size and inhibition of ISC proliferation. Reduction of endogenous Myc in TSC-deficient midguts restored normal ISC growth and division. These results might appear contradictory to the current work, where Myc is a positive regulator of ISC proliferation. However, in both scenarios, modulation of Myc levels restores the normal proliferative rate of ISCs (Cordero, 2012).

Previous work in mouse showed that Myc upregulation is essential for Wnt-driven ISC hyperproliferation in the intestine. However, Myc overexpression alone only recapitulates some of the phenotypes of hyperactivated Wnt signaling. This study shows that overexpression of Myc is capable of mimicking some aspects of high Wnt signaling in the Drosophila midgut, such as the activation of Jak/Stat, but is not sufficient to drive ISC hyperproliferation. Multiple lines of evidence have shown that forced overexpression of Myc in Drosophila and vertebrate models results in apoptosis partly through activation of p53. Therefore, driving ectopic myc alone is unlikely to parallel Apc deletion in the intestine, where the activation of multiple pathways downstream of Wnt signaling is likely to contribute cooperatively to hyperproliferation (Cordero, 2012).

Understanding the contribution of Jak/Stat signaling to the Apc phenotype in the mammalian intestine has been complicated by genetic redundancy between Stat transcription factors. Constitutive deletion of Stat3 within the intestinal epithelium slowed tumor formation in the ApcMin/+ mouse, but the tumors that arose were more aggressive and ectopically expressed Stat1. Using the Drosophila midgut, direct in vivo evidence is provided that activation of Jak/Stat signaling downstream of Apc1/Myc mediates Apc1-dependent hyperproliferation (Cordero, 2012).

The data on the Drosophila midgut and in mouse and human tissue samples suggest that blocking Jak/Stat activation could represent an efficacious therapeutic strategy to treat CRC. Currently, there are a number of Jak2 inhibitors under development and it would be of great interest to examine whether any of these could modify the phenotypes associated with Apc loss (Cordero, 2012).

Previous studies have demonstrated that enterocytes (ECs) are the main source of Upds/interleukins in the midgut epithelium. The results show that activation of Wnt/Myc signaling in ISCs leads to non-autonomous upregulation of upd3 within ECs. Furthermore, Spitz/Egfr signaling appears to mediate the paracrine crosstalk between Wnt/Myc and Jak/Stat in the midgut. Overexpression of a dominant-negative Egfr in ECs blocks upd3 upregulation and ISC hyperproliferation in response to high Wnt signaling. A previous EC-specific role for Egfr has been demonstrated during midgut remodeling upon bacterial damage. Nevertheless, the downstream signaling that mediates such a role of Egfr remains unclear given that the activation of downstream MAPK/ERK occurs exclusively within ISCs. Therefore, the current evidence would suggest that Egfr activity in ECs does not involve cell-autonomous ERK activation. Consistent with these observations, p-ERK (Rolled -- FlyBase) localization was not detected outside ISCs in response to either Apc loss or overexpression of wg in the Drosophila midgut. Reports on the Apc murine intestine have also failed to detect robust ERK activation. Since MAPK/ERK is only one of the pathways activated downstream of Egfr, it is possible that ERK-independent mechanisms are involved. It is important to explore this further because ERK-independent roles of Egfr signaling have not yet been reported in Drosophila. Thus, what mediates Upd3 upregulation in ECs in response to Egfr signaling activation and whether Spitz-dependent upregulation of Upd3 involves a direct role of Egfr in ECs remain unclear. A potential alternative explanation is that intermediate factors induced in response to Spitz/Egfr activation in ISCs might drive Upd3 expression (Cordero, 2012).

In summary, this study has elucidated a novel molecular signaling network leading to Wnt-dependent intestinal hyperproliferation. Given the preponderance of APC mutations in CRC, the integration of Egfr and Jak/Stat activation might be a conserved initiating event in the disease (Cordero, 2012).

De-regulation of JNK and JAK/STAT signaling in ESCRT-II mutant tissues cooperatively contributes to neoplastic tumorigenesis

Multiple genes involved in endocytosis and endosomal protein trafficking in Drosophila have been shown to function as neoplastic tumor suppressor genes (nTSGs), including Endosomal Sorting Complex Required for Transport-II (ESCRT-II) components vacuolar protein sorting 22 (vps22), vps25, and vps36. However, most studies of endocytic nTSGs have been done in mosaic tissues containing both mutant and non-mutant populations of cells, and interactions among mutant and non-mutant cells greatly influence the final phenotype. Thus, the true autonomous phenotype of tissues mutant for endocytic nTSGs remains unclear. This study shows that tissues predominantly mutant for ESCRT-II components display characteristics of neoplastic transformation and then undergo apoptosis. These neoplastic tissues show upregulation of JNK, Notch, and JAK/STAT signaling. Significantly, while inhibition of JNK signaling in mutant tissues partially inhibits proliferation, inhibition of JAK/STAT signaling rescues other aspects of the neoplastic phenotype. This is the first rigorous study of tissues predominantly mutant for endocytic nTSGs and provides clear evidence for cooperation among de-regulated signaling pathways leading to tumorigenesis (Woodfield, 2013).

While it is well established how de-regulated signaling pathways in ESCRT-II mutant clones mediate non-cell autonomous interactions with neighboring non-mutant cells to contribute to hyperplastic overgrowth and increased cell survival, it was largely unknown which signaling pathways trigger neoplastic transformation autonomously. To address this question, predominantly mutant eye-antennal imaginal discs were generated in which competitive interactions are eliminated so that it was possible to examine the autonomous results of de-regulated signaling (Woodfield, 2013).

Overall, it appears that the same signaling pathways that are induced in mosaic clones are also activated in predominantly mutant tissues. However, two results of this study are noteworthy. First, it is surprising that JNK activity is strongly induced in tissues predominantly mutant for ESCRT-II genes. This is surprising because JNK signaling was believed to be induced by cell competition from neighboring non-mutant cells in mosaic tissues. However, non-mutant tissue is largely eliminated by the ey-FLP/cl method and thus competitive interactions are eliminated. Therefore, it is not known how JNK signaling is induced in these tissues. Nevertheless, JNK signaling is critical for the overgrowth phenotype of predominantly ESCRT-II mutant eye discs as inhibition of this pathway partially blocks cell proliferation. Second, de-regulation of the JAK/STAT signaling pathway is critical for the neoplastic transformation of vps22 mutant discs. Loss of JAK/STAT signaling dramatically normalizes the neoplastic phenotype of vps22 mutant cells. In addition to JNK and JAK/STAT activity, Notch activity was also found to be increased in discs predominantly mutant for ESCRT-II genes. Therefore, a genetic requirement of Notch signaling was tested for neoplastic transformation of ESCRT-II mutant cells. However, loss of Notch was inconclusive because even the wild-type control discs did not grow when Notch was inhibited (Woodfield, 2013).

Interestingly, although ESCRT-II mutant tissues undergo neoplastic transformation, they also show high levels of apoptosis. Animals with predominantly mutant eye-antennal imaginal discs die as headless pharate pupae, a phenotype likely caused by the apoptosis of the imaginal discs before the adult stage. Reduction of JNK signaling in vps22, vps25, or vps36 mutant discs leads to lower levels of apoptosis, supporting a role for JNK signaling in the cell death of the predominantly mutant tissues. More excitingly, JNK also controls proliferation in these tissues, as shown by the reduction of proliferation seen when JNK signaling was down-regulated. This observation is consistent with previous findings that JNK can induce non-cell autonomous proliferation and that apoptosis-induced proliferation is mediated by JNK activity. While inhibition of JNK signaling reduces proliferation in predominantly mutant ESCRT-II mutant discs, it does not affect other aspects of the neoplastic phenotype (Woodfield, 2013).

The role of JAK/STAT signaling in these mutants is complex. In mutant clones of ESCRT-II mosaic discs, Notch-induced secretion of the JAK/STAT ligand Upd triggers non-cell autonomous proliferation. However, autonomous de-regulated JAK/STAT signaling observed in predominately mutant discs is critical for the neoplastic transformation of vps22 mutants. In vps22 Stat92E double mutant discs, organization of cellular architecture is definitively rescued with the layout of the tissue closely resembling that of a wild-type eye-antennal imaginal disc. In addition, apical-basal polarity markers are localized more-or-less correctly in these tissues, indicating that epithelial polarity is more intact. Finally, differentiation in the posterior portion of the eye disc is preserved when JAK/STAT signaling is inhibited. Thus, de-regulation of JAK/STAT signaling in vps22 mutant discs contributes to the cellular disorganization and the lack of differentiation seen in the tissues, which is consistent with a previous study that implicated JAK/STAT signaling in cell cycle control, cell size, and epithelial organization in tsg101 mutant tissues (Woodfield, 2013).

It was recently shown that cells with strong gain of JAK/STAT activity transform into supercompetitors and eliminate neighboring cells with normal JAK/STAT activity by cell competition. However, in mosaic discs, a supercompetitive behavior of ESCRT-II mutant cells has not been observed. In fact, these mutant cells are eliminated by apoptosis. Only if apoptosis is blocked in these cells, is a strong overgrowth phenotype with neoplastic characteristics observed. Thus, apoptosis can serve as a tumor suppressor mechanism to remove cells with potentially malignant JAK/STAT activity (Woodfield, 2013).

How endosomal trafficking specifically regulates JAK/STAT signaling and, thus, how blocking trafficking leads to increases in signaling pathway activity are interesting questions to answer in the future. It is possible that, like endocytic regulation of the Notch receptor, the endosomal pathway tightly regulates Domeless (Dome), the JAK/STAT pathway receptor. It has been shown previously that Dome is trafficked through the endocytic machinery and that this trafficking of Dome can affect the downstream output of the JAK/STAT signaling pathway. It is also possible that Notch-induced Upd secretion causes autocrine JAK/STAT signaling in these mutants. However, technical problems (knocking down Notch function both in wild-type and mutant tissue causes general problems in tissue growth) prevented examination of this possibility (Woodfield, 2013).

It will be important to examine how de-regulated JAK/STAT signaling in ESCRT-II mutants causes neoplastic transformation. JAK/STAT signaling is known to be an oncogenic pathway in Drosophila and in humans but its downstream targets that promote tumorigenesis are not yet clear. JAK/STAT signaling may be feeding into other pathways that promote tumorigenesis, such as dpp signaling, or may be targeting other proteins involved in transformation, such as Cyclin D (Woodfield, 2013).

A number of studies have implicated genes that function in endocytosis and endosomal protein sorting as tumor suppressors in human cancers. Most well known is Tsg101, as early studies showed that downregulation of Tsg101 (see Drosophila TSG101) promotes the growth of mouse 3T3 fibroblasts in soft aga. When these cells were injected into nude mice, they formed metastatic tumors. However, later studies have shown conflicting results, and it is still unclear if Tsg101 functions as a tumor suppressor in metazoans. Importantly, a number of studies have shown changes in expression of ESCRT components in human cancer cells, including changes in expression of ESCRT-I components Tsg101 and Vps37A and ESCRT-III components Chmp1A and CHMP3. Since the primary proteins that function in endocytosis and endosomal trafficking are conserved from yeast to humans, it is likely that these findings in Drosophila may have important implications for human disease (Woodfield, 2013).

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

JAK/STAT signaling is localized to the wing hinge, but its function there is not known. The Drosophila STAT Stat92E is downstream of Homothorax and is required for hinge development by cell-autonomously regulating hinge-specific factors. Within the hinge, Stat92E activity becomes restricted to gap domain cells that lack Nubbin and Teashirt. While gap domain cells lacking Stat92E have significantly reduced proliferation, increased JAK/STAT signaling there does not expand this domain. Thus, this pathway is necessary but not sufficient for gap domain growth. Reduced Wingless (Wg) signaling dominantly inhibits Stat92E activity in the hinge. However, ectopic JAK/STAT signaling does not perturb Wg expression in the hinge. Negative interactions occur between Stat92E and the notum factor Araucan, resulting in restriction of JAK/STAT signaling from the notum. In addition, this study found that the distal factor Nub represses the ligand unpaired as well as Stat92E activity. These data suggest that distal expansion of JAK/STAT signaling is deleterious to wing blade development. Indeed, mis-expression of Unpaired within the presumptive wing blade causes small, stunted adult wings. It is concluded that JAK/STAT signaling is critical for hinge fate specification and growth of the gap domain and that its restriction to the hinge is required for proper wing development (Ayala-Camargo, 2013).


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

date revised: 20 January 2018
 

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