InteractiveFly: GeneBrief

unpaired 1, unpaired 2 & unpaired 3: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

According to FlyBase, the small eye (sy) mutation was described by Bridges on July 3, 1919. Originally considered two separate loci, outstretched (od) and small eye, Muller proposed a single gene based on the recovery of a mutant with both phenotypes, which was not resolvable into two components by recombination, as well as on the failure to recover recombinants between os and sy in transheterozygotes (Verderosa, 1954). The original os mutation was described by Abrahamson in 1953 (Verderosa, 1954). The os mutation gave a phenotype of wings held virtually at right angles to body and small and rounded eyes. The unpaired term for this gene originated with Carroll (1986) in a description of zygotically active genes that affect the spatial expression of the fushi tarazu during early Drosophila embryogenesis. Referred to as sisterless C (Cline, 1993), the gene is considered as an X:A numerator element in sex determination. Finally cloned and described in 1998, outstretched, termed unpaired by Harrison, 1998, has been found to be a secreted protein, associated with the extracellular matrix, that activates the JAK pathway. This essay will conform to the FlyBase convention and refer to this much encountered protein is Outstretched.

Our current understanding of the JAK/STAT pathway is deduced primarily from studies in mammals. JAKs are a novel class of nonreceptor tyrosine kinases, of which four mammalian members have been characterized. The unique feature of these molecules is the presence of two tandem kinase-homologous domains. The carboxy-terminal domain of these proteins has been shown to catalyze tyrosine phosphorylation, whereas the more amino-terminal domain is apparently catalytically inactive. The major substrates for JAK tyrosine phosphorylation are the STATs. This family of molecules, with seven identified members in mammals, is able to bind to specific DNA sequences and activate transcription. Together, the JAKs and STATs comprise a simple intracellular signal relay that can be activated by a number of extracellular factors. In vertebrates, the cytokines are the largest class of molecules to utilize the cascade. Although the cytokines have significantly different amino acid sequences, they are divided into two categories on the basis of their structures. The larger class, type I cytokines, have a characteristic up-up-down-down helical structure, referring to the arrangement of alpha helices in the protein. The receptors for these molecules are heteromultimeric subunits, with many cytokines using a common subunit in the receptor complex. Whereas the type II cytokines have different structure than the type I class, they also bind to heteromultimeric receptor complexes (Harrison, 1998 and references).

Mechanistically, the JAK/STAT pathway provides a direct means to respond to extracellular signals. The first step in activation is binding of the extracellular ligand to the appropriate transmembrane receptor. Ligand binding typically induces receptor dimerization or multimerization. Current models hold that receptor dimerization brings their associated JAK molecules into close proximity to each other, presumably facilitating trans-phosphorylation of the JAKs. Signals are then transmitted directly to the nucleus through the activation of appropriate STATs (The term STAT stands for signal transducer and activator of transcription). Inactive STATs are found in the cytoplasm and, upon JAK activation, are recruited to the inner surface of the cell membrane. Here, they are tyrosine phosphorylated by the JAKs, then translocate to the nucleus and bind specific DNA sequences to regulate transcription. In this manner, direct response to signals is achieved via transcriptional activation of downstream target genes by activated STATs (Harrison, 1998 and references).

Recently, a Drosophila paradigm for the JAK-STAT pathway has emerged in which a single JAK, termed hopscotch (hop) and a STAT, termed stat92E or marelle, have been identified. Drosophila hop was originally identified as a gene involved in embryonic pattern formation. Embryos derived from females that do not express the hop gene product in their germ lines show novel embryonic defects characterized by the loss and/or fusion of a specific subset of body segments. This phenotype is unusual because it does not resemble any of the stereotypical classes of segmentation genes (gap, pair-rule, or segment polarity genes). This same embryonic phenotype is also seen in animals derived from females that lack stat92E gene activity. A third gene, outstretched or unpaired (upd), with the distinctive embryonic mutant phenotype of hop and stat92E, has now been cloned. It is suggested that this gene encodes a ligand for the JAK/STAT pathway during Drosophila segmentation. The conceptual translation product does not resemble that of any of the cytokines (Harrison, 1998). It is similar to only one other identified protein, the Om(1E) protein of Drosophila ananassae, a very closely related species (Juni, 1996).

The similarity of the os mutant phenotype with that of hop and stat92E, along with the genetic interactions that exist between them, suggest that all three genes are involved in the same developmental process. A prediction of this hypothesis is that mutations in os would affect the expression of segmentation genes in the same manner as hop and stat92E. Analysis of the expression of pair-rule genes in os mutant embryos confirms this prediction. The removal of os results in the stripe-specific loss of expression of several pair-rule genes. Specifically, in os mutants, the fifth stripes of expression of the genes even-skipped (eve), fushi tarazu (ftz), and runt are reduced or absent. Additionally, the third stripes of eve and ftz, and the second stripe of runt are variably reduced. These stripe-specific effects are identical to those described for maternal loss of hop and stat92E activities (Binari, 1994 and Hou, 1996).

The enhancer elements responsible for control of the third stripe of eve expression have been mapped to a 500-bp element upstream of the eve transcriptional start site. A reporter construct carrying 5.2 kb of eve upstream sequence, including this enhancer region, fused to the lacZ gene drives expression of lacZ in the second, third, and seventh stripes of eve. Previous work has shown that this fragment contains sequences that bind Stat92E protein in vitro. Removal of maternal activity of either hop or stat92E results in the loss of the third stripe from the reporter construct. Similarly, zygotic mutation of os also causes the specific loss of the third stripe, without affecting the second or seventh stripes (Harrison, 1998 and references).

The lack of sequence similarity between Os and any known vertebrate activators is intriguing. But like cytokines, the predicted secondary structure for Os includes several stretches of alpha-helical regions. Whereas Os has no sequence homology with cytokines, perhaps the alpha-helices fold into a structure reminiscent of cytokines. There is precedent among mammalian ligands for structural similarities in molecules that do not have sequence homology with type I cytokines. These molecules also utilize JAKs for signal transduction. Although there may not have been strong evolutionary conservation of specific sequences, there may be conservation of general structure in ligands because of functional requirement for binding to the appropriate receptor. On the other hand, some aspects of Os structure are less consistent with a cytokine-type molecule. First, the Os protein is extremely basic, with a predicted pI of nearly 12. Second, in contrast with many cytokines, Os is associated with the ECM, which may limit the range of activity of the ligand. These characteristics suggest that Os may be representative of a new family of ligands that stimulates the JAK pathway. Determination of the relationship between Os and other JAK activating ligands must wait for the identification of Os homologs in other species. Finally, characterization of the Os receptor will reveal whether Os signals through a conventional receptor molecule or whether it defines a novel mechanism by which the JAK/STAT pathway becomes activated (Harrison, 1998).

Polarity determination in the Drosophila eye: a novel role for Unpaired and JAK/STAT signaling

Polarity of the Drosophila compound eye is established at the level of repeating multicellular units (known as ommatidia), which are organized into a precise hexagonal array. The adult eye is composed of ~800 ommatidia, each of which forms one facet. Sections through the eye reveal that each ommatidium contains eight photoreceptor cells in a stereotypic trapezoidal arrangement that has two mirror-symmetric forms: a dorsal form present above the dorsoventral (DV) midline, and a ventral form below. An axis of mirror-image symmetry, known as the midline, runs along the DV midline (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). By analogy to the terrestrial equator, the extreme dorsal and ventral points of the eye are referred to as the poles. Differentiation of ommatidia begins during the third instar larval stage when a furrow moves from posterior to anterior over the epithelium of the eye imaginal disc. Each ommatidial unit is born as a bilaterally symmetrical cluster of photoreceptor precursors, that is polarized on its anteroposterior axis. The clusters then become polarized on the DV (or equatorial-polar) axis, by the process of proto-ommatidium rotation via two 45° steps away from the DV midline, forming the equator. It has been suggested that the direction of this rotation is a consequence of a gradient of positional information emanating from either the midline or the polar regions of the disc (Zeidler, 1999 and references).

A number of recent studies have shed light on some of the mechanisms involved in the positioning of the equator on the DV midline of the eye imaginal disc. It is now clear that a critical step is the activation of Notch activity in a line of cells along the midline, and that this localized activation of Notch is a consequence of the restricted expression of the fringe (fng) gene product in the ventral half of the disc and the homeodomain transcription factor Mirror (Mirr) in the dorsal half of the disc. Furthermore, an important role for Wingless (Wg) in polarity determination on the DV axis has been demonstrated. Wg is a secreted protein (and the founder member of the Wnt family of morphogens) that is expressed at the poles of the eye disc. Wg has been shown to act as an activator of mirr expression; increasing the levels of Wg expression in the eye disc shifts mirr expression and the position of the equator ventrally, whereas reduction of wg function shifts mirr expression dorsally. Additionally, it has been shown convincingly that a gradient of Wg signaling across the DV axis of the eye disc regulates ommatidial polarity such that the lowest point of Wg signaling coincides with the equator (Zeidler, 1999 and references).

The JAK/STAT pathway is central to the establishment of planar polarity during Drosophila eye development. A localized source of the pathway ligand, Unpaired/Outstretched, present at the midline of the developing eye, is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror (Zeidler, 1999).

The data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localized activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, since fng loss of function clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity. Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals (most likely detected by the receptor Frizzled) that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal (Zeidler, 1999 and references).

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

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

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

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

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

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

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

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

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

Regulation of circadian behavioral output via a MicroRNA-JAK/STAT circuit

Although molecular components of the circadian clock are known, mechanisms that transmit signals from the clock and produce rhythmic behavior are poorly understood. This study found that the microRNA miR-279 regulates the JAK/STAT pathway to drive rest:activity rhythms in Drosophila. Overexpression of microRNA miR-279 or miR-279 deletion attenuates rest:activity rhythms. Oscillations of the clock protein Period are normal in pacemaker neurons lacking miR-279 , suggesting that miR-279 acts downstream of the clock. The JAK/STAT ligand, Upd, was identified as a target of miR-279 , and it was shown that knockdown of Upd rescues the behavioral phenotype of miR-279 mutants. Manipulations of the JAK/STAT pathway also disrupt circadian rhythms. In addition, central clock neurons project in the vicinity of Upd-expressing neurons, providing a possible physical connection by which the central clock could regulate JAK/STAT signaling to control rest:activity rhythms (Luo, 2012).

Although microRNAs play a critical role in most biological processes, few have been specifically implicated in circadian behavioral rhythms. In mammals, miR-219 is involved in central clock function and miR-132 in light input to the central clock. In Drosophila, bantam miRNA affects free-running circadian rhythms by targeting the Clock gene. This study has isolate miR-279 as an effecter of clock-controlled behavioral output, and Upd, the ortholog of the JAK/STAT ligand, was identified as the circadian-relevant target of this miRNA. It was further shown that manipulations of other components of the JAK/STAT pathway also disrupt behavioral rhythms, and expression of STAT92E was found to be rhythmic. Given the critical need for appropriate expression levels of Upd, it is speculate that rhythms in STAT92E derive from circadian regulation at the level of Upd. Athough levels of UPD do not appear to cycle in whole brains, cycling in specific cells cannot be excluded and secretion of UPD could be cyclic. Cyclic release of UPD would lead to rhythmic activation of Dome and thereby STAT92E. Because STAT92E regulates its own expression through feedback, rhythmic STAT92E activity is expected to result in cyclic expression. PDF is also thought to be released cyclical, and it regulates cycling of STAT92E. This suggests control of the JAK/STAT pathway by central clock cells, which is supported by the proximity of PDF projections and Upd neurons. However, rhythmic regulation of the JAK/STAT pathway could be reinforced by clocks in other cells, e.g., those in which Per and Upd-GAL4 are coexpressed. In addition, miR-279 may regulate more than just Upd in the JAK/STAT pathway (Luo, 2012).

These data demonstrate a role of the JAK/STAT pathway in circadian rhythms. To date, most studies of signaling in the circadian system have focused on cAMP or mitogen-activated protein kinase (MAPK) pathways, which may function in all aspects of circadian timekeeping (input to the clock, clock function, and output). In the Drosophila circadian system, MAPK activity cycles in parts of the fly brain, and this appears to be related to the function of the Neurofibromatosis 1 (NF1) protein in circadian rhythms. Studies have shown that NF1 is required downstream of the clock for rhythmic rest:activity, and its effects on rhythms are mediated by the RAS/MAPK pathway (Luo, 2012).

Interestingly, in mammalian glia and neural stem cells, NF1 influences STAT3 activity such that STAT3 signaling is hyperactivated in NF1-deficient cells. It is not known if there is a connection between NF1 and JAK/STAT in Drosophila, but since STAT92E fluctuates with a circadian rhythm in the fly brain, it is an intriguing possibility that the two are linked in the circadian system (Luo, 2012).

Upd and its receptor, Dome, are expressed widely in the fly brain. It is interesting, however, that Upd is coexpressed with Per in cells where Per cycles with a shifted phase under LD conditions. In these cells, Per cycles but is out of phase with other neurons, including those comprising the central clock. Two sites of Upd and Per coexpresssion are in some of the DNs and in neurons are termed LLNs because of their lateral location. The function of these double-positive neurons is unclear at this time. The dorsal neurons are unlikely to be the DN2s because although Per expression in DN2s is antiphase in larvae, it cycles with a normal phase in adult flies. Likewise, the LLNs are probably not lateral posterior neurons (LPNs), located in the same region, because those are apparently three in number in each hemisphere and show normal Per cycling. Based upon their position, the DNs that express Upd could represent a subset of the dorsal neuron cluster 1 (DN1s). Regardless of the precise cell types in which they are expressed, Upd and Dome expression provide tools to map the cellular circuitry of the output pathway that drives rhythmic rest:activity (Luo, 2012).

It is likely that many other microRNAs are involved in circadian rhythms. Because identification of such miRNAs will be difficult through traditional genetic screens, other approaches will have to be devised. Bioinformatics to identify miRNAs that target clock genes is obviously a viable method, but, as noted in this study, this approach typically identifies many possible targets for each miRNA, only some of which are biologically relevant. Thus, future computational analyses will need to be followed by many additional tests. Although this is doable, another caveat of this approach is that it will restrict analysis to miRNAs that target clock genes. Another approach is to identify miRNAs that are expressed cyclically. This was done recently in Drosophila, and it identified some miRNAs, which may turn out to be important regulators of circadian rhythms. In addition to these approaches, it will be important to completely dissect the circadian circuitry. Although there is an understanding of the components of the central clock and the mechanisms that entrain the clock to light, how clock signals are transduced to produce overt rhythms, in particular behavioral rhythms, is less well understood. Advances in this area, including those reported here, will provide a framework upon which whole circuits can be assembled (Luo, 2012).

Fat body p53 regulates systemic insulin signaling and autophagy under nutrient stress via Drosophila Upd2 repression

The tumor suppressor p53 regulates multiple metabolic pathways at the cellular level. However, its role in the context of a whole animal response to metabolic stress is poorly understood. Using Drosophila, this study shows that AMP-activated protein kinase (AMPK)-dependent Dmp53 activation is critical for sensing nutrient stress, maintaining metabolic homeostasis, and extending organismal survival. Under both nutrient deprivation and high-sugar diet, Dmp53 activation in the fat body represses expression of the Drosophila Leptin analog, Unpaired-2 (Upd2), which remotely controls Dilp2 secretion in insulin-producing cells. In starved Dmp53-depleted animals, elevated Upd2 expression in adipose cells and activation of Upd2 receptor Domeless in the brain result in sustained Dilp2 circulating levels and impaired autophagy induction at a systemic level, thereby reducing nutrient stress survival. These findings demonstrate an essential role for the AMPK-Dmp53 axis in nutrient stress responses and expand the concept that adipose tissue acts as a sensing organ that orchestrates systemic adaptation to nutrient status (Ingaramo, 2020).

The ability of an organism to sense nutrient stress and coordinate metabolic and physiological responses is critical for its survival. Over the last years, the p53 tumor suppressor has emerged as an important regulator of cellular metabolism, and its activation has been regularly observed in response to diverse metabolic inputs, such as changes in oxygen levels or nutrient availability. It has been shown that p53 interacts with main players in key nutrient-sensing pathways, such as mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK), leading to modulation of autophagy and lipid and carbohydrate metabolism. p53 restricts tumor development partially by inhibiting glycolysis, limiting the pentose phosphate pathway, and promoting mitochondrial respiration. Conversely, p53 activation can benefit tumor growth by stimulating adaptive cellular responses in nutrient-deficient conditions. p53 activation is known to induce cell-cycle arrest and promote cell survival in response to transient glucose deprivation, regulate autophagy and increase cell fitness upon fasting, and promote cancer cell survival and proliferation after serine or glutamine depletion. Therefore, p53 plays a pivotal role in the ability of cells to sense and respond to nutrient stress, functions that are important not only to control cancer development but also to regulate crucial aspects of animal physiology. Further studies concerning p53 regulation and function in response to nutrient and metabolic challenges at an organismal level would expand understanding on the role of p53 in normal animal physiology, aging, and disease (Ingaramo, 2020).

The single Drosophila ortholog of mammalian p53 (Dmp53) has also been shown to regulate tissue and metabolic homeostasis. Dmp53 regulates energy metabolism through induction of cell-cycle arrest and cell growth inhibition in response to mitochondrial dysfunction by regulating glycolysis and oxidative phosphorylation to promote cell fitness in dMyc-overexpressing cells and by modulating autophagy protecting the organism from oxidative stress. Studies in Drosophila have also identified tissue-specific roles of Dmp53 in regulating lifespan and adaptive metabolic responses impacting on animal aging and stress survival, evidencing conserved functions of p53, and positioning Drosophila p53 studies as a valuable alternative providing relevant insights on mammalian health and disease (Ingaramo, 2020).

The insulin pathway is highly conserved from mammals to Drosophila and regulates carbohydrate and lipid metabolism, tissue growth, and longevity in similar ways. Drosophila insulin-like peptides (Dilps) promote growth and maintain metabolic homeostasis through activation of a unique insulin receptor (dInR) and of a conserved intracellular insulin and insulin-like growth factor (IGF) signaling pathway (IIS). Dietary conditions tightly regulate Dilp2 production and/or secretion from the insulin-producing cells (IPCs), neuroendocrine cells analogous to pancreatic β-cells located in the fly brain. Interestingly, a nutrient-sensing mechanism in the fat body (FB), a functional analog of vertebrate adipose and hepatic tissues, non-autonomously regulates Dilp2 secretion and couples systemic growth and metabolism with nutrient availability. According to the nutritional status, the FB produces signaling molecules capable of promoting or inhibiting insulin secretion from the IPCs. Thus, a simple integrated system composed of various organs and conserved signaling pathways regulates metabolic homeostasis and organismal growth in response to nutrient availability (Ingaramo, 2020).

The FB also functions as the organism's main energy reserve and is responsible for coupling energy expenditure to nutrient status. In well-fed animals, circulating insulin activates insulin receptors in the FB and promotes energy storage in the form of glycogen and triacylglycerol (TAG). Upon limited nutrient availability, stored lipids and glycogen are broken down to supply energy for the rest of the body. Previous work showed that FB-specific inhibition of Dmp53 activity accelerated the consumption of main energy stores, reduced sugar levels, and compromised organismal survival during nutrient deprivation. The mechanism by which Dmp53 regulates metabolic homeostasis and organismal survival under nutrient stress is not entirely understood and might involve regulation of specific signaling and metabolic pathways (Ingaramo, 2020).

This study provides evidence that AMPK-dependent Dmp53 activation in the FB non-cell-autonomously regulates TOR signaling and autophagy induction upon acute starvation, which is essential for organismal survival. Dmp53 activation in response to nutritional stress is required for proper communication between the FB and IPCs by modulating the expression of the Drosophila Leptin analog, Unpaired-2 (Upd2). Elevated Upd2 levels in adipose cells of starved Dmp53-depleted animals result in sustained Dilp2 circulating levels, activation of insulin/TOR signaling, and impaired autophagy induction in the whole animal, therefore reducing survival rates upon nutrient deprivation. These results indicate that Dmp53 plays an essential role in Drosophila, integrating nutrient status with metabolic homeostasis by modulating Dilp2 circulating levels, systemic insulin signaling, and autophagy (Ingaramo, 2020).

Even though progress has been made in understanding p53 metabolic functions at the cellular level, its role in the context of a whole animal response to metabolic stress is poorly understood. This study provides evidence that Drosophila p53 is critically involved in nutrient sensing and in the orchestration of an organismal response to nutrient stress. AMPK-dependent Dmp53 activation in the FB in response to nutritional stress is required for proper communication between the FB and the IPCs by modulating the expression of Drosophila Leptin analog, Upd2. Elevated Upd2 levels and activation of JAK/STAT signaling in the brain of starved Dmp53-depleted animals result in sustained Dilp2 circulating levels, activation of insulin signaling, and impaired autophagy induction in various tissues, therefore reducing survival rates upon nutrient deprivation. These results position the AMPK-p53 axis as a key player in nutrient sensing and in regulating adaptive physiological responses to low nutrient availability by remotely controlling insulin secretion and autophagy (Ingaramo, 2020).

Studies in mice have also shown that p53 is activated under several nutrient stress conditions, such as nutrient deprivation, high-caloric diet, and high-fat diet (HFD). p53 becomes activated under nutrient deprivation and regulates expression of genes involved in mitochondrial fatty acid uptake and oxidative phosphorylation. In turn, pharmacological or genetic inhibition of p53 prevented excessive fat accumulation commonly observed under HFD and resulted in decreased expression of proinflammatory cytokines and improved insulin resistance in mice with type 2 diabetes (T2D)-like disease. Conversely, upregulation of p53 in adipose tissue caused an inflammatory response that led to insulin resistance. These results show that both mice and Drosophila p53 activation in individuals exposed to challenging nutrient conditions regulate global metabolism and directly contribute to diet-associated phenotypes (Ingaramo, 2020).

Leptin is mainly produced by adipose tissue in mice and humans, and regulates food intake, energy expenditure, and metabolism acting mostly on neuronal targets in the brain. This study has shown that Dmp53 activation in the FB under nutrient stress impacts systemic insulin signaling and autophagy induction via regulation of Upd2/Leptin expression. Notably, reduced survival of Dmp53-depleted animals to nutrient deprivation was highly reverted when inhibiting either Upd2 expression in the FB or JAK/STAT signaling in GABAergic neurons in the fly brain. Similar to Upd2, Leptin circulating levels decline during fasting conditions and are increased in animals fed with a HFD. Low Leptin levels during starvation trigger adaptive metabolic and hormonal responses, such as increased appetite and decreased energy expenditure. In HFD-fed mice, p53 activation is necessary for fat accumulation in the liver and adipose tissue, indicating that p53 is essential for coordinating energy expenditure and storage in response to nutrient availability (Liu, 2017). Reduced expression of p53 target genes, such as GLUT4 and SIRT1, has been proposed to reduce NAD+ levels and energy expenditure, leading to obesity (Liu, 2017). Alternatively, p53 activation in adipose cells could regulate Leptin expression, which is known to act on the CNS to reduce food intake and enhance energy expenditure, thus limiting obesity in times of nutrient abundance. Further investigations into the role of adipose tissue p53 activity in modulating physiological and metabolic responses to stress will be necessary to have a better picture of the role p53 plays in the development of metabolic disorders, such as obesity and T2D. Of importance, based on conserved adipose tissue-specific functions of p53 and signaling pathways involved, studies in Drosophila are likely to provide insights relevant to mammalian health and disease (Ingaramo, 2020).

In the past decade, significant interest has been raised in understanding non-canonical functions of p53 that might have potential roles in tumor suppression. The fact that p53 is activated in the adipose tissue of obese animals, along with the results presented concerning a putative direct role of p53 in controlling Upd2/Leptin expression, demonstrates the importance of p53 in regulating metabolism. This is particularly interesting given that epidemiological studies over the last few decades have shown a strong influence of obesity on cancer risk and that increased Leptin can have hormone-like functions affecting tumor development. In this context, the results give insights toward the molecular understanding of p53 activation under metabolic stress and its possible role in tumor suppression acting at either local or organismal level (Ingaramo, 2020).

TOR and AMPK play essential roles in nutrient sensing, are important regulators of energy balance at both cellular and whole-body levels, and have been shown to interact with p53. Previously showed that TOR inhibition following long starvation treatments (24-48 h) contributes to Dmp53 activation, mainly by alleviating miRNA-mediated targeting of Dmp53 in the FB (Barrio, 2014). This work, demonstrated that rapid activation of Dmp53 is dependent on AMPK and absolutely required for metabolic and physiological changes that promote organismal resistance to nutrient deprivation. This short-term activation of Dmp53 by AMPK could be part of a dual mechanism along with previously demonstrated long-term activation by lack of TOR, and both of these regulating mechanisms may be important for establishing a rapid response to transient acute nutrient stress also guaranteeing a sustained response when facing a much longer nutrient-deprived period. Given that activated Dmp53 reduces Upd2 expression, systemic insulin, and TOR signaling, it would be reasonable to speculate that Dmp53-dependent TOR inhibition constitutes a positive feedback loop to reinforce Dmp53 activation upon long-term starvation conditions. Therefore, the results place p53 in a crucial position connecting nutrient sensing pathways to endocrine mechanisms, as part of a possible physiological feedback mechanism (Ingaramo, 2020).

Drosophila AMPK activation has been shown to extend lifespan and promote tissue proteostasis through non-cell-autonomous regulation of autophagy. Given that Dmp53, acting downstream of AMPK under nutrient stress, non-cell-autonomously regulates Dilp2 levels and autophagy, it will be interesting to determine whether p53, and perhaps its direct phosphorylation by AMPK, is also required for extending organismal lifespan upon tissue-specific AMPK activation (Ingaramo, 2020).

Gut cytokines modulate olfaction through metabolic reprogramming of glia

Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. This study shows that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. These findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies (Cai, 2021).

Olfactory perception influences nutrition and promotes physiological and mental well-being. In flies, a dedicated olfactory circuit elicits avoidance behaviours towards geosmin-a volatile compound that is released by mould and some bacteria. Olfactory receptors also mediate an initial attraction to food that contains certain enteropathogens. After infection with these pathogens, however, an avoidance behaviour is triggered by immune receptors in the brain, gustatory bitter neurons, and the neuropeptide leukokinin. Whether changes in olfactory perception contribute to this behavioural switch from attraction to avoidance remains unclear (Cai, 2021).

In Drosophila, odorants are sensed by olfactory receptor neurons in the head, the antenna and the maxillary palp. Olfactory receptor neurons synapse into projection neurons at the antennal lobe (AL), where the signal is converted into a spatiotemporal code in 50 glomerular compartments. Projection neurons axons project to higher olfactory centres to instruct innate and learned behaviour. In this system, glia and neurons operate as a tightly coupled unit to maintain olfactory sensitivity (Cai, 2021).

In ageing flies, olfactory perception of both aversive and attractive odours declines, but the mechanism(s) of this decline remain unclear. Olfactory perception and other neurological processes also decline in ageing mammals, often influenced by gastrointestinal signals (Cai, 2021).

This study investigated the communication between the gut and the brain, and how it influences infection-induced avoidance behaviour, infection tolerance, and olfactory decline during ageing (Cai, 2021).

A modified capillary feeder (CAFE) assay was used to measure choice between food that did or did not contain Erwinia carotovora carotovora 15 (Ecc15), a non-lethal enteropathogen that causes intestinal inflammation. Consistent with recent reports, naive flies consumed more Ecc15-containing food than normal food. However, when orally infected with Ecc15 for 24 h before the feeding assay, flies developed a distinct aversion to food that contained Ecc15. To assess whether this involved changes in olfactory perception, the 'preference index' for attractive (such as putrescine) or aversive (such as 3-octanol) odours was determined in T-maze assays. Preference or aversion for attractive or aversive odours, respectively, declined after infection, which indicates that infection causes a non-selective decline in olfactory discrimination. This was transient, as olfactory discrimination recovered 5 days after infection, coincident with the clearance of bacteria and epithelial regeneration in the intestine. Olfactory discrimination was not influenced by starvation or exposure to heat-killed Ecc15. Consistent with their reported role in sensing pathogenic bacteria, the CO2 receptor Gr63a or the odorant receptor co-receptor Orco was required for the attraction to Ecc15 food: Orco1 and Gr63a1 mutants ingested less Ecc15 food under naive conditions, and when infected, failed to further reduce ingestion of Ecc15-containing food. Together, these observations suggest that after an initial odorant-mediated attraction, flies develop aversion to enteropathogens, through a concerted activation of gustatory and immune receptors and suppression of olfaction (Cai, 2021).

After oral infection with Ecc15, damaged intestinal enterocytes produce the inflammatory IL-6-like cytokines Unpaired 2 and 3 (Upd2 and Upd3) to stimulate intestinal stem-cell proliferation and epithelial regeneration. Proteins of the Upd family activate the JAK-STAT signalling pathway through the receptor Domeless (Dome) and the JAK homologue Hopscotch (Hop). Using the 2xSTAT::GFP reporter for JAK-STAT pathway activity, this study found upregulated GFP expression in the brain 4 h after oral Ecc15 infection, as well as after oral infection with the more lethal enteropathogen Pseudomonas entomophila (PE) that damages the gut epithelium. JAK-STAT activity was observed in a sparse population of cells of the brain that stained positive for the glial marker Repo. Subtype-specific Gal4 drivers revealed that among the five subtypes of Drosophila glia (astrocytes, ensheathing, perineural, subperineural and cortex glia), ensheathing glia (EG) were the main population that upregulate STAT activity in response to Ecc15 infection. This was confirmed using four different Gal4 drivers to label EG and by flow cytometry. Infection did not influence numbers and membranous processes (labelled using UAS::mCD4GFP) of EG at the AL, and glomerular compartmentalization in the AL and lobe size remained unaffected. JAK-STAT activation in EG was sufficient and required for infection-induced changes in olfactory discrimination, as overexpression of constitutively active Hop (hop^tuml) in EG reduced olfactory discrimination, whereas loss of Dome or STAT in all glia (repo::Gal4), or specifically in EG (GMR56F03::Gal4), rescued the decline of olfactory discrimination caused by Ecc15 infection. Overexpression of hop^tuml in EG also reduced ingestion of Ecc15-containing food and promoted survival of flies fed PE-containing food, whereas knockdown of Dome or STAT in EG increased ingestion of Ecc15-containing food in infected flies and increased mortality on PE-containing food. It is proposed that the corresponding changes in ingestion of PE-laced food contribute to reduced mortality, but it is possible that additional genetic background conditions influence mortality, as seen, for example, in Orco-mutant flies, which ingest less bacteria but show increased susceptibility to PE (Cai, 2021).

To test whether gut-derived Upd proteins directly contribute to the infection-induced activation of JAK-STAT signalling in EGs, intestinal enterocyte-specific perturbations were performed using Mex1::Gal4, an enterocyte driver with no expression in the brain. Indeed, JAK-STAT activation in glia at the AL could be triggered in naive flies or prevented in infected flies by overexpression or knockdown, respectively, of Upd2 and Upd3 in enterocytes. Consistently, enterocyte-derived Upd2 and Upd3 were sufficient and required for the modulation of olfactory discrimination caused by infection. Knockdown of Upd2 or Upd3 did not affect olfaction in naive flies, and perturbing these ligands in fatbody (cg::Gal4) or haemocytes (hml::Gal4), tissues that are sources for Upd proteins in other contexts, did not significantly affect STAT activity in glia at the AL (Cai, 2021).

Loss of olfactory sensitivity is an early sign of normal ageing and neurodegeneration. In ageing Drosophila, olfactory perception has been reported to deteriorate before vision, a decline that was possible to recapitulate in T-maze assays. Glomerular compartments in the AL became less organized and less distinct in geriatric (60-70-day-old) flies and AL size increased with age. This correlates with a reduction in the number of EG and of glial membranous processes, changes that are expected to affect AL structure, and thus probably contribute to the age-related decline in olfaction (Cai, 2021).

Ageing in Drosophila is accompanied by the development of intestinal inflammation, and is associated with the constitutive expression and release of Upd cytokines. Consistently, JAK-STAT activity in the AL of EG was increased in old flies, and knockdown of Dome or STAT by RNA interference (RNAi) in EG specifically or in all glia rescued the decline of olfactory discrimination in old flies. The loss of Dome in EG also rescued the age-related decline of EGs and restored the size of the AL. JAK-STAT activation in the EG of old flies is a consequence of intestinal Upd release, as knocking down Upd2 and Upd3 in enterocytes alleviated STAT activation in the AL, and prevented the age-related decline of olfactory discrimination (Cai, 2021).

This age-related decline of olfactory discrimination was independent of the microbiota, as germ-free old flies still exhibited reduced olfaction sensitivity, increased JAK-STAT signalling in the AL, decreased numbers of EG, loss of glial cellular processes, and an enlarged AL. These results are consistent with the observation that the age-related increase in Upd released from the gut is also independent of the microbiota (Cai, 2021).

To understand why EG but not other glia selectively respond to Upd ligands and activate JAK-STAT signalling during ageing or infection, single-cell RNA sequencing (scRNA-seq) was performed on purified glia from young and old flies. Either all glia (labelled using repo::Gal4) or EG selectively (labelled using GMR56F03::Gal4) were profiled using Smart-seq2. The expression of dome was significantly higher in EG than in other glia, consistent with the specific upregulation of socs36E, a known target of JAK-STAT signalling, in EG but not in other glia during ageing. These results are supported by a similar upregulation of Socs36E in EG of old flies observed in a previous scRNA-seq dataset (Cai, 2021).

Bulk RNA sequencing analysis on glia (repo::Gal4, UAS::tdTomato) purified from central brains of flies expressing a 10xSTAT::GFP reporter revealed that the transcriptomes of STAT::GFP⁺ glia from Ecc15 infected and uninfected flies were more similar to each other than to STAT::GFP^- glia of either condition, which indicates that JAK-STAT induction has a stronger influence on glial transcriptomes than other infection-related changes. Differentially expressed genes (866 genes using a cut-off of twofold change, P < 0.001, false discovery rate (FDR) < 0.01 and reads > 0.5) were significantly enriched in genes that encode proteins involved in lipid metabolism and carbohydrate transmembrane transport. These included the lipid binding protein Glial lazarillo (GLaz, a homologue of apolipoprotein D in mammals), which facilitates lipid transport from neurons to glia in flies; the lipid droplet surface binding proteins Lsd-1 and Lsd-2; the diacylglycerol O-acyltransferase Midway, which is a central regulator of triacylglycerol biosynthesis, and Coatomer, which is responsible for protein delivery to lipid droplets (LDs). This induction of lipid storage genes was coupled with induction of the monocarboxylate transporter (MCT) Outsiders (Out), and the MCT accessory protein Basigin (Bsg), sugar transporters (Tret1-1 and Tret1-2), and 17 enzymes involved in β-oxidation (Cai, 2021).

Glial MCTs promote lipid production in neurons and LD accumulation in glia by establishing a neuron and glia 'lactate shuttle'. To test a potential role for STAT signalling in influencing this shuttle at the AL, the accumulation of LDs was assessed at the AL in infected young flies using a combination of a neutral lipid probe (LipidTox, deep red) and a lipid peroxidation probe (C11-Bodipy, 581/591). A transient accumulation of LDs was observed 24 h after infection that decreased 4 days after infection, possibly owing to increased levels of β-oxidation. Overexpression of hop^tuml in the EG of young flies also promoted LD accumulation, whereas knocking down Dome or STAT rescued infection-induced accumulation. GLaz and Out were required for LD accumulation after infection, and overexpression of Upd2 and Upd3 in the gut induced LD accumulation at the AL, whereas knockdown of Upd2 or Upd3 alleviated LD accumulation in infected flies. Infection or JAK-STAT perturbation did not influence lipid peroxidation in LDs in young flies (Cai, 2021).

During neuronal stress, neurons can preferentially transfer fatty acids to glia, causing lipid accumulation and increasing fatty acid β-oxidation in glia. This study observed a significant induction of LDs specifically in EG at the AL in old flies, phenocopying hop^tuml overexpression. As fatty acid β-oxidation is a source of reactive oxygen species (ROS) that can result in lipid peroxidation, and lipid peroxidation in pigment cells (glia of the retina) promotes the demise of photoreceptors in the retina (whereas oxidative stress contributes to age-related dysfunction of cholinergic projection neurons within the olfactory circuit) it was reasoned that overall levels of ROS might increase in glia with age. Various genetically encoded ROS sensors were expressed in all glia (repo::Gal4) or in EG only (GMR56F03::Gal4) to measure levels of hydrogen peroxide (H2O2; measured by RoGFP2_Orp1) or the glutathione redox potential (measured by RoGFP2_Grx1) within the mitochondria or cytosol, respectively. Cytosolic levels of H2O2 were increased in the EG of old flies (both cytosolic and mitochondrial H2O2 levels were increased in all glia), whereas the cytosolic glutathione redox potential remained unchanged. In contrast to acute intestinal infection in young flies, lipids were peroxidated in LDs of old flies. Knocking down STAT specifically in EG, or knocking down Upd2 and Upd3 in gut enterocytes, inhibited LD accumulation and alleviated lipid peroxidation in old flies (Cai, 2021).

Olfactory discrimination was partially rescued in old and young infected flies after knockdown of GLaz and Out in EG. Knockdown of GLaz and Out also led to more Ecc15 food consumption, increased mortality after PE exposure, and reduced LD accumulation in the glia of old flies (Cai, 2021).

To confirm that infection or ageing-induced metabolic changes in EG affect neuron or glia metabolic coupling at the AL, the consequences of perturbing projection neurons directly were assessed using GH146::Gal4. Knocking down Out but not lactate dehydrogenase (Ldh) in projection neurons rescued olfactory discrimination of infected or aged flies, whereas food preference or mortality was not influenced. Overexpression of lipase 4 (Lip-4) in projection neurons, or knockdown of the neuronal lipid binding protein Neural lazarillo (NLaz), significantly improved olfactory discrimination in infected or old flies, and overexpression of Lip-4 increased Ecc15 food consumption and increased mortality after PE exposure (Cai, 2021).

This work suggests that gut-derived inflammatory cytokines modulate the metabolic coupling of glia and neurons in the brain of Drosophila to induce an adaptive temporary halt of olfactory discrimination after intestinal infection, but also contribute to age-related olfactory decline. It is proposed that gut-derived Upd2 and Upd3 reprogram lipid metabolism in EG, increasing lactate and lipid transport between glia and olfactory neurons, resulting in LD accumulation and upregulation of mitochondrial β-oxidation, potentially a source of increased ROS production. Chronic activation of this metabolic shift in old flies results in the accumulation of peroxidated lipids in EG, promoting their decay and contributing to the previously described functional decline of olfactory neurons. Detailed characterization of this metabolic reprogramming, and further exploration of the role of lipid synthesis in projection neurons for glial lipid accumulation and for olfactory discrimination are important avenues for further study (Cai, 2021).

These findings further determine the regulation of avoidance behaviour against enteropathogens in insects. In addition to gustatory bitter neurons and immune receptors in octopaminergic neurons, Upd proteins constitute a direct endocrine signal from the damaged intestinal epithelium in this complex but essential behaviour. It is proposed that Upd-mediated suppression of olfactory discrimination is required to prevent olfaction-mediated attraction to a food source after pathogenicity has been established and aversion is induced by gustatory neurons. It remains unclear, however, whether gustatory neurons are also affected by JAK-STAT signalling in EG. Whether similar mechanisms are conserved and control infection-induced loss of sensory perception in vertebrates including humans will be interesting to explore (Cai, 2021).

Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells

Paternal environmental factors can epigenetically influence gene expressions in offspring. This study demonstrates that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes (Seong, 2020).

This study indicates that paternal restraint stress induces epigenetic status of offsprings via a p38-MEKK1-dATF-2 pathway and restraint stress-induced upd3 might have a role in transmission of the stress information from somatic to germline cells. Notably, it was observed that paternal restraint stress reduces energy metabolism activity in offspring and sensitizes to rotenone toxicity (Seong, 2020).

Restraint stress information, received by the central nervous system (CNS) and/or other sensing tissues, promotes expression of upd3 in peripheral somatic tissues. In germ cells, accumulated humoral upd3 activates the JAK/STAT pathway, which subsequently activates the p38-dATF-2 pathway. Phosphorylated dATF-2 may be released from the promotor regions of its target genes, resulting in decreased H3K9me2 levels. Epigenetic marks may be retained in mature sperm and inherited by offspring. After fertilization, histone marks may act as inheritable epigenetic memory and regulate gene expression. It was observed that genes involved in the one-carbon metabolic pathway were upregulated in offspring from the paternal restraint stress-exposed fathers, while genes involved in the respiratory metabolic pathway were downregulated. It is assumed that paternal restraint stress-induced- and dATF-2-mediated upregulation of the one-carbon cycle induces downregulation of respiratory metabolism due to a trade-off relationship between these two metabolic processes (Seong, 2020).

The stress response, orchestrated by the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system via stress hormones (adrenaline, noradrenaline, and cortisol), plays a key role in physiology and in several diseases including depression. Accumulated evidence has also implicated inflammatory cytokines in depression. A peripheral infection can induce sickness behaviors, including depression-like behavior, which is caused by the immune-to-brain communication via proinflammatory cytokines produced from innate immune cells. Previous meta-analytic reports indicate that depressed patients possess significantly higher concentrations of IL-6. It was also reported that restraint stress activates the HPA axis and induces higher levels of IL-6 in a rodent model. Furthermore, IL-6 knockout mice exhibit resistance to stress-induced depression-like behaviors, suggesting that IL-6 plays a key role in eliciting depression. The present results suggest that upd3, the Drosophila homolog of IL-6, is also induced by restraint stress. Studies on the inflammatory status of the human HMC-1 cell line showed that adrenaline enhances IL-6, IL-8, and IL-13 production, mediated in part by the p38 signaling pathway. The Drosophila model of myeloproliferative neoplasm indicates that p38 pathway contributes to feed-forward loop in JAK pathway by regulating upd3 gene expression. In Drosophila, restraint stress-dependent upd-3 induction in peripheral tissue may also be mediated by the p38 pathway in response to stress hormones such as adrenaline, as occurs in mammalian systems (Seong, 2020).

The present study showed that upd3 has a role in transmitting restraint stress information to germ cells. It was observed that overexpression of upd3 in neuronal somatic tissues affected the heterochromatic status of offspring in a dATF-2 dependent manner. These results indicate that humoral Upd3, secreted from somatic cells, may be able to affect the epigenetic status of germ cells via the dATF-2 pathway. Moreover, p38 can be activated in response to several extracellular factors, such as TNF, IL-1, growth factors, insulin, salt concentration, and reactive oxygen species (ROS), either directly or indirectly. Thus, humoral components can act as key players to regulate the epigenetic status of germline cells. Upd3, a JAK ligand, is expressed in somatic gonads and activates the JAK/STAT pathway in male germ cells suggesting that testicular germ cells are responsive to the restraint stress-induced increase in Upd3. The activation of p38 by JAK signaling has been demonstrated in the Drosophila immune system, as well as in various vertebrate systems59. Somatic tissue-specific overexpression of upd3 clearly alters the heterochromatin status in the next generation. dATF-2 is highly expressed in germ cells in testes, and expression of TotA, a known upd3 signal target gene, is regulated by the MEKK1-dATF-2 pathway. Therefore, restraint stress-induced humoral upd3 in somatic tissues may control the epigenetic status of germ cells via the p38-dATF-2 signaling pathway in Drosophila. Interestingly, restraint stress-dependent activation of p38 in testes was mainly induced during the latter stages of restraint stress treatment for 10 h. A relatively long time may therefore be necessary to accumulate sufficient humoral upd3 for activation of p38 in germ cells (Seong, 2020).

In restraint stress experiments, animals may also receive mechanical stress, and it may be difficult to completely differentiate this from restraint stress. The relationship between restraint stress and mechanical stress is poorly understood. Mechanical stress induces different sets of cytokines in different types of tissues and IL-6 is one of the major components induced by mechanical stress in mammals. In Drosophila, a recent report showed that forceps squeezing of larvae induces upd3 gene expression. IL-6 induction by either restraint stress or mechanical stress may therefore reflect a common mechanism for sensing and communicating stress information between restraint stress in the nervous system and mechanical stress in the peripheral tissues (Seong, 2020).

In Drosophila, the transit time from spermatagonial stem cells to mature sperm available for insemination at 2 °C is ~260 h. During spermatogenesis, transcription mainly occurs in spermatagonial stem cells and spermatocytes. Herein, when fathers were exposed to restraint stress only once, only the first brood, but not successive broods, exhibited paternal restraint stress-induced heterochromatin disruption. However, exposure to restraint stress three times induced heterochromatin disruption not only in the first brood but also in successive broods. These results suggest that single restraint stress exposure induces heterochromatin disruption only in spermatocytes, whereas repeated restraint stress exposure also causes heterochromatin disruption in spermatagonial stem cells. Interestingly, it has been indicated that long-term memory is required for the maintained effect of the maternal transmission of ethanol preference after 24 h of wasp exposure. There is a possibility that the establishment of neuronal memory by restraint stress may be important for the stable transmission of the epigenetic status in germ cells. Further analysis is needed to elucidate the mechanistic aspect of maintenance of epigenetic change induced by restraint stress in germ cells (Seong, 2020).

Increasing evidence indicates that the paternal environment affects the transcriptome and epigenetic status of their offsprings in several animal models. Although the mechanism of paternal inheritance of epigenetic status remains unclear, some evidence suggests that histone modification is an important player. A recent study indicated that histone replacement-completed sperm retain histone H3 mainly on specific promoter regions in mice. In Drosophila, epiallelic status of H3K27me3 can be dominantly transmitted to the progeny. Drosophila paternal Cid/Cenp-A, a centromere histone protein, can be transmitted to progeny embryos. Similarly, human spermatozoa retain and transmit nucleosomes with constitutive heterochromatin containing H3K9me3 to embryos. A previous report showed that dATF-2 is required for the establishment and maintenance of heterochromatin by regulating the H3K9me2 level, and heat shock stress induces dATF-2 phosphorylation by p38 and its release from heterochromatin, and disrupts the heterochromatin status in germ cells. This causes heterochromatin disruption, which is inherited by the next generation. Based on these observations, it is likely that restraint stress disrupts heterochromatin by decreasing H3K9me2 levels in testicular germ cells, and this is transmitted to offspring. Via a similar mechanism, restraint stress may reduce H3K9me2 on dATF-2 target genes, and this may be transmitted to offspring to modulate metabolism. However, it was not possible to analyze dATF-2 target genes in testicular germ cells by ChIP because anti-dATF-2 antibodies worked only on embryo chromatin but not testis chromatin. This could be due to the presence of non-specific proteins that bind to anti-dATF-2 antibodies in testes. To detect epigenetic effect of paternal restraint stress, restraint stress-treated w1118 males were crossed with wm4 females. The level of histone H3K9me2 was observed to be slightly but significantly decreased in the w gene locus of wm4 offsprings from restraint stress-treated fathers. Although the X chromosome harboring the wm4 gene in the male offspring was derived from the unstressed female, paternal restraint stress increased w expression in male offspring, which is reminiscent of paramutation. It has been reported that paternal stress can influence epigenetic status on maternally supplied X chromosome using the wm4 strain. A previous report also indicated that quantitative content of heterochromatin on the Y chromosome affects PEV phenotypes of wm4 flies. Physical pairing between the heterochromatic regions of X chromosomes and other chromosomes, or the trans-action of some molecules, such as noncoding RNA, may induce partial disruption of the heterochromatin on the X chromosome. Small noncoding RNAs have been also reported as a factor of transgenerational transmission of paternal phenotypes in several model animals. Noncoding RNAs in sperm may also influence the transgenerational effect by paternal restraint stress. Further analysis is required to elucidate the detailed mechanism by which restraint stress affects metabolism in offspring via dATF-2 in testicular germ cells (Seong, 2020).

Consistent with a previous report on mice, the present study indicates that paternal restraint stress affects metabolism in offspring, albeit differently, possibly due to differences among species and experimental conditions. In the present study, paternal restraint stress enhanced the expression of genes involved in one-carbon metabolism, while paternal restraint stress decreased the expression of genes involved in glycolysis and the TCA cycle in the previous work on mice. Restraint stress disrupts heterochromatin possibly by decreasing the H3K9me2 level, which is thought to directly enhance the transcription of dATF-2 target genes. Therefore, downregulation of genes by paternal restraint stress, such as glycolysis-related genes, may occur indirectly via upregulation of one-carbon metabolism-associated genes. The phosphoglycerate dehydrogenase (PHGDH) gene is important in balancing glycolysis and one-carbon metabolism, and its amplification in some cancer cells causes the diversion of a relatively large amount of glycolytic carbon into serine and glycine metabolism. The upregulation of one-carbon metabolism genes, including PHGDH, enhances the levels of glutathione and taurine, which are major factors for reducing ROS during detoxification. ROS stabilizes the transcription factor hypoxia-inducible factor-1α (HIF-1α), which activates genes related to glycolysis. Therefore, one possibility is that indirect downregulation of glycolysis genes may be due to decreased activity of HIF-1 following the lowering of ROS levels by paternal restraint stress (Seong, 2020).

Multiple studies have shown that psychological stress affects metabolism, and metabolism conversely modulates the response to psychological stress. The incidence of both depression and diabetes increases with age, and inflammatory cytokines play a key role in eliciting both diseases. Growing evidence indicates that depression and type 2 diabetes share biological origins, particularly overactivation of a cytokine-mediated inflammatory response, potentially through dysregulation of the HPA axis. Thus, psychological stress and metabolism are tightly connected, and this study indicates that psychological stress modulates metabolism in offspring through intergenerational inheritance via sperm. Some previous evidence showed that diet composition may attenuate stress-induced symptoms, and the present results, including precise gene expression changes induced by paternal restraint stress, may contribute to the development of useful foods or supplements with therapeutic benefits (Seong, 2020).

Fat- and sugar-induced signals regulate sweet and fat taste perception in Drosophila

This study investigated the interplay between taste perception and macronutrients. While sugar's and protein's self-regulation of taste perception is known, the role of fat remains unclear. In Drosophila, fat overconsumption reduces fatty acid taste in favor of sweet perception. Conversely, sugar intake increases fatty acid perception and suppresses sweet taste. Genetic investigations show that the sugar signal, gut-secreted Hedgehog, suppresses sugar taste and enhances fatty acid perception. Fat overconsumption induces Unpaired 2 (Upd2) secretion from adipose tissue to the hemolymph. This study revealed that taste neurons take up Upd2, which triggers Domeless suppression of fatty acid perception. It was further shown that the downstream JAK/STAT signaling enhances sweet perception and, via Socs36E, fine-tunes Domeless activity and the fatty acid taste perception. Together, these results show that sugar regulates Hedgehog signaling and fat induces Upd2 signaling to balance nutrient intake and to regulate sweet and fat taste perception (Zhao, 2023).

This study investigated how sugar and fat overconsumption regulate taste perception in Drosophila melanogaster. Consistent with previous work, it was found that gut-derived Hh serves as a proxy for sugar. In Drosophila, fat and sugar activate sweet taste cells, and it was expected that this signal would suppress both sugar and fatty acid taste perception and thus regulate caloric intake. However, it was demonstrated that the sugar-induced Hh signal increases fatty acid taste sensitivity and suggests a nutrient-balancing effect. The results also show that the gut Hh signal shifts from enhancing to suppressing fatty acid taste perception under an HFD. Thus, sugar and the gut-released Hh regulate taste to balance fat and sugar nutrient intake and not caloric intake (Zhao, 2023).

Diet experiments further demonstrated a reciprocal relationship between the two macronutrients in which fat consumption increases sweet sensation and sugar consumption enhances fat taste perception. It was also demonstrated that fat overconsumption suppresses fatty acid perception much like sugar overconsumption suppressed sweet taste. The results demonstrate that Upd2 is the fat taste suppressive signal. Fat overconsumption induces fat body expression and secretion of Upd2 that activates JAK/STAT signaling in the sweet taste neurons. This mechanistic study further shows that fatty acid taste suppression is directly downstream of Dome, whereas the sweet taste regulation requires JAK/STAT signaling. Together these results demonstrate that there are two proxy signals, Hh for sugar and Upd2 for fat, that both regulate fatty acid and sweet taste (Zhao, 2023).

The results show that Upd2, similar to Hh, is taken up and stored by the taste neurons. For both ligands, the reservoir increases the ligand’s local concentration. The increase in ligand concentration is likely vital because most signaling molecules must meet a specific threshold concentration for effectiveness. The gut secretion of Hh during development also does not affect wing development, which together with the current data suggests that the endocrine signals may require local accumulation to achieve effective levels. Another reason for the reservoir is likely to reduce fluctuations in ligand and taste regulation. Accumulation of the ligand likely takes time, and uptake can be regulated, and the reservoir levels do not necessarily reflect the hemolymph levels. Thus, the reservoir increases ligand levels and at the same time makes ligand levels and the taste signal robust to quick metabolic changes (Zhao, 2023).

This mechanistic analysis further shows that the fatty acid taste perception level is regulated by negative feedback on Dome. Two negative factors in the Upd2 pathway were discovered, Et">and Socs36E, dramatically affect fatty acid perception. The two have different effects on JAK/STAT signaling, with Et sequestering Dome in signaling-incompetent complexes and Socs36E inhibiting both Dome and STAT activation. Et also only regulates fatty acid perception, whereas Socs36E regulates both fat and sugar taste. Simple negative feedback systems, like the Dome-Stat92E-Socs36E-Dome, tend to slow down regulation and make it more robust to changes. Thus, the storage and feedback loops of Hh and Upd2 suggest that many cycles of consumption and taste are continuously interacting to form a sort of "memory" of the sugar-respective fat state. This it was also observed in flies raised on the balanced low-fat control diet. In this condition, although Upd2:GFP is only found at low levels in the hemolymph, Socs36E is still required to avoid loss of fatty acid taste. This suggests that Upd2 levels are not limiting and cause a sufficient activation of Dome even on balanced diets. It also suggests that Upd2 and Hh, rather than acting as a gradual signal, communicate the presence of fat and sugar in the diets (Zhao, 2023).

Collectively, these findings underscore taste sensitivity as a product of both immediate feeding experience and cumulative nutritional history. The taste regulatory mechanisms between sugar and fat have similarities, in that they are slow and act over several meals, functioning as a body memory of the feeding and nutritional status. The signals form a closed regulatory loop in which the final component, the nutrient level in the body, feeds back on the start, taste and food identification. Thus, this body-taste axis when in balance probably sets taste perception levels that best support the metabolic needs of the animal. Consequently, if intake becomes dysregulated, it might reach a point where the taste regulatory mechanisms described here can speed up rather than inhibit sugar and fat malnutrition and advance to obesity and metabolic disease (Zhao, 2023).

The following are limitations to this study. (1) Only males were studied; how females and the changes in metabolism after mating are linked to Upd2 and Hh remain for investigation. (2) The secretion of the endogenous Upd2 was not studied. Thus, it was not possible to show the full magnitude of the Upd2 uptake and dynamics. (3) How Upd2 regulates food intake or foraging strategies was not studied. Feeding and free running behavior studies are needed for such conclusions. (4) Only fat and sweet taste sensation was studied. It seems likely that micronutrients (vitamins, salts, and minerals) also have dedicated signals that would be interesting to identify (Zhao, 2023).

REGULATION

Transcriptional Regulation

Notch (N) signal is activated at the dorsoventral (DV) border of the Drosophila eye disc and is important for growth of the eye disc. In this study, the Pax protein Eyg is shown to be a major effector mediating the growth promotion function of N. eyg transcription is induced by N signaling occurring at the DV border. Like N, eyg controls growth of the eye disc. Loss of N signaling can be compensated by overexpressing eyg, whereas loss of the downstream eyg blocks the function of N signaling. In addition, N and eyg can induce expression of upd, which encodes the ligand for the Jak/STAT pathway and acts over long distance to promote cell proliferation. Loss of eyg or N can be compensated by overexpressing upd. These results suggest that upd is a major effector mediating the function of eyg and N. The functional link from N to eyg to upd explains how the localized Notch activation can achieve global growth control (Chao, 2004).

Notch is activated at the DV boundary of the early eye disc. This equatorial N signal then activates eyg expression at the transcriptional level. When N signal is reduced, eyg expression is reduced. When N signal is elevated, eyg expression is induced. Induction of eyg expression occurs at the DV border between the dorsal Dl-expressing and the ventral Ser-expressing cells. Furthermore, when the upstream N signal is blocked, overexpression of eyg can rescue the growth defect in the eye, whereas increasing N signaling cannot rescue the eye-growth defect caused by the downstream eyg gene. This analysis shows that the induction of eyg by N is dependent on the ligands Dl and Ser, and involves the effector Su(H) and the antagonist Hairless. Thus, the localized activation of N signal is transmitted to the induction of a transcription factor, Eyg, which then promotes cell proliferation. A recent paper (Dominguez, 2004) has come to the same conclusion (Chao, 2004).

Eyg is a transcription factor, so must activate the transcription of some genes that promote cell proliferation. Upd is reported to act through the Jak/STAT signaling pathway to promote cell proliferation. upd expression is dependent on eyg and N signaling. Furthermore, when the upstream N signaling or eyg is reduced, overexpression of upd can rescue the growth defect. The overgrowth effect due to overexpression of the upstream N or eyg is blocked when the downstream upd is defective. The results suggest that upd is a major effector for the growth promotion by N and eyg (Chao, 2004).

These results have demonstrated the functional link from Notch to eyg to upd in the promotion of eye growth. The link to upd solved a long-standing problem. N signaling is activated locally at the border between the dorsal Dl-expressing cells and the ventral Ser-expressing cells. How does a localized activation of N signal promote cell proliferation throughout the entire eye disc? The finding of eyg as the major mediator of N function did not solve the problem, since Eyg is a transcriptional factor and is expected to affect target gene expression autonomously. The link from eyg to upd provided a solution, because Upd is a diffusible signaling molecule. Upd protein can distribute over a long distance and exert long-range non-autonomous effect to promote cell proliferation (Tsai, 2004). So the localized N activation can locally activate eyg, which then turns on upd expression, probably through a short-range signal. The Upd signal then acts over a long range to promote cell proliferation in the early eye disc (Chao, 2004).

Although N activates eyg, and eyg activates upd, these transcriptional activation may be direct or indirect. When novel DV borders were created by ectopic expressing Dl or Ser, eyg is induced non-autonomously at the border of these clones. It is also noted that in Su(H) mutant clones, mutant cells at the border of the clone can still express eyg-lacZ. These observations suggest that N may induce a short-range signal, which then activates eyg expression. Alternatively, the apparent non-autonomous induction may be due to perdurance of the reporter protein in cells that were once close to the clone border. The induction of upd by eyg also may be indirect. Clonal expression of eyg also induced upd expression non-autonomously. In addition, based on RNA in situ hybridization, eyg expression in the eye disc does not extend to the posterior margin, so does not overlap with the expression domain of upd (Tsai, 2004). These observations suggested that the effect of eyg on upd expression may be indirect. However, an eyg enhancer trap line showed reporter expression extending to the posterior margin (Dominguez, 2004). Thus, the possibility that Eyg can directly activate the expression of upd cannot be excluded (Chao, 2004).

The activation of eyg and upd are context dependent. N^act does not induce eyg expression in antenna and wing discs. In the eye disc, N^act induces eyg expression only in the region anterior to the MF, and not within the wg expression domain in the lateral margin. Similarly, N^act and eyg can only induce upd expression at the margin, but not in the center of the eye disc. N^act induces upd at the posterior margin but not lateral margins, while eyg can induce upd in the lateral margins but not in the posterior margin. The context dependence indicates that additional factors are involved to determine the specificity of induction (Chao, 2004).

In a late third instar eye disc, eyg is expressed in an equatorial domain that does not overlap with the disc margin, so eyg cannot induce upd. In early eye disc, eyg expression domain comes closer to the posterior margin. Thus, the induction of upd by eyg is likely at second instar, which is consistent with the timing of upd expression (Chao, 2004).

Although eyg plays an important role in mediating the growth-promoting N signal, it is probably not the only effector. In the eyg^M3-12 null mutant, ey>N^act does not rescue the endogenous eye field, but can still induce proliferation to provide the antennal disc and an extra eye field. Thus, N can induce proliferation by an eyg-independent mechanism. The effect on antenna and on eye seems to be separate, because ey>N^act can induce a large antenna disc with duplicate or triplicate antennal field without rescue of the eye disc. Because N can induce upd, but not eyg, in the posterior margin, the induction of upd can also be through an eyg-independent mechanism (Chao, 2004).

N^act can induce overgrowth in the central domain of the eye disc. In this case, eyg, but not upd, is induced. In addition, the overgrowth does not extend much beyond the clone. Ectopic eyg in the central domain also induces proliferation without inducing upd. In eyg¹ mutant, there is no upd-lacZ expression in eye disc, but the eye is only slightly reduced. These results suggest that the N signaling and eyg can induce local proliferation independent of upd (Chao, 2004).

Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth caused by increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired

The reproducible pattern of organismal growth during metazoan development is the product of genetically controlled signaling pathways. Patterned activation of these pathways shapes developing organs and dictates overall organismal shape and size. Patches of tissue that are mutant for the Drosophila Tsg101 ortholog, erupted, cause dramatic overexpression of adjacent wild-type tissue. Tsg101 proteins function in endosomal sorting and are required to incorporate late endosomes into multivesicular bodies. Drosophila cells with impaired Tsg101 function show accumulation of the Notch receptor in intracellular compartments marked by the endosomal protein Hrs. This causes increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired (upd), which encodes the secreted ligand of the JAK-STAT pathway. Activation of JAK-STAT signaling in surrounding wild-type cells correlates with their overgrowth. These findings define a pathway by which changes in endocytic trafficking can regulate tissue growth in a non-cell-autonomous manner (Moberg, 2005). Tsg101 possesses the ability to bind monoubiquitinated substrates. These substrates are predicted to be the ubiquitinated cytoplasmic tails of membrane bound proteins, and this interaction is predicted to deliver cargos to the lysosome via multivesicular bodies (Moberg, 2005 and reference therein).

The Notch receptor has two properties that implicate it in a pathway by which ept mutations non-cell-autonomously promote tissue growth. (1) The restricted activation of Notch in cells along the dorsoventral (D/V) boundary of the eye imaginal disc is required for growth of the entire eye. (2) Ub-dependent endocytosis plays an important role in regulating Notch activity in vivo. In mammalian cells, ubiquitination and endocytosis contributes to Notch1 activation, and, in Drosophila, there is evidence to suggest that the ubiquitin ligase Deltex may be required for endocytosis-dependent Notch activation. Further, alleles of the endosomal sorting gene Hrs, the homolog of yeast Vps27, affect Notch localization in imaginal disc cells, indicating that Notch is a physiological target of the MVB pathway (Moberg, 2005).

In light of these observations, ept mosaic eye discs were stained with an antibody specific to the Notch cytoplasmic domain (anti-N^cyto). Notch protein is detected in wild-type eye discs most prominently in a stripe of cells within the morphogenetic furrow (MF) and is concentrated at the apical cell surface. In contrast, ept cells contain elevated levels of Notch. This increase occurs in ept clones throughout the eye disc, but it is most apparent in clones that lie within or posterior to the MF. Moreover, the Notch in ept cells accumulates in nonnuclear, intracellular puncta that also stain positive for Ub, and for the endosomal protein Hrs. Together, these data indicate that ept mutations block the routing of ubiquitinated cell surface proteins, among them Notch, in an Hrs-positive endosomal compartment (Moberg, 2005).

Notch is normally processed in cells by a series of cleavage events required for receptor maturation and presentation at the cell surface, and for ligand-stimulated activation of the Notch pathway. Because ubiquitination and endocytosis have been shown to affect Notch cleavage, attempts were made to determine if ept mutations also affect Notch processing. Eye-antennal discs composed of ept mutant cells [ept/M(3)] or FRT80B control cells (FRT80B/M(3)) were generated by the eyFLP/Minute technique. Immunoblot of tissue extracts with the anti-N^cyto antibody confirms that Notch levels are increased considerably in eye-antennal discs composed of ept mutant cells, and shows that ept mutant cells are enriched in a ~120 kDa form of Notch. The molecular identity of this fragment has not been determined, but its size appears similar to certain processed forms of Notch. Indeed, while no one form of Notch predominates in wild-type cells, this species appears to be the most abundant Notch species in ept cells (Moberg, 2005).

To examine Notch activation, clones of ept mutant cells were generated in the presence of the Notch-inducible transgene E(spl)mβ-CD2, a Suppressor of Hairless (Su(H))-dependent transcriptional reporter that has been used to detect equatorial Notch activation in the developing eye. Posterior to the MF, CD2 expression is detected in the interommatidial cells, and outlines a single cell from each photoreceptor cluster in a mirror-image pattern along the equator. Thus, in addition to equatorial activation, the reporter detects Notch activation in postmitotic interommatidial cells, and in the R3-R4 cell fate choice. In ept mutant clones, reporter activity is strongly elevated. The degree of activation exceeds that observed in wild-type eye discs, and it does not appear to depend upon the location of ept cells within the disc, occurring on either side of the MF and in the antennal disc. Some ept cells within a single optical section appear not to activate the Notch reporter. However, in most of these cases, CD2, which localizes to cell membranes, can be detected in a focal plane slightly offset from that of the nuclear green fluorescent protein (GFP). Thus, these data show that defects in Notch regulation in ept cells are accompanied by ectopic and excessive activation of the Notch pathway (Moberg, 2005).

The requirement for Notch in eye disc growth has been linked to its ability to induce expression of the eyegone (eyg) gene at the D/V boundary of the eye disc. eyg encodes a Pax6-like transcription factor (Eyg) required for disc growth, and, like Notch, ectopic expression of eyg is able to induce growth nonautonomously. Consistent with its effect on Notch, it was found that ept mutant cells express elevated levels of Eyg compared to surrounding wild-type cells. Thus, Eyg may function downstream of Notch within ept cells to promote the growth of surrounding cells in a manner similar to its normal growth-promoting role at the D/V boundary (Moberg, 2005).

Recent work suggests that the unpaired (upd) gene may be an important growth regulatory target of Notch. upd encodes the secreted ligand (Upd) of the Domeless (Dome) receptor, which signals through the JAK-STAT pathway. JAK-STAT signaling is implicated in many processes during Drosophila development, including the control of cell proliferation, cell motility, stem cell renewal, and planar cell polarity. upd is required for normal growth of the eye, and ectopic expression of upd in the larval eye nonautonomously promotes cell proliferation and produces enlarged and misshapen eyes similar to those observed in ept mosaics. Significantly, Notch is both necessary and sufficient to activate upd transcription along the posterior margin of the eye disc (Moberg, 2005).

When ept mosaic eye discs were stained with an anti-Upd antiserum, a dramatic increase was observed in the level of Upd protein in ept mutant cells compared to adjacent wild type cells. Consistent with a transcriptional link between Notch and upd, Upd protein accumulation appears coincident with expression of the Notch reporter, and ept mosaic eye-antennal discs contain clones of cells expressing very high levels of upd mRNA. Together, these observations suggest that Notch, perhaps acting via Eyg, promotes ectopic upd expression in ept mutant cells (Moberg, 2005).

Clonal overexpression of upd induces localized tissue outgrowths and deregulates the division of surrounding cells. This mitogenic activity is linked to induction of cyclin D, and to accelerated progression through the G1 phase of the cell cycle. ept mutant clones can produce phenotypes quite similar to clonal overexpression of upd. In one example of an ept clone, lower half of the disc appeared morphologically normal, while the other half, despite being composed largely of wild-type cells, was misshapen and enlarged. This localized effect correlated with proximity to a large ept mutant clone expressing Upd. Similar hyperplastic growth was associated with clones of upd-expressing cells in the antennal disc. The patterns of BrdU incorporation in ept mosaic eye discs are disorganized, and the number of BrdU-labeled nuclei increases in proximity to Upd-expressing ept mutant cells in the eye and antenna. This aberrant cell proliferation occurs in GFP-positive wild-type cells. Hence, the growth-promoting activity of ept mutations is likely mediated by a diffusible extracellular signal like Upd (Moberg, 2005).

Receipt of the Upd signal via Domeless initiates a signaling cascade that activates a transcription factor encoded by the stat92E gene. stat92E encodes the Drosophila ortholog of the mammalian signal transducers and activators of transcription (STAT) family of transcriptional regulators, which function in diverse processes such as immunity and oncogenesis, and is the only member of this gene family in Drosophila. Heterozygosity for a stat92E loss-of-function allele (stat92E⁰⁶³⁴⁶) strongly suppresses the nonautonomous eye overgrowth associated with mosaicism for ept mutations, such that ept-mosaic;stat92E⁰⁶³⁴⁶/+ eyes are comparable in size to control FRT80B mosaic eyes. Thus, nonautonomous overgrowth elicited by ept mutations is sensitive to the genetic dosage of the Upd-responsive transcription factor stat92E. In light of the effect on Upd, these data strongly indicate that the growth-promoting activity of ept mutant cells requires Upd-dependent activation of the JAK-STAT pathway in adjacent tissue (Moberg, 2005).

ept mutant clones in mosaic eye discs are small and survive poorly into adulthood. It is possible that this is the result of cell competition, a process by which slow-growing cells in the vicinity of wild-type cells are eliminated. If so, then the poor survival of ept cells might be rescued by eliminating competing cells. Therefore the growth characteristics were examined of ept/M(3) discs, which are composed almost entirely of cells lacking Tsg101 function. ept/M(3) animals reach the larval 'wandering' stage 4 days later than control larvae, and, when they do, they are enlarged. A small fraction of these animals pupate and die before becoming pharate adults. The remainder die as giant larvae containing high levels of Upd (Moberg, 2005).

Allowing ept mutant cells to grow in epithelia lacking wild-type cells also uncovers a context-dependent cell-autonomous overgrowth phenotype. Rather than surviving poorly as they do in mosaic discs, ept/M(3) eye discs overgrow into large masses that lack normal disc morphology. These masses are composed of folded and convoluted sheets of cells fused together, and they often include a distended sac-like structure. The overgrowth phenotypes of ept/M(3) animals and discs do not reflect an increased rate of growth: control L3 larvae are the same size as ept/M(3) larvae of the same temporal age, and the ept/M(3) eye discs, while mispatterned, are not obviously increased in size. Thus, the ept/M(3) masses are the result of an extended larval phase, and a failure of the disc to stop growing when it reaches the appropriate size. Thus, cells lacking Tsg101 may be unable to respond to signals that normally sense and restrict organ size (Moberg, 2005).

Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development

The TOR and Jak/STAT signal pathways are highly conserved from Drosophila to mammals, but it is unclear whether they interact during development. The proline-rich Akt substrate of 40 kDa (PRAS40) mediates the TOR signal pathway through regulation of TORC1 activity, but its functions in TOR complex 1 (TORC1, a rapamycin-sensitive form of Tor in mice that consists of mTOR, raptor, and mLST8) proved in cultured cells are controversial. The Drosophila gene Lobe (L) encodes the PRAS40 ortholog required for eye cell survival. L mutants exhibit apoptosis and eye-reduction phenotypes. It is unknown whether L regulates eye development via regulation of TORC1 activity. This study found that reducing the L level, by hypomorphic L mutation or heterozygosity of the null L mutation, resulted in ectopic expression of unpaired (upd), which is known to act through the Jak/STAT signal pathway to promote proliferation during eye development. Unexpectedly, when L was reduced, decreasing Jak/STAT restored the eye size, whereas increasing Jak/STAT prevented eye formation. Ectopic Jak/STAT signaling and apoptosis are mutually dependent in L mutants, indicating that L reduction makes Jak/STAT signaling harmful to eye development. In addition, genetic data suggest that TORC1 signaling is downregulated upon L reduction, supporting the idea that L regulates eye development through regulation of TORC1 activity. Similar to L reduction, decreasing TORC1 signaling by dTOR overexpression results in ectopic upd expression and apoptosis. A novel finding from these data is that dysregulated TORC1 signaling regulates the expression of upd and the function of the Jak/STAT signal pathway in Drosophila eye development (Wang, 2009).

The target of rapamycin (TOR) and Jak/STAT signal pathways are highly conserved in animals and important in many developmental processes. Dysregulation of these pathways can lead to cancer formation. This study presents data showing that TOR regulates the function of Jak/STAT signaling during Drosophila eye development (Wang, 2009).

The gene unpaired (upd) encodes a ligand that activates Drosophila Jak/STAT signaling. It is expressed in the posterior margin of the dorsal/ventral (D/V) boundary, the posterior center (PC), in the larval eye imaginal disc at second and early third instar stages. Notch at the D/V boundary activates the transcription of eye gone (eyg), which activates upd expression at the PC. Expression of upd is also regulated by Hedgehog (Hh) signaling. The cells of Drosophila compound eyes are derived from the eye-antennal disc, which develops from ectoderm of the embryo and grows inside the larva. These cells proliferate rapidly during the first and second instar stage. In early third instar larvae, morphogenetic furrow (MF) that arise at the posterior margin progresses in a wave-like manner toward the anterior margin of the eye disc. Jak/STAT signaling is known to promote proliferation during eye development, and is required for MF initiation; a loss of Jak/STAT function results in reduced eyes. Therefore, Jak/STAT signaling is regulated by Notch/Eyg and the Hh signaling pathways, and plays positive roles in eye development (Wang, 2009).

TOR signaling is one of the downstream branches of insulin signal pathway. Insulin and insulin-like growth factor elicit a signal cascade involving phosphatidyl-inositol 3-kinase (PI3K) that stimulates PDK-mediated Akt phosphorylation. Phosphorylated Akt can activate TOR, which nucleates the TOR complex 1 (TORC1), allowing it to phosphorylate the downstream targets, the translational repressor eukaryotic initiation factor (4EBP) and the ribosomal protein S6 kinase (S6k). Phosphorylation of 4EBP and S6K promotes CAP-dependent translation and thereby increases protein synthesis. In addition, activation of TOR can also promote ribosome biogenesis via Myc. Loss of the Drosophila TOR (dTOR) function reduces eye size, indicating that TOR signaling is required for eye development (Wang, 2009).

PRAS40 mediates the insulin signal pathway from Akt to TORC1. Upon insulin stimulation, activated Akt phosphorylates PRAS40 and causes it to dissociate from TORC1, allowing TORC1 signaling to proceed. Thus, PRAS40 can apparently act as an inhibitor of TORC1. However, it has been reported that PRAS40 is required for TORC1 activity, and thus the interactions of PRAS40 with TORC1, based on studies in cultured cells are controversial. The effect of PRAS40 on TORC1 signaling in vivo is still unclear (Wang, 2009).

The Drosophila Lobe (L) protein shares high sequence conservation with PRAS40. L mutants have reduced adult eyes and exhibit ectopic apoptosis during eye development, indicating that L is required for eye development. But whether it regulates eye development via regulation of TORC1 activity is unknown (Wang, 2009 and references therein).

This study identified a new L allele, L^fee. Quantitative RT-PCR and genetic analysis revealed that L^fee is a hypomorphic allele. The eye defect was mediated by ectopic Jak/STAT signaling and cell apoptosis. In L mutants, the ectopic Jak/STAT signaling had a negative effect on eye development, but not a positive one as previously reported. It was also found that TORC1 signaling was hypoactivated in L mutants, suggesting that, like PRAS40, L is required for TORC1 activity. This study suggests that hypoactivated TORC1 signaling in L mutants result in ectopic Jak/STAT signaling and apoptosis, impairing eye development (Wang, 2009).

The spontaneous mutant fly, freaky eye (fee), is homozygously viable and has abnormal adult eyes. The eyes of most fee flies are smaller than those of wild type flies because of a nick at the anterior border of the eye. At the nicked region, extra hairs and/or rod-like tissues are usually present. Overgrowth of eye tissue occasionally occurs, resulting in eye enlargement. The eyes of fee flies were categorized into six classes depending on their size relative to the eyes of the wild type. The various eye-reduction phenotypes of fee flies were similar to those of L mutants. For example, the L^si heterozygote exhibits slightly reduced eyes that are nicked near the anterior D/V boundary, similar to the major fee phenotype. In the L^si homozygote, the ventral eye is absent, which is also reminiscent of the fee phenotype (Wang, 2009).

Whether fee is a mutant of L was investigated; the trans-heterozygotes for fee and the null mutant L^rev6-3 had smaller eyes than fee flies. In addition, fee could to be recombined with L^rev6-3, suggesting that fee is allelic to L. Quantitative RT-PCR showed that the L mRNA levels were highly reduced in fee flies, suggesting that fee is a L hypomorphic mutant; therefore these were designated L^fee (Wang, 2009).

This study shows that reduction of L phenocopies overexpression of dTOR. Overexpression of dTOR has been reported to produce phenotypes similar to that of loss of dTOR, because excess dTOR may titrate cofactors and thereby decrease TOR activity. This suggests that TOR signaling is downregulated by L reduction. Consistent with this, genetic analysis of L mutants and TOR signal pathway component suggest TORC1 hypoactivity in L mutants. PRAS40 has been proposed to function in the assembly of TORC1. It is possible that, in similar way to dTOR overexpression, reducing L impairs TORC1 assembly, thus decreasing TORC1 signaling. Reduction of L may disrupt eye development through downregulation of TORC1 signaling, supporting the idea that PRAS40 is required for TORC1 activity (Wang, 2009).

Drosophila eye development requires the TOR and Jak/STAT signal pathways, but it is not know whether an interaction between these two signal pathways occurs. Endogenous upd expression is present in the posterior center (PC) of the eye disc, but not in the interior eye disc. This study demonstrated that L reduction can induce ectopic upd expression in the interior eye disc, indicating that L is a negative regulator for upd expression. The data show that L reduction-mediated eye disruption is due to hypoactivation of TORC1 signaling, suggesting that hypoactivity of TORC1 is responsible for inducing upd expression (Wang, 2009).

Ectopic upd expression is induced by reduction of L (L^fee and L^rev/+), but not by its complete loss (L^rev homozygous clones), suggesting that different L levels may cause distinct effects. As PRAS40 acts to transmit the Akt signal to TORC1, complete loss, but not reduction, of L could result in an uncoupling between Akt and TORC1. This would release the Akt-mediated inhibition of TORC1, resulting in increased TORC1 activity. Thus, complete loss of L or PRAS40 may increase TORC1 activity. It is possible that the opposite functions of PRAS40 reported in cultured cells could be due to different PRAS40 levels remaining after knockdown. Whether complete loss of L function inhibits or promotes TORC1 signaling in Drosophila eyes remains to be investigated (Wang, 2009).

Mosaic analysis data showed that dTOR homozygote clones did not induce ectopic upd expression, suggesting that complete loss of dTOR function has a different effect from that of L reduction. Overexpression of dMyc can completely restore the eye size in the L^fee flies, but only partially represses the eye defect of dTOR overexpression. These data support the idea that L reduction may not equate to loss of dTOR. It was reasoned that as TOR is involved in TORC1 and TORC2, its loss should eliminate the functions of both TORC1 and TORC2. Because L participates only in TORC1 signaling, reduction of L would affect TORC1 signaling only. The regulation of TORC1 and TORC2 signaling by L needs further investigation (Wang, 2009).

It was found that suppressing apoptosis can decrease ectopic upd expression upon L reduction, suggesting that apoptosis is a cause of ectopic upd expression. It has been reported that apoptosis can activate ectopic upd expression and Jak/STAT signaling via Notch signaling in apoptosis-induced compensatory proliferation. However, ectopic upd expression on L reduction is not likely to be mediated by Notch activity, and no ectopic proliferation occurs. Thus, apoptosis due to L reduction is different from apoptosis-induced compensatory proliferation. Further, TOR hypoactivation may trigger ectopic upd expression independent of apoptosis; suppression of apoptosis did not eliminate all ectopic upd expression. Further investigation of how hypoactivated TORC1 regulates upd expression is needed (Wang, 2009).

The Drosophila Upd acts through Jak/STAT signaling to promote proliferation during eye development. However, this study found that on L reduction, decreasing Jak/STAT signaling could restore the eye defect, whereas increasing the upd expression level could completely abolish eye development. Thus, an unexpected finding was that ectopic Jak/STAT signaling in L mutants is harmful to eye development (Wang, 2009).

The fact that decreasing Jak/STAT signaling can reduce apoptosis in L mutants indicates that the induction of ectopic Jak/STAT signaling is required for apoptosis. It was reasoned that the apoptosis-promoting ability of Jak/STAT is possibly due to its repression of Serrate (Ser) expression. Ser expression is inhibited by L mutation, and loss of Ser function during eye development causes apoptosis. The current data showed that heterozygosity for Ser can reduce eye size in L^fee heterozygotes, but not in the wild type, suggesting that decreased Ser expression may play a role in eye reduction. Whether Ser repression mediates the apoptosis remains to be investigated. In addition, because inhibition of apoptosis does not strongly restore the L eye defect, but decreasing Jak/STAT activity fully restores it (comparing ey > p35 and Stat92E^ts), there is the possibility that the ectopic Jak/STAT activity affects eye development via an apoptosis-independent mechanism. Thus, a novel finding from the data is that Jak/STAT signaling can negatively regulate eye development (Wang, 2009).

An important issue is the control over the positive and negative roles of Jak/STAT signaling during eye development. Overexpression of upd driven by ey-GAL4 in the wild type produces adult with enlarged eyes, but it eliminates eye formation in L mutants. Because L reduction exhibits hypoactivation of TORC1 signaling, it is speculated that TORC1 signaling plays a role in controlling the balance between the opposing functions of Jak/STAT signaling (Wang, 2009).

In summary, reduction of the Drosophila PRAS40 L results in hypoactivation of TORC1 signaling. This leads to apoptosis and ectopic Jak/STAT activation, both of contribute to disruption of eye development. The data indicate that TORC1 signaling is able to regulate the expression and functions of the Jak/STAT signal pathway during eye development. Further studies using L mutants may uncover the mechanisms by which L regulates TORC1 signaling, and how TOR controls the Jak/STAT signal pathways. Also noteworthy is the report that decreasing PRAS40 can increase apoptosis of tumor cells, and it is therefore of interest to investigate whether PRAS40 and TORC1 can regulate the Jak/STAT signal pathway in tumors (Wang, 2009).

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 Apc^Min/+ 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).

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).

The transcriptional response to tumorigenic polarity loss in Drosophila

Loss of polarity correlates with progression of epithelial cancers, but how plasma membrane misorganization drives oncogenic transcriptional events remains unclear. The polarity regulators of the Drosophila Scribble (Scrib) module are potent tumor suppressors and provide a model for mechanistic investigation. RNA profiling of Scrib mutant tumors revealed multiple signatures of neoplasia, including altered metabolism and dedifferentiation. Prominent among these was upregulation of cytokine-like Unpaired (Upd) ligands, which drive tumor overgrowth. This study identified a polarity-responsive enhancer in upd3, which was activated in a coincident manner by both JNK-dependent Fos and aPKC-mediated Yki transcription. This enhancer, and Scrib mutant overgrowth in general, were also sensitive to activity of the Polycomb Group (PcG), suggesting that PcG attenuation upon polarity loss potentiated select targets for activation by JNK and Yki. These results link epithelial organization to signaling and epigenetic regulators that control tissue repair programs, and provide insight into why epithelial polarity is tumor-suppressive (Bunker, 2015).

JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling

A stable pool of morphogen-producing cells is critical for the development of any organ or tissue. This study presents evidence that JAK/STAT signalling in the Drosophila wing promotes the cycling and survival of Hedgehog-producing cells, thereby allowing the stable localization of the nearby BMP/Dpp-organizing centre in the developing wing appendage. The inhibitor of apoptosis dIAP1 and Cyclin A were identified as two critical genes regulated by JAK/STAT and contributing to the growth of the Hedgehog-expressing cell population. JAK/STAT was found to have an early role in guaranteeing Wingless-mediated appendage specification, and a later one in restricting the Dpp-organizing activity to the appendage itself. These results unveil a fundamental role of the conserved JAK/STAT pathway in limb specification and growth by regulating morphogen production and signalling, and a function of pro-survival cues and mitogenic signals in the regulation of the pool of morphogen-producing cells in a developing organ (Recasens-Alvarez, 2017).

Morphogens of the Wnt/Wg, Shh/Hh and BMP/Dpp families regulate tissue growth and pattern formation in vertebrate and invertebrate limbs. This study has unraveled a fundamental role of the secreted Upd ligand and the JAK/STAT pathway in facilitating the activities of these three morphogens in exerting their fate- and growth-promoting activities in the Drosophila wing primordium. Early in wing development, two distinct mechanisms ensure the spatial segregation of two alternative cell fates. First, the proximal-distal subdivision of the wing primordium into the wing and the body wall relies on the antagonistic activities of the Wg and Vn signalling molecules. While Wg inhibits the expression of Vn and induces the expression of the wing-determining genes, Vn, through the EGFR pathway, inhibits the cellular response to Wg and instructs cells to acquire body wall fate. Second, growth promoted by Notch pulls the sources of expression of these two morphogens apart, alleviates the repression of wing fate by Vn/EGFR, and contributes to Wg-mediated appendage specification. Expression of Vn is reinforced by a positive amplification feedback loop through the activation of the EGFR pathway. This existing loop predicts that, in the absence of additional repressors, the distal expansion of Vn/EGFR and its targets would potentially impair wing development. The current results indicate that Upd and JAK/STAT restrict the expression of EGFR target genes and Vn to the most proximal part of the wing primordium, thereby interfering with the loop and allowing Wg to correctly trigger wing development. Evidence is presented that JAK/STAT restricts the expression pattern and levels of its own ligand Upd and that ectopic expression of Upd is able to bypass EGFR-mediated repression and trigger wing development de novo. This negative feedback loop between JAK/STAT and its ligand is of biological relevance, since it prevents high levels of JAK/STAT signalling in proximal territories that would otherwise impair the development of the notum or cause the induction of supernumerary wings, as shown by the effects of ectopic activation of the JAK/STAT pathway in the proximal territories. Thus, while Wg plays an instructive role in wing fate specification, the Notch and JAK/STAT pathways play a permissive role in this process by restricting the activity range of the antagonizing signalling molecule Vn to the body wall region (Recasens-Alvarez, 2017).

Later in development, once the wing field is specified, restricted expression of Dpp at the AP compartment boundary organizes the growth and patterning of the whole developing appendage. Dpp expression is induced in A cells by the activity of Hh coming from P cells, which express the En transcriptional repressor. This study shows that JAK/STAT controls overall organ size by maintaining the pool of Hh-producing cells to ensure the stable and localized expression of the Dpp organizer. JAK/STAT does so by promoting the cycling and survival of P cells through the regulation of dIAP1 and CycA, counteracting the negative effects of En on these two genes. Since the initial demonstration of the role of the AP compartment boundary in organizing, through Hh and Dpp, tissue growth and patterning, it was noted that high levels of En interfered with wing development by inducing the loss of the P compartment. The capacity of En to negatively regulate its own expression was subsequently shown to be mediated by the Polycomb-group genes and proposed to be used to finely modulate physiological En expression levels. Consistent with this proposal, an increase was observed in the expression levels of the en-gal4 driver, which is inserted in the en locus and behaves as a transcriptional reporter, in enRNAi-expressing wing discs. The negative effects of En on cell cycling and survival reported in this work might also contribute to the observed loss of the P compartment caused by high levels of En. As is it often the case in development, a discrete number of genes is recurrently used to specify cell fate and regulate gene expression in a context-dependent manner. It is proposed that the capacity of En to block cell cycle and promote cell death might be required in another developmental context and that this capacity is specifically suppressed in the developing Drosophila limbs by JAK/STAT, and is modulated by the negative autoregulation of En, thus allowing En-dependent induction of Hh expression and promoting Dpp-mediated appendage growth. It is interesting to note in this context that En-expressing territories in the embryonic ectoderm are highly enriched in apoptotic cells. Whether this apoptosis plays a biological role and relies on En activity requires further study (Recasens-Alvarez, 2017).

Specific cell cycle checkpoints appear to be recurrently regulated by morphogens and signalling pathways, and this regulation has been unveiled to play a major role in development. Whereas Notch-mediated regulation of CycE in the Drosophila eye and wing primordia is critical to coordinate tissue growth and fate specification by pulling the sources of two antagonistic morphogens apart, the current results indicate that JAK/STAT-mediated regulation of CycA is critical to maintain the pool of Hh-producing cells in the developing wing and to induce stable Dpp expression. The development of the wing hinge region, which connects the developing appendage to the surrounding body wall and depends on JAK/STAT activity, has been previously shown to restrict the Wg organizer and thus delimit the size and position of the developing appendage. The current results support the notion that JAK/STAT and the hinge region are also essential to restrict the organizing activity of the Dpp morphogen to the developing appendage. Taken together, these results reveal a fundamental role of JAK/STAT in promoting appendage specification and growth through the regulation of morphogen production and activity, and a role of pro-survival cues and mitotic cyclins in regulating the pool of morphogen-producing cells in a developing organ. The striking parallelisms in the molecules and mechanisms underlying limb development in vertebrates and invertebrates have contributed to the proposal that an ancient patterning system is being recurrently used to generate body wall outgrowths. Whether the conserved JAK/STAT pathway plays a developmental role also in the specification or growth of vertebrate limbs by regulating morphogen production or activity is a tempting question that remains to be elucidated (Recasens-Alvarez, 2017).

Muscle directs diurnal energy homeostasis through a Myokine-dependent hormone module in Drosophila

Inter-tissue communication is critical to control organismal energy homeostasis in response to temporal changes in feeding and activity or external challenges. Muscle is emerging as a key mediator of this homeostatic control through consumption of lipids, carbohydrates, and amino acids, as well as governing systemic signaling networks. However, it remains less clear how energy substrate usage tissues, such as muscle, communicate with energy substrate storage tissues in order to adapt with diurnal changes in energy supply and demand. Using Drosophila, this study shows that muscle plays a crucial physiological role in promoting systemic synthesis and accumulation of lipids in fat storage tissues, which subsequently impacts diurnal changes in circulating lipid levels. The data reveal that the metabolic transcription factor Foxo governs expression of the cytokine unpaired 2 (Upd2) in skeletal muscle, which acts as a myokine to control glucagon-like adipokinetic hormone (AKH) secretion from specialized neuroendocrine cells. Circulating AKH levels in turn regulate lipid homeostasis in fat body/adipose and the intestine. The data also reveal that this novel myokine-dependent hormone module is critical to maintain diurnal rhythms in circulating lipids. This tissue crosstalk provides a putative mechanism that allows muscle to integrate autonomous energy demand with systemic energy storage and turnover. Together, these findings reveal a diurnal inter-tissue signaling network between muscle and fat storage tissues that constitutes an ancestral mechanism governing systemic energy homeostasis (Zhao, 2017).

Targets of activity

The genetic and molecular data regarding outstretched and its relationship to Hopscotch and Stat92E are all consistent with the predicted role of Os as a ligand that activates the JAK signaling cascade. To directly investigate this hypothesis, os was expressed in Drosophila cells, which were then assayed for tyrosine phosphorylation of Hop. The cell line chosen for this experiment is the Clone 8 (Cl.8) line, derived from developing wing imaginal disc. For cells to respond to Os by phosphorylating Hop, it was hypothesized that some transmembrane receptor would be required to bind Os by an extracellular domain, and be associated with Hop on the intracellular domain. Since no such receptor has yet been identified in flies, cells were chosen that were derived from a tissue known to be responsive to such a signal. It has been shown that Om(1E) protein from Drosophila ananassae, overexpressed in the wing disc, causes defects (Juni, 1996), suggesting that a receptor for this Os homolog must be present in the wing discs of D. ananassae. Thus, the D. melanogaster wing disc-derived Cl.8 cell line seemed a likely candidate to express a receptor for Os. To show Os-dependent tyrosine phosphorylation of Hop, anti-Hop immunoprecipitates from os-transfected cells were prepared and tested for reactivity with the anti-phosphotyrosine antibody 4G10. Although Hop protein is detectable in all samples, Hop is tyrosine phosphorylated only in immunoprecipitates prepared from os-transfected cells. Transfection of cells with os lacking a signal sequence does not result in Hop phosphorylation, consistent with the notion that Os is required extracellularly for signaling to occur. To further demonstrate that extracellularly provided Os is necessary and sufficient to observe Hop phosphorylation, Cl.8 cells were cocultured with S2 cells transiently transfected with Os. After thorough removal of the nonadherent S2 cells, Hop immunoprecipitates were prepared from Cl.8 cell lysates and analyzed. Hop phosphorylation is only seen when Cl.8 cells are cultured in the presence of os-transfected S2 cells. Identical results are obtained when Cl.8 cells are grown in the presence of conditioned medium taken from os-transfected 293T cells. These data are consistent with the hypothesis that Os is an extracellular ligand that binds a membrane-bound receptor to activate the JAK signaling pathway (Harrison, 1998).

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

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

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

Interaction between Ras^V12 and scribbled clones induces tumour growth and invasion

Human tumours have a large degree of cellular and genetic heterogeneity. Complex cell interactions in the tumour and its microenvironment are thought to have an important role in tumorigenesis and cancer progression. Furthermore, cooperation between oncogenic genetic lesions is required for tumour development; however, it is not known how cell interactions contribute to oncogenic cooperation. The genetic techniques available in the fruitfly Drosophila melanogaster allow analysis of the behaviour of cells with distinct mutations, making this the ideal model organism with which to study cell interactions and oncogenic cooperation. In Drosophila eye-antennal discs, cooperation between the oncogenic protein Ras^V12 and loss-of-function mutations in the conserved tumour suppressor scribbled (scrib) gives rise to metastatic tumours that display many characteristics observed in human cancers. This study shows that clones of cells bearing different mutations can cooperate to promote tumour growth and invasion in Drosophila. The Ras^V12 and scrib^- mutations can also cause tumours when they affect different adjacent epithelial cells. This interaction between Ras^V12 and ^scrib- clones involves JNK signalling propagation and JNK-induced upregulation of JAK/STAT-activating cytokines, a compensatory growth mechanism for tissue homeostasis. The development of ^RasV12 tumours can also be triggered by tissue damage, a stress condition that activates JNK signalling. Given the conservation of the pathways examined in this study, similar cooperative mechanisms could have a role in the development of human cancers (Wu, 2010).

Clones of mutant cells marked with green fluorescent protein (GFP) can be generated in the eye-antennal imaginal discs of Drosophila larvae by mitotic recombination. Clones expressing Ras^V12, an oncogenic form of the Drosophila Ras85D protein, moderately overgrow. Clones mutant for scrib lose apico-basal polarity and die. In contrast, scrib clones simultaneously expressing Ras^V12 grow into large metastatic tumours. To understand better the cooperation between these two mutations, animals were produced in which cell division after a mitotic recombination event creates two daughter cells: one expressing Ras^V12 and the other mutant for scrib. Discs containing adjacent Ras^V12 (GFP-positive) and scrib^- clones developed into large tumours, capable of invading the ventral nerve cord. This shows that Ras^V12 and scrib also cooperate for tumour induction when they occur in different cells. These tumours are referred to as Ras^V12//scrib^- tumours, to denote interclonal oncogenic cooperation and distinguish them from Ras^V12scrib^- tumours, in which cooperation occurs in the same cells intraclonally (Wu, 2010).

This study has used Drosophila to investigate how oncogenic cooperation between different cells can promote tumour growth and invasion. These experiments, addressed to understanding interclonal cooperation in Ras^V12//scrib^- tumours, uncovered a two-tier mechanism by which scrib^- cells promote neoplastic development of Ras^V12 cells: (1) propagation of stress-induced JNK activity from scrib^- cells to Ras^V12 cells; and (2) expression of the JAK/STAT-activating Unpaired cytokines downstream of JNK. These findings, therefore, highlight the importance of cell interactions in oncogenic cooperation and tumour development. It was also shown that stress-induced JNK signalling and epigenetic factors such as tissue damage can contribute to tumour development in flies. Notably, tissue damage caused by conditions such as chronic inflammation has been linked to tumorigenesis in humans. Furthermore, expression of the Unpaired cytokines promotes tumour growth as well as an antitumoural immune response, which parallels the situation in mice and humans. Future research into phenomena such as compensatory growth and interclonal cooperation in Drosophila will provide valuable insights into the biology of cancer (Wu, 2010).

Asymmetric localisation of cytokine mRNA is essential for JAK/STAT activation during cell invasiveness

The transition from immotile epithelial cells to migrating cells occurs in all organisms during normal embryonic development, as well as during tumour metastasis. During Drosophila oogenesis, border cells (BCs) are recruited and delaminate from the follicular epithelium. This process is triggered by the polar cells (PCs), which secrete the cytokine Unpaired (Upd) and activate the JAK/STAT pathway in neighbouring cells, turning them into invasive BCs. Interestingly, either a decrease or an increase in BC number alters migration, indicating that mechanisms controlling the level of JAK/STAT signalling are crucial in this process. This study shows that PCs have a highly stable and polarised network of microtubules along which upd transcripts are asymmetrically transported in a Dynein-dependent manner. In the absence of upd mRNA localisation the ligand is no longer efficiently secreted, leading to a loss of signalling strength as well as recruitment and migration defects. These findings reveal a novel post-transcriptional regulatory mechanism of JAK/STAT signalling in the control of epithelial cell invasiveness (Van de Bor, 2011).

mRNA subcellular localisation prior to translation is a conserved mechanism that restricts protein function both spatially and temporally. It plays an important role in the establishment of cell polarity and in the specification of embryonic axes. This study shows that mRNA localisation also plays a role in the fate determination of migratory cells within an epithelium. Transcripts that are known to be transported encode a wide variety of proteins, including transcription factors, components of the cytoskeleton and signalling molecules. Some well-characterised examples include Veg1 and An1 mRNAs in Xenopus, the localisation of which determines the vegetal and animal poles of the embryo, the Ash1 mRNA which localises to the bud site in yeast, and Actin mRNA localisation at the leading edge of migrating fibroblasts and growth cones. Transcript localisation can also act as a mechanism to regulate signalling pathways. One example is the Wnt-like protein encoded by the Drosophila wg gene, which plays an important role in segment patterning; apical localisation of wg transcripts in syncytial embryos restricts the diffusion of the Wg protein and therefore contributes to the definition of sharp boundaries. The current data show that, within the follicular epithelium, mRNA transport and localisation regulate the JAK/STAT signalling cascade to provide a peak of ligand activity that is important for follicle cell patterning and oBC specification. Furthermore, the results show that upd mRNA localisation is essential for accumulation of the Upd protein at the apical side of epithelial cells. Hence, as for Wg, Upd is a secreted protein that needs to be restricted to a narrow region, a process that depends on BicD and Egl function. BicD and Egl form a complex that binds the Dynein motor as well as various minus end-directed mRNA signals. Interestingly, unlike BicD, Egl is specifically required for mRNA transport and for no other cargoes in Drosophila, confirming that upd mRNA localisation is a key factor in the control of JAK/STAT pathway activity. It was shown previously that the Drosophila JAK/STAT pathway receptor Dome is apically enriched in the follicular epithelium and that Dome undergoes ligand-dependent endocytosis apically. In addition, a pool of STAT (Stat92E - FlyBase) protein is apically enriched in a Par3-dependent manner in the Drosophila embryonic ectoderm. These observations suggest that polarisation and/or subcellular localisation represent a key aspect of JAK/STAT signalling efficiency that serves to avoid misrouting and dilution of the ligand. This is likely to represent a general mechanism utilised in other major signalling pathways, as it has been observed that the Notch and EGF receptors, as well as Patched and Frizzled, also localise apically and/or basolaterally (Van de Bor, 2011).

Proper patterning of the anterior follicular epithelium by the JAK/STAT pathway requires the establishment of a robust gradient, with an appropriate ligand concentration for follicle cells to become properly determined. The transport of upd mRNA and the subsequent accumulation of the Upd protein apically contribute to setting the high concentration of ligand required for oBC determination. Recently, a new negative-feedback mechanism has been identified in outer border cells that involves a regulatory circuit between Slbo, STAT and Apt. This feedback mechanism inhibits JAK/STAT activity in cells that receive low levels of Upd ligand, i.e. those distant from the source. Therefore, JAK/STAT signalling requires several regulatory mechanisms to select the correct number of migratory BCs. First, in cells sending the signal (PCs), post-transcriptional control of upd mRNA and asymmetric protein accumulation are important for setting the correct ligand levels. It is possible that subcellular localisation of the transcript could also control the translation of the protein spatially, which in turn could affect translation efficiency, post-translational modifications such as glycosylation, co-factor association and/or degradation. Second, in receiving cells mechanisms are required to read the Upd gradient and limit the number of migratory cells, which involves spatial regulation of JAK/STAT signalling in anterior follicle cells receiving the Upd ligand through Apt. Additional mechanisms that involve sequestration of the ligand after its secretion might also exist. Indeed, in tissue culture, Upd is mainly found associated with the extracellular matrix, which might help to limit Upd diffusion in vivo. Interestingly, it has been reported that a transient cap of extracellular matrix forms at the apical side of PCs when BCs are being recruited, raising the possibility of an association of Upd with the extracellular matrix cap for the purposes of building up the gradient (Van de Bor, 2011).

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)Espl^δmδ-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)Espl^δmδ-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).

Post-transcriptional Regulation

The let-7-Imp axis regulates aging of the Drosophila testis stem-cell niche

Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. This study demonstrates that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals (Toledano, 2012).

Many stem cells lose the capacity for self-renewal when removed from their local microenvironment (or niche), indicating that the niche has a major role in controlling stem-cell fate. Changes to the local and systemic environments occur with age that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. The stem-cell niche in the Drosophila testis is located at the apical tip, where both germline stem cells (GSCs) and somatic cyst stem cells are in direct contact with hub cells. Hub cells express the self-renewal factor Upd, which activates the JAK-STAT signalling pathway to regulate the behaviour of adjacent stem cells. Ageing results in a progressive and significant decrease in the levels of upd in hub cells. However, constitutive expression of upd in hub cells was sufficient to block the age-related loss of GSCs, suggesting that mechanisms might be in place to regulate upd and maintain an active stem-cell niche (Toledano, 2012).

To identify potential regulators of upd, a collection of transgenic flies carrying green fluorescent protein (GFP)-tagged proteins was screened for expression in hub cells. The Drosophila homologue of Imp protein is expressed throughout the testis tip in young flies (Fabrizio, 2008); however, antibody staining revealed a decrease (~50%) in Imp expression in the hub cells of aged males. Imp is a member of a conserved family of RNA-binding proteins that regulate RNA stability, translation and localization (Yisraeli, 2005). Given the similarity in the ageing-related decline in Imp protein and upd mRNA in hub cells, it is proposed that Imp could be a new regulator of upd (Toledano, 2012).

To address whether Imp acts in hub cells to regulate upd, the bipartite GAL4-UAS system was used in combination with RNA-mediated interference (RNAi) to reduce Imp expression exclusively in hub cells. Fluorescence in situ hybridization (FISH) to detect upd mRNA was used in combination with immunofluorescence microscopy to determine whether the loss of Imp expression affects upd levels. The loss of Imp specifically in hub cells resulted in reduced expression of upd, as well as a significant reduction in GSCs and hub cell), when compared with controls. Consistent with a reduction in JAK-STAT signalling, decreased accumulation of STAT was observed when Imp levels were reduced by RNAi in hub cells (Toledano, 2012).

RNA-binding proteins characteristically target several RNAs; therefore, it was of interest to determine whether upd is a physiologically relevant target of Imp. Expression of upd together with an Imp RNAi construct was sufficient to completely rescue the defects caused by reduced Imp expression in hub cells, suggesting that Upd acts downstream of Imp to maintain GSCs and niche integrity. Importantly, the constitutive expression of upd alone in hub cells did not lead to an increase in GSCs in testes from 1-day-old males. These data suggest that Imp acts in hub cells to promote niche integrity and GSC maintenance, at least in part, by positively regulating upd (Toledano, 2012).

If Imp acts in hub cells in adult testes to regulate upd mRNA, it is speculated that the loss of Imp function during development might lead to a decrease in upd and a subsequent reduction in GSCs. Null mutations in Imp result in lethality at the pharate adult stage; therefore, testes from third instar larvae (L3) carrying Imp null alleles, Imp⁷ and Imp⁸, were examined. Deletion of the Imp locus was verified by PCR of genomic DNA. Combined immunofluorescence and FISH showed that although Fas3⁺ hub cells were easily detected, the expression of upd was significantly reduced: 24% of Imp⁷ mutants and 15% of Imp⁸ mutants had no detectable upd at this stage. In addition, the average number of GSCs and hub cells in testes from Imp mutants was significantly reduced when compared with control L3 testes. Notably, the re-expression of Imp in somatic niche cells was sufficient to rescue upd expression in Imp mutants to comparable levels to controls, and the reduction in the average number of GSCs and hub cells in Imp mutants was also reversed (Toledano, 2012).

Imp family members contain conserved KH domains that mediate direct binding to RNA targets. To determine whether Imp could associate directly with upd mRNA in vivo, testes were dissected from young flies expressing GFP-tagged Imp. Immunoprecipitation of Imp with anti-GFP antibodies, followed by quantitative reverse transcriptase PCR (qRT-PCR) analysis, showed a significant enrichment (~208-fold) of associated upd mRNA relative to control antibodies. Minimal enrichment for the ubiquitously expressed RNAs rp49 (also known as RpL32; ~4-fold) and GapDH (also known as Gapdh1; ~8 fold) or for the negative control med23 (~4-fold), was observed after Imp immunoprecipitation, indicating that the interaction between Imp and upd mRNA in hub cells is specific. Consistent with these observations, Imp protein and upd RNA co-localized in hub cells within perinuclear foci, probably ribonucleoprotein particles (Toledano, 2012).

An in vitro protein-RNA binding assay showed that Imp associates with the upd 3' untranslated region (UTR), specifically the first 250 base pairs (region 1), as no substantial binding to other portions of the upd 3'UTR was detected. Moreover, Imp did not bind the 5' untranslated or coding regions of upd or to the med23 3'UTR. Notably, a putative consensus binding sequence CAUH (in which H denotes A, U or C) for the mammalian IMP homologues (IGF2BP1- 3) occurs 22 times within the upd 3'UTR, including a cluster of four tandem repeats within the first 35 nucleotides of region 1. To test whether this motif mediates binding between Imp and upd, the first 33 nucleotides were removed to generate a sequence excluding the CAUH repeats, which resulted in a reduction in binding, compare domain 1 with domain 2. Point mutations in the third nucleotide of each motif (U = G) did not abolish the binding; however, point mutations in the consensus motif of MRPL9 RNA, a target of mammalian IGF2BPs, also did not abolish binding, suggesting that secondary structures probably mediate the association between IGFBP family members and their target RNAs. Altogether, the data identify the first 33 base pairs of the upd 3'UTR as a putative target sequence for Imp, and support observations that Imp associates specifically with upd in vivo (Toledano, 2012).

To gain further insight into the mechanism by which Imp regulates upd, a GFP reporter was constructed that contained the 3'UTR from either upd or med23. Transcript levels for gfp were fivefold higher in Drosophila Schneider (S2) cells that co-expressed Imp with the gfp-upd-3'UTR reporter than in cells that co-expressed Imp with the gfp-med23-3'UTR reporter. The significant increase in reporter mRNA levels indicates that it is likely that Imp regulates upd mRNA stability (Toledano, 2012).

RNA-binding proteins, including mammalian IGF2BP1, have been shown to counter microRNA (miRNA)-mediated targeting of mRNAs. However, no consensus miRNA seeds were located within the first 34 base pairs of domain 1 of the upd 3'UTR. It is speculated that if Imp binding blocks small RNA-mediated degradation of upd, polyadenylated, cleaved upd degradation intermediates would be detected in the testes of older males, when Imp expression in hub cells is reduced. Using a modified rapid amplification of complementary DNA ends (RACE) technique, a specific cleavage product was identifed starting at nucleotide 33 of the upd 3'UTR in the testes of 30-day-old flies, but not in RNA extracts from the testes of 1-day-old males. Importantly, the same degradation product of upd was also detected in the testes of young flies when Imp was specifically depleted from hub cells using RNAi-mediated knockdown. As a positive control, the esi-2-mediated cleavage product of mus308 was detected in testes from both 1- and 30-day-old flies (Toledano, 2012).

To test whether small RNAs might mediate upd cleavage, small RNA libraries generated from the testes of 1- and 30-day-old flies were cloned and deep-sequenced. Although no small RNAs with exact pairing to the upd degradation product were identified, two short interfering RNAs (siRNAs; termed siRNA1 and siRNA2) with high sequence complementarity to the predicted target site in the upd 3'UTR were present in the testis library generated from 30-day-old males. Using qRT- PCR for mature small RNAs, it was found that the siRNA2 levels in the testes, relative to the levels of the control small RNAs bantam and mir-184, were similar in young and old males (deep sequencing analysis demonstrated that expression of these two control miRNAs did not change with age). The source of siRNA2 is the gypsy5 transposon, which is inserted at several loci throughout the fly genome and is conserved in numerous Drosophila species (Toledano, 2012).

To gain further insight into the mechanism by which Imp and siRNA2 regulate upd, the levels of the upd GFP reporter (gfp-upd-3'UTR) in the presence or absence of Imp and siRNA2 was investigated in S2 cells. To generate a reporter that should not be susceptible to siRNA-mediated degradation, the cleavage site in the upd 3'UTR that was identified by RACE (³²AUU = CGG; gfp-upd-3'UTR^mut) was mutated. Cells were transfected with either of the GFP reporter constructs, with or without haemagglutinin-tagged Imp (Imp- HA), and subsequently transfected with siRNA2; gfp expression was quantified by qRT- PCR (Toledano, 2012).

The co-expression of siRNA2 and the gfp-upd-3'UTR reporter resulted in a significant decrease in gfp transcript levels. Conversely, the co-expression of Imp blocked siRNA2-mediated reduction of gfp mRNA such that gfp levels were higher than in control cells. Furthermore, mutation of the putative cleavage site rendered the upd 3'UTR resistant to siRNA2-mediated degradation. These data, in combination with the in vitro binding data, suggest that Imp binds to and protects the upd 3'UTR from endogenous and exogenous siRNA2 in S2 cells. Thus, endo-siRNA2 is a bona fide candidate that could direct upd degradation when Imp is absent or its levels are reduced, although targeting by other small RNAs cannot be excluded (Toledano, 2012).

In Drosophila, Argonaute-1 (AGO1) is the principle acceptor of miRNAs and primarily regulates targets in a cleavage-independent mode, whereas AGO2 is preferentially loaded with siRNAs and typically regulates targets by mRNA cleavage. AGO2 expression was detected throughout the tip of the testis, as verified by immunostaining of testes from transgenic flies expressing 3×Flag-HA-tagged AGO2. To test whether AGO2 binds to upd mRNA in vivo, thereby potentially regulating upd levels directly, testes were dissected from aged (30-day-old) 3×Flag- HA- AGO2 males. Immunoprecipitation of AGO2, followed by qRT- PCR, showed significant enrichment (~102-fold) of upd mRNA bound to AGO2. Negligible binding of a negative control, rp49, to AGO2 was detected, suggesting specific association of AGO2 with upd mRNA in vivo and supporting a previous findings that upd is probably targeted by the siRNA pathway (Toledano, 2012).

To test whether Imp can impede the binding of AGO2 to the upd 3'UTR, S2 cells stably expressing Flag-tagged AGO2 were transfected with the gfp-upd-3'UTR reporter. Consistent with our previous observations, transcript levels of gfp-upd-3'UTR increased ~18-fold when Imp was co-expressed. Despite increases in the overall levels of gfp mRNA, the presence of Imp markedly reduced the association of AGO2 with the upd 3'UTR, indicating that Imp antagonizes the ability of AGO2 to bind the upd 3'UTR (Toledano, 2012).

Similar to the AGO family, Drosophila encodes two Dicer proteins that seem to have distinct roles in small RNA biogenesis. Dicer-1 (Dcr-1) is essential for the generation of miRNAs, and Dcr-2 is required for siRNA production from exogenous and endogenous sources. If siRNAs were involved in upd degradation in older males, it would be predicted that the loss of Dcr-2 would suppress the ageing-related decline in upd and GSCs. Consistent with a role for Dcr-2 in the generation of siRNAs, siRNA2 levels were significantly reduced in Dcr-2 homozygous mutants relative to heterozygous controls. Testes from 30- and 45-day-old Dcr-2 mutant flies showed increased levels of upd by qRT- PCR when compared with controls. Whereas a ~90% reduction of upd is observed in the testes from aged Dcr-2 heterozygous controls, only a ~45% reduction in upd was observed in testes from age-matched, Dcr-2 homozygous mutants, indicating that upd levels are higher when Dcr-2 function is compromised. Furthermore, the testes from aged Dcr-2 mutants contained more GSCs, on average, when compared with controls. Conversely, the forced expression of Dcr-2 in hub cells resulted in a reduction in the average number of GSCs and led to a significant reduction in upd levels, as detected using qRT- PCR and combined immunofluorescence and FISH, which seemed to be specific, as no significant change in Imp transcript levels was observed. Expression of Imp in combination with Dcr-2 resulted in a significant increase in upd levels. These observations indicate that Imp can counter the decrease in upd levels resulting from forced Dcr-2 expression, providing further evidence that Imp protects upd from targeted degradation by the siRNA pathway (Toledano, 2012).

The data suggest that Imp has a role in stabilizing upd in hub cells; therefore, the ageing-related decline in Imp would be a major contributing factor to the decrease in upd mRNA in the hub cells of aged males. To investigate the mechanism that leads to the decline in Imp expression with age, the Imp 3'UTR was examined for potential instability elements. Within the first 160 base pairs there is a canonical seed sequence for the heterochronic miRNA let-7. Expression of a reporter gene under the control of the let-7 promoter showed that let-7 expression increases in hub cells of ageing male, which was confirmed by let-7 FISH of testes from aged males. In addition, mature let-7 miRNA was enriched twofold in the testes from 30-day-old flies, relative to 1-day-old males. Therefore, an age-related increase in let-7 is one mechanism by which Imp expression could be regulated in an ageing-dependent manner in testes from older males (Toledano, 2012).

Consistent with these observations, the forced expression of let-7 specifically in hub cells led to a decrease in Imp. In addition, let-7 expression in S2 cells reduced the levels of a heterologous gfp-Imp-3'UTR^WT reporter. S2 cells were transfected with a let-7 mimic or with negative control miRNA, and gfp expression was quantified by qRT- PCR. There was a 70% reduction in gfp-Imp-3'UTR^WT expression in the presence of let-7, relative to control miRNA. A gfp-Imp-3'UTR^mut reporter with mutations in the canonical seed for let-7 (at nucleotide 137) was unaffected by let-7 expression, indicating that mutation of the let-7 seed rendered the RNA resistant to degradation. These data confirm that let-7 can destabilize Imp through sequences in the 3'UTR. However, further increasing the levels of let-7 resulted in a decrease in gfp expression from the mutated 3'UTR, indicating that other, putative let-7 seeds in the Imp 3'UTR can be targeted by let-7 (Toledano, 2012)

If the age-related decrease in Imp contributes to a decline in upd and subsequent loss of GSCs, it is proposed that re-expression of Imp in hub cells would rescue the ageing-related decrease in upd. Therefore, flies in which Imp was constitutively expressed in hub cells were aged, and upd levels were quantified by qRT- PCR. The expression of an Imp construct containing a truncated 3'UTR (Imp-KH- HA) lacking let-7 target sequences specifically in hub cells was sufficient to suppress the ageing-related decline in upd, with concomitant maintenance of GSCs, similar to what was observed by re-expressing upd in the hub cells of aged males. Maintenance of Imp-KH- HA expression in aged males was verified by staining with an anti-HA antibody. Conversely, the expression of an Imp construct that is susceptible to degradation by let-7 (Imp^T21) did not lead to an accumulation of Imp in the testes of 30- and 50-day-old flies, as levels were similar to the levels of endogenous Imp at later time points. Consequently, the expression of this construct was not sufficient to block the ageing-related decline in GSCs. These data indicate that let-7-mediated regulation of Imp contributes to the decline in Imp protein in older flies, and supports a model in which an ageing-related decline in Imp, mediated by let-7, exposes upd to degradation by siRNAs. Thus, both the miRNA and siRNA pathways act upstream to regulate the ageing of the testis stem-cell niche by generating let-7 and siRNA2, which target Imp and upd, respectively (Toledano, 2012).

Drosophila has proven to be a valuable model system for investigating ageing-related changes in stem-cell behaviour. Cell autonomous and extrinsic changes contribute to altered stem-cell activity; thus, determining the mechanisms underlying the ageing-related decline of self-renewal factors, such as the cytokine-like factor Upd, may provide insight into strategies to maintain optimal niche function (Toledano, 2012).

The data indicate that Imp can regulate gene expression by promoting the stability of selected RNA targets by countering inhibitory small RNAs. Therefore, rescue of the aged niche by Imp expression may be a consequence of effects on Imp targets, in addition to upd, in somatic niche cells. Furthermore, as Imp is expressed in germ cells, it could also act in an autonomous manner to regulate the maintenance of GSCs. The canonical let-7 seed in the Imp 3'UTR is conserved in closely related species, and reports have predicted that the let-7 family of miRNAs target mammalian Imp homologues (IGF2BP1- 3). Given the broad role of the let-7 family in ageing, stem cells, cancer and metabolism, the regulation of Imp by let-7 may be an important, conserved mechanism in numerous physiological processes (Toledano, 2012).

Non-coding RNAs can ensure biological robustness and provide a buffer against relatively small fluctuations in a system. However, after a considerable change, a molecular switch is flipped, which allows a biological event to proceed unimpeded. In the current model, Imp preserves niche function in young flies until a time at which miRNAs and siRNAs act together to trigger an 'ageing' switch that leads to a definitive decline in upd and, ultimately, in stem-cell maintenance. Therefore, targeting signalling pathways at several levels using RNA-based mechanisms will probably prove to be a prevalent theme to ensure robustness in complex biological systems (Toledano, 2012).

Protein Interactions

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

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

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

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

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

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

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

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

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

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

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

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

To study the interaction between Mom/Domeless and Unpaired, their cDNAs were subcloned into epitope-tagged mammalian expression vectors. 293T cells were cotransfected with V5-tagged Upd and HA-tagged Mom and expression was detected by immunofluorescence and Western blot with specific mouse monoclonal anti-tag antibodies. To examine the direct binding of Upd to Mom, 293T cells were transfected with Upd-V5. The ligand was released to the medium by treating the cells with heparin. Subsequently, the concentrated conditioned medium was applied to 293T cells nontransfected and transfected with HA-Mom and with a truncated form containing the N-terminal domain, Mom-N. Of note, given that there are no specific antibodies available for Mom, and that both anti-HA and anti-V5 antibodies have the same animal origin, (which prohibits double staining), an indirect approach was used to ascertain evidence of their presence in transfected cells. Since it is known that cells transfected with two DNAs will incorporate both at the same time, 293T cells were transfected with HA-Mom along with Stat92E. Therefore, cells stained with rabbit anti-STAT antibody should be those also expressing Mom. As expected, V5 staining was detected in cells containing Stat92E and transfected with Mom or Mom-N. These data show that Upd can be detected in 293T cells only when Mom is present, which indicates a physical interaction between these two molecules (Chen, 2002).

The presence of a putative signal sequence and five amino-linked glycosylation sites in Os suggests that it is post-translationally modified in the secretory pathway. To examine the possibility of amino-linked glycosylation, a mammalian expression plasmid containing os was transfected into human 293T cells. The cells were metabolically labeled with [35S]methionine in the presence or absence of tunicamycin, a potent inhibitor of amino-linked glycosylation. Os protein was recovered by immunoprecipitation, and the sizes of resultant Os proteins were compared. Several bands of 45-65 kD are seen in the untreated cells, whereas only the smallest 45-kD band is observed when the cells are treated with tunicamycin. The absence of the larger products in the treated cells suggests that the larger proteins are glycosylated forms. The presence of multiple forms of Os in the untreated cells most likely reflects partially glycosylated intermediates. These results suggest that Os contains a functional signal sequence that targets Os to the endoplasmic reticulum for glycosylation and secretion. Therefore, removal of the putative signal sequence should eliminate targeting of the Os protein to the endoplasmic reticulum and prevent glycosylation. As predicted, when the signal sequence was removed from Os, only the 45-kD band was observed (Harrison, 1998).

Comigration of the smallest Os protein species with a os product lacking a signal sequence suggests that the signal sequence is normally cleaved in the mature protein. To examine cleavage of the signal sequence, a plasmid was constructed in which a hemagglutinin (HA) epitope tag was fused to the amino terminus of Os. Previous work has shown that amino-terminal extensions on signal sequences do not abrogate in vivo function. The plasmid encoding the tagged Os was transfected into S2 cells and the resulting protein was precipitated with anti-Os antisera. The precipitated protein was then detected by Western blotting by use of either anti-Os or anti-HA antisera. The protein is detectable with the anti-Os serum, but not with the anti-HA antisera. These results suggest that the amino terminus of Os, including the HA tag, is proteolytically removed (Harrison, 1998).

The presence of modifications to Os that take place in the secretory pathway are consistent with the hypothesis that Os is a secreted protein. However, in initial experiments, Os could not be detected in the medium of transfected cells despite its high level of expression. Therefore, to determine the localization of Os, culture medium and extracellular matrix (ECM) were harvested separately from os-transfected S2 cells and tested for the presence of Os. To avoid contamination of the ECM fraction with cells and cell fragments, culture dishes were washed extensively prior to harvesting. The majority of Os protein is found to be associated specifically with the ECM, with only a small quantity free in the medium (Harrison, 1998).

Binding of proteins to the ECM is often mediated by glycosaminoglycans, such as heparan sulfate. Therefore, free heparin was added to the medium from os-transfected cells to determine whether heparan could prevent association of Os with the ECM. Addition of heparin releases nearly all of the Os protein into the medium. The ability of heparin to compete with ECM suggests that Os normally binds to the ECM through association with glycosaminoglycans. As expected, Os lacking a signal sequence is not secreted and cannot be detected in either the medium or ECM (Harrison, 1998).

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).

Dome is not uniformly distributed at the membrane but is restricted to apicolateral regions. Other receptor molecules have been shown to preferentially localize to apicolateral membranes, such as the EGF and Notch receptors, suggesting that the apical region is an active signaling interface for several receptors in follicle cells. Indeed, the apical localization of upd mRNA, membrane Dome and Dome-containing vesicles support a model in which ligand-receptor interactions take place apically in follicle cells, to activate the JAK/STAT pathway (Ghiglione, 2002).

Dome is a transmembrane protein with both extracellular and intracellular domains whose functions are unknown. The extracellular part contains a cytokine-binding module (CBM) and 3 fibronectin-type III domains likely participating in ligand binding, while the intracellular domain presumably interacts with Hop, through binding to one or several potentially phosphorylated tyrosines. Using truncated forms of Dome it has been shown that both the extracellular and intracellular domains are essential for BC migration and signal transduction. The dominant negative phenotypes that are observed are consistent with a model in which DomeDeltaCYT would titrate the ligand Upd, and DomeDeltaEXT titrates Hop, therefore inducing a dramatic reduction in signaling strength. Both constructs may also lead to the formation of non-functional Dome-Dome dimers by capturing the wild-type Dome protein in an inactive complex. Further biochemical work will be necessary to understand the molecular mechanisms underlying Dome signal transduction (Ghiglione, 2002).

The pattern of epithelial markers in dome mutant cells indicates that the JAK/STAT pathway is active in all follicle cells, a notion that is reinforced by the wide expression of nuclear Stat92E. How is Dome activated during egg chamber development and does this activation follow the same profile at all stages? Given the restricted pattern of upd expression in the egg chamber and its dramatic effect upon overexpression, it is unlikely that Upd is able to signal long distances in the follicular epithelium of late stage egg chambers. Rather, a model by which the JAK/STAT pathway plays a pre-patterning function is favored, acting early during egg chamber development to activate DE-cadherin and Crumbs expression. This view is consistent both with the expression pattern of upd and the distribution of Dome-containing vesicles described in this study. The formation of endogenous vesicles can be promoted by Upd, and a gradient of such vesicles is present around polar cells. Strikingly, these vesicles, which likely indicate active signaling through Dome, are widespread at early stages and become more restricted later on. It is proposed that during early development, the Upd signal produced by anterior and posterior polar cells contributes to the differentiation of all follicle cells. At this stage, Upd would be more diffusible than later, as suggested by the pattern of Dome intracellular vesicles. The study of the mechanisms controlling Dome activation and Upd activity will require additional tools to directly detect Upd, as, for example, Upd-GFP fusion proteins (Ghiglione, 2002).

This study has revealed several new findings about the function of dome and the JAK/STAT pathway during oogenesis. Future work will help to understand how Upd and Dome initially interact at the cell surface and transduce the signal to downstream JAK/STAT pathway members (Ghiglione, 2002).

It is commonly accepted that activation of most signalling pathways is induced by ligand receptor dimerization. This belief has been challenged for some vertebrate cytokine receptors of the JAK/STAT pathway. This study addresses the question of whether DOME, the Drosophila receptor of the JAK/STAT pathway, can dimerize and whether the dimerization is ligand-dependent. To analyze DOME homo-dimerization, a ß-gal complementation technique was applied that allows the detection of protein interactions in situ. This technique has been used in cell culture but this is the first time that it has been applied to whole embryos. This technique, which has been rename ßlue-ßlau technique, can be used to detect DOME homo-dimerization in Drosophila developing embryos. Despite DOME being ubiquitously expressed, dimerization is developmentally regulated. The state of DOME dimerization was investigated in the presence or absence of ligand; DOME dimerization is not ligand-induced, indicating that ligand independent cytokine receptor dimerization is a conserved feature across phyla. The functional significance of ligand-independent receptor dimerization was further analyzed by comparing the effects of ectopic ligand expression in cells in which the receptor is, or is not, dimerized. Ligand expression can only activate STAT downstream targets or affect embryo development in cells in which the receptor is dimerized. These results suggest a model in which ligand-independent dimerization of the JAK/STAT receptor confers cells with competence to activate the pathway prior to ligand reception. Thus, competence to induce the JAK/STAT signalling pathway in Drosophila can be regulated by controlling receptor dimerization prior to ligand binding. These results reveal a novel level of JAK/STAT signalling regulation that could also apply to vertebrates (Brown, 2003).

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-ras^V12, 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-ras^V12 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 (N^intra) 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. hop^Tum-l and hop^T42 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 glypicans Dally and Dally-like are essential regulators for JAK/STAT signaling and Unpaired distribution in eye development

The highly conserved JAK/STAT pathway is a well-known signaling system that is involved in many biological processes. In Drosophila, this signaling cascade is activated by ligands of the Unpaired (Upd) family. Therefore, the regulation of Upd distribution is one of the key issues in controlling the JAK/STAT signaling activity and function. Heparan sulfate proteoglycans (HSPGs) are macromolecules that regulate the distribution of many ligand proteins including Wingless, Hedgehog and Decapentaplegic (Dpp). This study shows that during Drosophila eye development, HSPGs are also required in normal Upd distribution and JAK/STAT signaling activity. Loss of HSPG biosynthesis enzyme Brother of tout-velu (Botv), Sulfateless (Sfl), or glypicans Dally and Dally-like protein (Dlp) led to reduced levels of extracellular Upd and reduction in JAK/STAT signaling activity. Overexpression of dally resulted in the accumulation of Upd and up-regulation of the signaling activity. Luciferase assay also showed that Dally promotes JAK/STAT signaling activity, and is dependent on its heparin sulfate chains. These data suggest that Dally and Dlp are essential for Upd distribution and JAK/STAT signaling activity (Zhang, 2013).

Upd distribution is essential for the JAK/STAT signaling pathway, but its regulation is largely unknown. This study has shown that the glypicans Dally and Dlp are required for the normal Upd distribution and JAK/STAT signaling activity. Overexpression of dally activated the JAK/STAT signal in eye discs. Dally and Dlp also up-regulated JAK/STAT signaling activity in cell culture, and its function was dependent on their attached HS chains. Together, these data indicate that Dally and Dlp are essential regulators for JAK/STAT signaling activity (Zhang, 2013).

One of the most important findings in this work is that Dally and Dlp regulate Upd distribution and may be required for the retention of Upd on the cell surface. Previous studies of Upd mainly focused on its transcriptional regulation and function, but little is known about the regulation of Upd distribution. It is known that Upd has a long-range effect on JAK/STAT signaling but the mechanism(s) behind is unknown. This study found that the long-range effect of Upd in eye development was due to the long-range extracellular distribution of Upd. Loss of dally or dlp led to reduction of extracellular Upd levels, while overexpression of dally or dlp was able to increase the extracellular levels of Upd. Therefore, it is concluded that the long-range distribution of Upd and subsequent JAK/STAT signaling activity are controlled by Dally and Dlp in Drosophila eye discs (Zhang, 2013).

Data from cell culture experiments suggested that dally and dlp may be required for the retention of Upd on the cell surface and subsequent signaling activity. With high levels of Upd, Dally and Dlp had mild effects on JAK/STAT signaling activity. With low levels of Upd, Dally and Dlp dramatically up-regulated JAK/STAT signaling activity. These results indicate that dally and dlp help to retain Upd on the cell surface and activate JAK/STAT signaling activity (Zhang, 2013).

Although both Dally and Dlp play roles in the regulation of Upd distribution, these studies show that Dally is likely the major regulator of JAK/STAT signaling activity. First, loss of dally showed a stronger reduction in JAK/STAT signaling activity than loss of dlp. Second, expression of dally rescued the reduction of JAK/STAT signaling in dally–dlp double mutant clones, but expression of dlp alone did not. Third, overexpression of dally but not dlp induced ectopic JAK/STAT signaling activity. Fourth, in cell culture, with high levels of Upd Dally but not Dlp can further up-regulate JAK/STAT signaling activity. All of these data support the view that Dally is the major regulator of JAK/STAT signaling activity in eye development. More experiments are needed to figure out why Dally has higher signaling activity in JAK/STAT pathway than Dlp (Zhang, 2013).

These studies also show that HS chains of HSPGs play important roles in the regulation of JAK/STAT signaling activity. In eye discs, the loss of botv or sfl led to the reduction in JAK/STAT signals, indicating that biosynthesis of HS chains is required for normal JAK/STAT signaling activity. Previous studies in cell culture showed that Upd may be associated with HS chains, and this association can be released by the addition of heparin. This study shows that in the presence of heparin, the activation of JAK/STAT by Dally and Dlp was compromised. These findings support the view that the activation of JAK/STAT signaling is dependent on the association of Upd with HS chains (Zhang, 2013).

The conserved Misshapen-Warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila

Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. This study shows that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP (Li, 2014).

Previous studies have shown that endothelial cells (ECs) produce regulatory factors in response to infection and damage and function as part of the niche to regulate intestinal stem cell (ISC)-mediated regeneration. Meanwhile, recent reports show that enteroblasts (EBs) can also produce growth factors including EGF receptor ligands, Wingless and Upd3, although the pathways that regulate their production are not known. The current results demonstrate that differentiating EBs also function as an important part of the niche to regulate ISC division via the Msn pathway. EB-specific knockdown of msn leads to highly increased Upd3 expression and midgut proliferation. A previous report suggests that undifferentiated EBs if remain in contact with the mother ISC can inhibit proliferation. Although the hyperproliferating midguts after loss of Msn contain many EBs, these EBs do go into normal differentiation and express high level of Upd3, which may overcome any inhibitory effect of undifferentiated EBs on ISC proliferation (Li, 2014).

Msn is known to regulate a number of biological processes. During embryonic dorsal closure the MAP kinase pathway Slipper-Hemipterous-JNK is downstream of Msn, and Slipper is able to bind to Msn in vitro. In the adult midgut, JNK is a mediator of aging-related intestinal dysplasia and is a stress-activated kinase in ECs to positively regulate ISC division. While the current RNAi experiments show that JNK has a function in EBs to negatively regulate ISC proliferation, this phenotype is not dependent on Upd3 or Yki. No change of JNK phosphorylation was detected after loss of Msn. Mammalian MAP4K4 has also been shown to function independently of JNK in some biological contexts. Therefore, Msn and JNK probably have independent functions in the midgut (Li, 2014).

This study has instead uncovered an interaction of Msn with Wts and subsequently regulation of Yki. Hpo-Wts-Yki has been demonstrated to have a function in ECs for stress and damage-induced response. Gal4 driven experiments have many caveats including cell-type specificity, differences in promoter strengths, and knockdown efficiency in different cell types. Nonetheless, the results of many parallel experiments that this study conducted strongly suggest that Msn and Hpo independently regulate Wts-Yki in EBs and ECs, respectively. How the Msn and Hpo pathways in the two cell types are coordinately regulated to produce an appropriate amount of Upd3 to achieve desirable intestinal growth under different circumstances remains an important question to be answered (Li, 2014).

Previous experiments in developing discs suggest that Wts and Yki but not Hpo act downstream of cytoskeleton regulators. Similarly, the mammalian Hpo homologs MST1/2 appear not to be involved in LATS regulation after cytoskeletal perturbation in some cell types. In vivo assay in midgut suggests a function for Msn, Yki and Upd3 downstream of actin capping proteins in EBs. Similarly, the Latrunculin B effect on MEFs suggests that MAP4K4 is required for cytoskeleton-regulated LATS and YAP phosphorylation. The situation in mammalian cells may be more complicated because the Msn/MAP4K4 subfamily also includes two other closely related kinases TNIK and MINK1. Proper regulation of Wts by the cytoskeleton may require both positive and negative regulators, because recent work in flies identified the LIM-domain protein Jub as a negative regulator of Wts in response to cytoskeletal tension. It will be interesting in future studies to determine how positive and negative regulators of Wts act in a coordinated manner to regulate cell fate and proliferation in response to cytoskeletal tension (Li, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic

The spatial distribution of the 2.2-kb os transcript is consistent with a role in embryonic segmentation. In situ hybridization to RNA in whole mount embryos has revealed a dynamic and segmentally repetitive pattern of expression. During the syncytial blastoderm stage, the 2.2-kb transcript is not expressed at levels that are detectable above background. Shortly before cellularization, the RNA becomes abundant, but is absent from the termini, and is restricted to only the trunk of the embryo and a single incomplete head stripe. At cellularization, the trunk expression resolves into ~7 stripes, then into 14 stripes, a phenomenon seen in some pair-rule segmentation genes (Harrison, 1998).

Localized JAK/STAT signaling is required for oriented cell rearrangement in 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).

Polarized subcellular localization of Jak/STAT components is required for efficient signaling

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

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

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

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

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

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

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

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

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

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

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

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

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

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, 2009). 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).

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).

EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells

Tissue-specific adult stem cells are commonly associated with local niche for their maintenance and function. In the adult Drosophila midgut, the surrounding visceral muscle maintains intestinal stem cells (ISCs) by stimulating Wingless (Wg) and JAK/STAT pathway activities, whereas cytokine production in mature enterocytes also induces ISC division and epithelial regeneration, especially in response to stress. This study shows that EGFR/Ras/ERK signaling is another important participant in promoting ISC maintenance and division in healthy intestine. The EGFR ligand Vein is specifically expressed in muscle cells and is important for ISC maintenance and proliferation. Two additional EGFR ligands, Spitz and Keren, function redundantly as possible autocrine signals to promote ISC maintenance and proliferation. Notably, over-activated EGFR signaling could partially replace Wg or JAK/STAT signaling for ISC maintenance and division, and vice versa. Moreover, although disrupting any single one of the three signaling pathways shows mild and progressive ISC loss over time, simultaneous disruption of them all leads to rapid and complete ISC elimination. Taken together, these data suggest that Drosophila midgut ISCs are maintained cooperatively by multiple signaling pathway activities and reinforce the notion that visceral muscle is a critical component of the ISC niche (Xu, 2011).

Adult stem cells commonly interact with special microenvironment for their maintenance and function. Many adult stem cells, best represented by germline stem cells in Drosophila and C. elegans, require one primary maintenance signal from the niche while additional signals may contribute to niche integrity. ISCs in the Drosophila midgut do not seem to fit into this model. Instead, they require cooperative interactions of three major signaling pathways, including EGFR, Wg and JAK/STAT signaling, for long-term maintenance. Importantly, Wg or JAK/STAT signaling over-activation is able to compensate for ISC maintenance and proliferation defects caused by EGFR signaling disruption, and vice versa. Therefore, ISCs could be governed by a robust mechanism, signaling pathways could compensate with each other to safeguard ISC maintenance. The mechanisms of the molecular interactions among these pathways in ISC maintenance remains to be investigated. In mammals, ISCs in the small intestine are primarily controlled by Wnt signaling pathways, and there are other ISC specific markers not controlled by Wnt signaling. In addition, mammalian ISCs in vitro strictly depend on both EGFR and Wnt signals, indicating that EGFR and Wnt signaling may also cooperatively control mammalian ISC fate. It is suggested that combinatory signaling control of stem cell maintenance could be a general mechanism for ISCs throughout evolution (Xu, 2011).

The involvement of EGFR signaling in Drosophila ISC regulation may bring out important implications to understanding of intestinal diseases, in which multiple signaling events could be involved. For example, in addition to Wnt signaling mutation, gain-of-function K-Ras mutations are frequently associated with colorectal cancers in humans. Moreover, activation of Wnt signaling caused by the loss of adenomatous polyposis coli (APC) in humans initiates intestinal adenoma, but its progression to carcinoma may require additional mutations. Interestingly, albeit controversial, Ras signaling activation is suggested to be essential for nuclear β-catenin localization, and for promoting adenoma to carcinoma transition. In the Drosophila midgut, loss of APC1/2 genes also leads to intestinal hyperplasia because of ISC overproliferation. Given that EGFR signaling is generally activated in ISCs, it would be interesting to determine the requirements of EGFR signaling activation in APC-loss-induced intestinal hyperplasia in Drosophila, which might provide insights into disease mechanisms in mammals and humans (Xu, 2011).

Previous studies suggest that intestinal VM structures the microenvironment for ISCs by producing Wg and Upd maintenance signals. This study identified Vn, an EGFR ligand, as another important ISC maintenance signal produced from the muscular niche. Therefore, ISCs are maintained by multiple signals produced from the muscular niche. In addition, Spi and Krn, two additional EGFR ligands, were identified that function redundantly as possible autocrine signals to regulate ISCs. These observations are consistent with a previous observation that paracrine and autocrine EGFR signaling regulates the proliferation of AMPs during larval stages, suggesting that this mechanism is continuously utilized to regulate adult ISCs for their maintenance and proliferation. The only difference is that the proliferation of AMP cells is unaffected when without autocrine Spi and Krn, due to redundant Vn signal from the VM, whereas autocrine Spi/Krn and paracrine Vn signals are all essential in adult intestine for normal ISC maintenance and proliferation. It was found that Vn and secreted form of Spi have similar roles in promoting ISC maintenance and activation, but additional regulatory or functional relationships among these ligands require further investigation, as the necessity of multiple EGFR ligands is still not completely understood. It is known that secreted/activated Spi and Krn are diffusible signals, but clonal analysis data show that Spi and Krn can display autonomous phenotypes. This observation indicates that these two ligands could behave as very short range signals in the intestinal epithelium, or they could diffuse over long distance but the effective levels of EGFR activation could only be achieved in cells where the ligands are produced. Interestingly, palmitoylation of Spi is shown to be important for restricting Spi diffusion in order to increase its local concentration required for its biological function. Whether such modification occurs in intestine is unknown, but it is speculated that Vn, Spi and Krn, along with the possibly modified forms, may have different EGFR activation levels or kinetics, and only with them together effective activation threshold could be reached and sustained in ISCs to control ISC behavior. Therefore, a working model is proposed that ISCs may require both paracrine and autocrine mechanisms in order to achieve appropriate EGFR signaling activation for ISC maintenance and proliferation (XU, 2011).

Mechanisms of JAK/STAT signaling activation is rather complex. In addition to Upd expression from the VM, its expression could also be detected in epithelial cells with great variability in different reports, possibly due to variable culture conditions. Upon injury or pathogenic bacterial infection, damaged ECs and pre-ECs are able to produce extra cytokine signals, including Upd, Upd2 and Upd3, to activate JAK/STAT pathway in ISCs to promote ISC division and tissue regeneration. Several very recent studies suggest that EGFR signaling also mediates intestinal regeneration under those stress conditions in addition to its requirement for normal ISC proliferation. Therefore, in addition to basal paracrine and autocrine signaling mechanisms that maintain intestinal homeostasis under normal conditions, feedback regulations could be employed or enhanced under stress conditions to accelerate ISC division and epithelial regeneration (Xu, 2011).

Evidence so far has indicated a central role of N signaling in controlling ISC self-renewal. N is necessary and sufficient for ISC differentiation. In addition, the downstream transcriptional repressor Hairless is also necessary and sufficient for ISC self-renewal by preventing transcription of N targeting genes in ISCs. Therefore, N inhibition could be a central mechanism for ISC fate maintenance in Drosophila. High Dl expression in ISCs may lead to N inhibition, though how Dl expression is maintained in ISCs at the transcriptional level is not clear yet. Hyperactivation of EGFR, Wg or JAK/STAT signaling is able to induce extra Dl⁺ cells, suggesting that these three pathways might cooperatively promote Dl expression in ISCs. It is also possible that these pathways regulate Dl expression indirectly. As Dl-N could have an intrinsically regulatory loop for maintaining Dl expression and suppressing N activation, these pathways could indirectly regulate Dl expression by targeting any component within the regulatory loop. Identifying their respective target genes by these signaling pathways in ISCs would be an important starting point to address this question (Xu, 2011).

Warts and Yorkie mediate intestinal regeneration by influencing stem cell proliferation

Homeostasis in the Drosophila midgut is maintained by stem cells. The intestinal epithelium contains two types of differentiated cells that are lost and replenished: enteroendocrine (EE) cells and enterocytes (ECs). Intestinal stem cells (ISCs) are the only cells in the adult midgut that proliferate, and ISC divisions give rise to an ISC and an enteroblast (EB), which differentiates into an EC or an EE cell. If the midgut epithelium is damaged, then ISC proliferation increases. Damaged ECs express secreted ligands (Unpaired proteins) that activate Jak-Stat signaling in ISCs and EBs to promote their proliferation and differentiation]. This study shows that the Hippo pathway components Warts and Yorkie mediate a transition from low- to high-level ISC proliferation to facilitate regeneration. The Hippo pathway regulates growth in diverse organisms and has been linked to cancer. Yorkie is activated in ECs in response to tissue damage or activation of the damage-sensing Jnk pathway. Activation of Yorkie promotes expression of unpaired genes and triggers a nonautonomous increase in ISC proliferation. These observations uncover a role for Hippo pathway components in regulating stem cell proliferation and intestinal regeneration (Staley, 2010).

Hippo signaling can have both autonomous and nonautonomous effects on growth, and this study reports that in the adult Drosophila midgut, Yki has profound nonautonomous effects on growth via the Jak-Stat pathway. Jak-Stat signaling is important for proliferation control and stem cell biology, not only in the Drosophila intestine, but also in other tissues, both in Drosophila and in vertebrates. Members of the interleukin (IL) family of cytokines are homologous to Upd ligands, and a microarray study in cultured mammalian cells found that the Yki homolog Yap could regulate IL cytokines, which raises the possibility that a regulatory connection between Hippo signaling and Jak-Stat signaling might be conserved. Increased levels and nuclear localization of Yap have been reported in colon cancer patient samples, and ubiquitous Yap1 overexpression causes overproliferation of progenitor cells in the murine intestine. These observations suggest that future considerations of the potential contributions of Hippo signaling to colon cancer should include evaluations both of its possible regulation by Jnk signaling and of possible nonautonomous effects mediated by cytokines (Staley, 2010).

Inositol phosphate kinase 2 is required for imaginal disc development in Drosophila

Inositol phosphate kinase 2 (Ipk2), also known as IP multikinase IPMK, is an evolutionarily conserved protein that initiates production of inositol phosphate intracellular messengers (IPs), which are critical for regulating nuclear and cytoplasmic processes. This study reports that Ipk2 kinase activity is required for the development of the adult fruit fly epidermis. Ipk2 mutants show impaired development of their imaginal discs. Loss of Ipk2 activity results in increased apoptosis and impairment of proliferation during larval and pupal development. The proliferation defect is in part attributed to a reduction in JAK/STAT signaling, possibly by controlling production or secretion of the pathway's activating ligand, Unpaired. Constitutive activation of the JAK/STAT pathway downstream of Unpaired partially rescues the disk growth defects in Ipk2 mutants. These results demonstrate an essential role for Ipk2 in producing inositide messengers required for imaginal disc tissue maturation and subsequent formation of adult body structures and provides molecular insights to signaling pathways involved in tissue growth and stability during development (Seeds, 2015).

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 Stat92E^ts 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).

The adult Drosophila malpighian tubules are maintained by multipotent stem cells

All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable and no stem cells have been identified. This study has identified multipotent stem cells in the region of lower tubules and ureters of the Malpighian tubules. Using lineage tracing and molecular marker labeling, it was demonstrated that several differentiated cells in the Malpighian tubules arise from the stem cells and an autocrine JAK-STAT signaling regulates the stem cells' self-renewal. Identifying adult kidney stem cells in Drosophila may provide important clues for understanding mammalian kidney repair and regeneration during injury (Singh, 2008).

The regenerating renal cells may come from one of the three possible sources, based on previous studies. First, the circulating blood contains bone marrow-derived stem cells able to differentiate into non-haematopoietic cells, such as cells of the kidney. Second, the differentiated glomerular and tubular cells may also be able to dedifferentiate into stem-like cells to repair the damaged tissues. Third, large numbers of slowly cycling cells have recently been identified in the mouse renal papilla region; these cells may be adult kidney stem cells and may participate in renal regeneration after ischemic injury. Further, the ureter and the renal collecting ducts were formed from the epithelium originating from the ureteric bud, and the nephrons and glomeruli were formed from the metanephric mesoderm-derived portion during kidney development. Two distinguished stem cell types have been proposed as responsible for repairing the renal collecting tubules and the nephrons. This study identified a type of pluripotent stem cells (RNSCs) in the Drosophila renal organ. The stem cells are able to generate all cell types of the adult fly MTs. In the region of lower tubules and ureters, autocrine JAK-STAT signaling regulates the stem cell self-renewal. Weak JAK-STAT signaling may convert an RNSC into a renalblast (RB), which will differentiate into an RC in the region of lower tubules and ureters, and a type I or type II cell in the upper tubules. These data indicate that only one type of stem cell may be responsible for repair and regeneration of the whole damaged tissues in mammalian kidney (Singh, 2008).

The Drosophila RNSCs represent a unique model to study the molecular mechanisms that regulate stem cell or cancer stem cell behavior. In most of the stem cell systems that has been well characterized to date, stem cells always reside in a specialized microenvironment, called a niche. A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The stromal cells often secrete growth factors to regulate stem cell behavior. The stem cell niche plays an essential role in maintaining stem cells, and stem cells will lose stem cell status once they are detached from the niche. The niche often provides the balanced (proliferation-inhibiting and proliferation-stimulating) signals that keep the stem cells dividing slowly. The inhibitory signals keep the stem cell quiescent most of the time while the stimulating signals promote stem cell division, to replenish lost differentiated cells. Maintaining the balance between proliferation-inhibiting and proliferation-stimulating signals is the key to maintaining tissue homeostasis (Singh, 2008).

Drosophila RNSCs are controlled differently. This study has demonstrated that the JAK-STAT signaling regulates the stem cell self-renewal. Both the ligand Upd and the receptor Dome are expressed in the RNSCs and the autocrine JAK-STAT signaling regulates the stem cell self-renewal; thus, the self-sufficient stem cells control their self-renewal or differentiation and do not need to constrained to a fixed niche. However, the RNSCs are still confined to the region of lower tubules and ureters even in the Upd overexpressed flies, suggesting that some other factors besides the JAK-STAT signaling may restrict the RNSCs to the region of the lower tubules and ureters (Singh, 2008).

Recent studies also suggest that tumors may arise from small populations of so-called cancer stem cells (CSCs). The CSCs probably have arisen from mutations that dysregulate normal stem cell self-renewal. For example, mutations that block the proliferation-inhibiting signals or promote the proliferation-stimulating signals can convert the normal stem cells into CSCs. This study demonstrates that amplifying the JAK-STAT signaling by overexpressing its ligand Upd stimulates the RNSCs to proliferate and also to differentiate into RC, which results in tumorous overgrowth in the MT. Therefore, the Drosophila RNSC system may also be a valuable in vivo system in which to study CSC regulation (Singh, 2008).

The RNSCs are located in the region of the lower tubules and ureter of the MTs, while ISCs are located at the posterior midgut. The MTs' ureters connect to the posterior midgut. The two types of stem cells are at close anatomical locations in the adult fly digestion system and also share some properties. For example, both of them are small nuclear cells, Arm-positive, and express esg. However, RNSCs and ISCs produce distinctly different progenies. ISCs produce progenies that include either Su(H)GBE-lacZ- or Pros-positive cells, which are not among the progenies of RNSCs because Su(H)GBE-lacZ and Pros are not expressed in the MTs. RNSCs produce progenies that include Cut- or TSH-positive cells, which are not among the progenies of ISCs because Cut and TSH are not expressed in the posterior midgut. One possibility for this difference is that, although RNSCs and ISCs originate from the same stem cell pool, their particular environments restrict their differentiation patterns. Future experiments, such as transferring RNSCs to the posterior midgut and vice versa, should be able to test this model (Singh, 2008).

The JAK-STAT signaling regulates self-renewal of the male germline, the male somatic, female escort stem cells in fly. The signaling also regulates self-renewal and maintenance of mammalian embryonic stem cells. This study reports that the JAK-STAT signaling regulates self-renewal of RNSCs. The JAK-STAT signaling may be a general stem cell signaling and also regulate stem cell self-renewal in other, un-characterized stem cell systems (Singh, 2008).

esg has been used as a marker of both male germline stem cells. This study has demonstrated that the esg-Gal4. UAS-GFP transgene is specifically expressed in RNSCs. The function of the esg gene is to maintain cells as diploid in Drosophila imaginal cells. Stem cells may have to be diploid, and esg may be a general stem cell factor. Identifying a stem cell signaling pathway (such as the JAK-STAT signal transduction pathway) and a stem cell factor (such as esg) will provide useful tools for identifying stem cells in other systems and for understanding stem cell regulation in general (Singh, 2008).

Cell competition modifies adult stem cell and tissue population dynamics in a JAK-STAT-dependent manner

Throughout their lifetime, cells may suffer insults that reduce their fitness and disrupt their function, and it is unclear how these potentially harmful cells are managed in adult tissues. This question was addressed using the adult Drosophila posterior midgut as a model of homeostatic tissue and ribosomal Minute mutations to reduce fitness in groups of cells. A quantitative approach was taken, combining lineage tracing and biophysical modeling, and how cell competition affects stem cell and tissue population dynamics was addressed. Healthy cells were shown to induce clonal extinction in weak tissues, targeting both stem and differentiated cells for elimination. It was also found that competition induces stem cell proliferation and self-renewal in healthy tissue, promoting selective advantage and tissue colonization. Finally, winner cell proliferation was shown to be fueled by the JAK-STAT ligand Unpaired-3, produced by Minute^-/+ cells in response to chronic JNK stress signaling (Kolahgar, 2015).

Recent studies have shown that cell competition can also take place in adult tissues. This work has taken this notion forward and delineated quantitatively how adult stem cells and tissue population dynamics are affected by cell competition. In the subfit population, differentiated cells are killed by apoptosis followed by cell delamination; stem cells are also eliminated, possibly via induction of differentiation, as dying stem cells have not been detected. In parallel, as this study has shown, the healthy tissue expands due to an increase in stem cell proliferation and self-renewal. Indeed, biophysical modeling shows that changes in these parameters of a magnitude comparable to what observed experimentally is sufficient to recapitulate the stem cell dynamics of wild-type tissue undergoing Minute cell competition. Interestingly, accelerated proliferation of fitter stem cells has been seen in mouse embryonic stem cells using in vitro models of cell competition. However, in those studies, increased stem cell self-renewal has not been observed, probably because stemness in vitro is artificially maintained by exogenous factors in the culture medium (Kolahgar, 2015).

In many adult homeostatic tissues, stem cells stochastically differentiate or self-renew, and this leads to clonal extinction balanced by clonal expansion. This is known as neutral drift competition, because through this process, stem cell compartments stochastically tend toward monoclonality. It has also been shown that stem cell competition can be nonneutral (i.e., biased) when stem cells acquire a cell-autonomous advantage. In these cases, the bias derives from intrinsic differences (e.g., faster proliferation) and does not rely on cell interactions. This study shows instead that in adult homeostatically maintained tissues, competitive cell interactions can act as extrinsic cues that actively modify stem cell behavior, and that this confers on winners an advantage (e.g., as this study observed, increased proliferation rate and self-renewal) and on losers a disadvantage (e.g., as observed induced cell death), influencing tissue colonization. It is important to note that clones of wild-type cells that have lost proliferative capability because they are devoid of ISCs are equally able to induce death in neighboring M/+cells. This rules out the possibility that physical displacement due to a faster clonal expansion is the cause of cell competition in this case. This process instead, like the recent reports of cell competition in the mouse heart and fly nervous system, likely corresponds to the adult equivalent of the cellular competition observed in developing tissues (Kolahgar, 2015).

This work shows that M/+ midguts suffer from a chronic inflammatory response, which through JNK signaling activation and the ensuing production of the JAK-STAT ligand Upd-3 promotes wild-type tissue overgrowth. Thus, in this tissue, the overproliferation of winner cells stems from the increased availability of proliferative signals in the M/+ environment. The results suggest that wild-type cells respond more efficiently than M/+ cells to this proliferation stimulus, and that this difference results in their preferential overgrowth, contributing to cell competition. It has long been suggested that cell competition may result from the limiting availability of growth factors, which would compromise the viability of loser cells. Here it was instead found that excess production of a growth factor (Upd-3) can boost cell competition by promoting preferential proliferation of fitter cells. Given that JNK and JAK-STAT are frequently activated in response to stress or deleterious mutations, it would be interesting to test whether this is a general mechanism used by loser cells to promote the overgrowth of fitter neighbors. Notably, differences in JAK-STAT signaling are sufficient to trigger cell competition and, consistent with this, reducing JAK-STAT signaling in wild-type cells compromises their ability to eliminate scribble/losers. Thus, increased JAK-STAT signaling may in addition provide wild-type cells with a heightened fitness state and help promote the elimination of M/losers (Kolahgar, 2015).

Ribosomal mutations are linked with many adult disorders, not just in Drosophila but more importantly in humans, where they are associated with a number of severe pathologies, collectively known as ribosomopathies. Given that 79 proteins make up the eukaryotic ribosome (and several more are involved in ribosomal production) and that many Minute mutations are dominant, the sporadic insurgence of M/+ in adult tissues is likely to be one of the most common spontaneous generations of somatic mutant cells in our bodies. The elimination of these cells via cell competition is likely to play an unappreciated role in maintaining healthy adult tissues (Kolahgar, 2015).

A striking feature emerging from the results is that, in response to cell competition, normal cells can efficiently repopulate adult tissues, thus effectively replacing potentially diseased cells. This bears striking resemblance to the phenomenon of mosaic revertants, observed in a number of human skin and blood diseases. Spontaneous sporadic reversion of genetically inherited, disease-bearing mutations leads to the generation of revertant cells, which effectively repopulate tissues, at times ameliorating the condition. In some instances, the revertants' expansion is so efficient that selective advantage has been. Intriguingly, ichthyosis with confetti, a skin disease characterized by confetti-like appearance of revertant skin spots, is associated with a mutation in Keratin 10, which, due to its nucleolar mislocalization, could affect ribosome production similar to M-/+mutants. Thus, based on these findings, it is tentative to speculate that selective advantage in mosaic revertants could in some cases be driven by cell competition (Kolahgar, 2015).

The work shows that M/+ midguts suffer from a chronic inflammatory response, which through JNK signaling activation and the ensuing production of the JAK-STAT ligand Upd-3 promotes wild-type tissue overgrowth. Thus, in this tissue, the overproliferation of winner cells stems from the increased availability of proliferative signals in the M/+ environment. The results suggest that wild-type cells respond more efficiently than M/+ cells to this proliferation stimulus, and that this difference results in their preferential overgrowth, contributing to cell competition. It has long been suggested that cell competition may result from the limiting availability of growth factors, which would compromise the viability of loser cells. This study found instead that excess production of a growth factor (Upd-3) can boost cell competition by promoting preferential proliferation of fitter cells. Given that JNK and JAK-STAT are frequently activated in response to stress or deleterious mutations, it would be interesting to test whether this is a general mechanism used by 'loser' cells to promote the overgrowth of fitter neighbors. Notably, differences in JAK-STAT signaling are sufficient to trigger cell competition and, consistent with this, reducing JAK-STAT signaling in wild-type cells compromises their ability to eliminate scribble/ losers. Thus, increased JAK-STAT signaling may in addition provide wild-type cells with a heightened fitness state and help promote the elimination of M/+ losers (Kolahgar, 2015).

Ribosomal mutations are linked with many adult disorders, not just in but more importantly in humans, where they are associated with a number of severe pathologies, collectively known as ribosomopathies. Given that 79 proteins make up the eukaryotic ribosome (and several more are involved in ribosomal production) and that many Minute mutations are dominant, the sporadic insurgence of M/+ cells in adult tissues is likely to be one of the most common spontaneous generations of somatic mutant cells in our bodies. The elimination of these cells via cell competition is likely to play an unappreciated role in maintaining healthy adult tissues (Kolahgar, 2015).

A striking feature emerging from the current results is that, in response to cell competition, normal cells can efficiently repopulate adult tissues, thus effectively replacing potentially diseased cells. This bears striking resemblance to the phenomenon of mosaic revertants, observed in a number of human skin and blood diseases. Spontaneous sporadic reversion of genetically inherited, disease-bearing mutations leads to the generation of revertant cells, which effectively repopulate tissues, at times ameliorating the condition. In some instances, the revertants' expansion is so efficient that selective advantage has been proposed. Intriguingly, ichthyosis with confetti, a skin disease characterized by confetti-like appearance of revertant skin spots, is associated with a mutation in Keratin 10, which, due to its nucleolar mislocalization, could affect ribosome production similar to M/+ mutants. Thus, based on the current findings, it is tentative to speculate that selective advantage in mosaic revertants could in some cases be driven by cell competition (Kolahgar, 2015).

ROS-induced JNK and p38 signaling is required for Unpaired cytokine activation during Drosophila regeneration

Upon apoptotic stimuli, epithelial cells compensate the gaps left by dead cells by activating proliferation. This has led to the proposal that dying cells signal to surrounding living cells to maintain homeostasis. Although the nature of these signals is not clear, reactive oxygen species (ROS) could act as a signaling mechanism as they can trigger pro-inflammatory responses to protect epithelia from environmental insults. Whether ROS emerge from dead cells and what is the genetic response triggered by ROS is pivotal to understand regeneration of Drosophila imaginal discs. Cell death was genetically induced in wing imaginal discs, the production of ROS was monitored, and the signals required for repair were analyzed. Cell death was found to generate a burst of ROS that propagates to the nearby surviving cells. Propagated ROS activate p38 and induce tolerable levels of JNK. The activation of JNK and p38 results in the expression of the cytokines Unpaired (Upd), which triggers the JAK/STAT signaling pathway required for regeneration. These findings demonstrate that this ROS/JNK/p38/Upd stress responsive module restores tissue homeostasis. This module is not only activated after cell death induction but also after physical damage and reveals one of the earliest responses for imaginal disc regeneration (Santabarbara-Ruiz, 2015).

Oogenesis

Janus kinase (JAK) plays several signaling roles in Drosophila oogenesis. The gene for a JAK pathway ligand, unpaired (outstreched), 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).

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).

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).

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).

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 egg develops through closely coordinated activities of associated germline and somatic cells. An essential aspect of egg development is the differentiation of the somatic follicle cells into several distinct subpopulations with specific functions. The graded activity of the Janus kinase (JAK) pathway, stimulated by the Unpaired ligand, patterns the anterior-posterior axis of the follicular epithelium. Different levels of JAK activity instruct adoption of distinct anterior cell fates. Further, the coordinated activities of the JAK/STAT and epidermal growth factor receptor (EGFR) pathways are required to specify the posterior terminal cell fate. It is proposed that Upd secreted from the polar cells may act as a morphogen to stimulate A/P-derived follicular fates through JAK pathway activation (Xi, 2003).

The Drosophila egg is an intricately patterned structure with distinct specializations and polarities. These features are critical to subsequent embryonic development because the polarities of the egg are transmitted to the embryo, establishing the initial pattern in a developing zygote. The pattern of the mature egg is established by complex cellular interactions among and between both somatic follicle cells and germline cells. Each egg begins as a 16-cell germline cyst, from which one cell will become the oocyte and the remainder will become the supporting nurse cells. In the germarium, the anterior structure in which oogenesis is initiated, the germline cyst, is surrounded by a monolayer of somatic follicle cell precursors. As the encapsulated cyst exits from the germarium, approximately 10-14 of the somatic cells cease proliferation and differentiate. This group of cells forms two distinct populations: two polar cells at the anterior and posterior poles of each chamber and approximately seven stalk cells that form a bridge between the consecutive cysts. As the cyst exits the germarium, the other somatic cells covering each chamber, the epithelial follicle cells, remain undifferentiated (Xi, 2003 and references therein).

After pinching off from the germarium, each germline cyst grows, while the epithelial follicle cells proliferate. During this time, the anterior-posterior polarity that will ultimately determine all of the epithelial follicular fates is established. Elegant experiments have shown that the underlying prepattern of the follicular epithelium displays mirror image symmetry at the termini in the anterior-posterior (A/P) axis. Cells adopt one of three anterior terminal fates [border, stretched, and centripetal cells (terminal to central)], depending on proximity to the poles. In the intervening region between the terminal domains, cells will adopt a default 'main body' identity, and the posterior terminal cells form nearest the posterior pole. The symmetry of the A/P pattern is broken by EGFR signaling at the posterior. Secreted Grk from the posteriorly localized oocyte activates EGFR on the overlying follicle cells, establishing posterior terminal fate. In the absence of EGFR signaling, the anterior pattern is repeated at the posterior (Xi, 2003 and references therein).

By stage 7, the epithelial follicle cells cease proliferation and enter an endocycle. Afterward, these cells begin to show morphological and molecular signs of differentiation into the five epithelial fates: border, stretched, centripetal, posterior, and main body cells. Each of these subpopulations of follicle cells has a specific function with respect to the production of a mature egg, such that the correct number and position of each type is critical to ultimate egg morphology. These functions influence the production of structures that are essential to the egg, such as the dorsal respiratory appendages and the micropyle. These functions are also critical for proper anterior-posterior organization of the oocyte and, therefore, also for the resulting embryo (Xi, 2003 and references therein).

In early follicular differentiation, JAK activity is required for the production of stalk cells and the repression of polar cell fates. Later, JAK signaling is important for the proper recruitment and migration of border cells, a subpopulation of the follicular epithelium. Between these events, loss of JAK signaling in the follicular epithelium leads to persistent expression of Fas III, a marker for immature follicle cells (Xi, 2003 and references therein). The failure of these epithelial follicle cells to mature in hop mutant clones, as well as the persistent expression of upd in the polar cells, suggests that JAK signaling may have a role in differentiation of the entire follicular epithelium (Xi, 2003).

To address whether JAK signaling may play a role in distinguishing the terminal and main body domains, expression of mirror-lacZ (mirr-lacZ) was examined in egg chambers with aberrant JAK signaling. At ovarian stages 6-8, mirr-lacZ is strongly expressed in the main body follicle cells, with graded reduction toward the termini. In clones of hop mutant cells in the terminal regions, expression of mirr-lacZ is strongly induced, even prior to differentiation of those cells. It is concluded that JAK pathway activity distinguishes the terminal from the main body domains, at least as marked by the mirr-lacZ reporter. Specifically, JAK signaling is required to establish terminal identity and/or repress main body fate (Xi, 2003).

JAK activation is essential for specification of terminal fates, but is it also sufficient for terminal identity? To test this possibility, hop and upd were expressed in clones within the follicular epithelium, since either can activate JAK signaling. JAK pathway activation represses mirr-lacZ cell autonomously for Hop, but nonautonomously for the secreted Upd. This suggests that JAK pathway activity directly establishes the terminal domain. Furthermore, Upd expression causes graded repression of mirr-lacZ in the neighboring cells. Those cells closest to the Upd source have no expression of mirr-lacZ, but the amount of reporter in the neighboring cells increases with the distance from the Upd-expressing cells. It is concluded that JAK signaling must be activated in a graded fashion around the source of the Upd ligand, presumably because of extracellular diffusion of Upd. Moreover, the range over which the ectopic Upd can suppress mirr-lacZ is greater near the poles than in the center of the egg. Because endogenous Upd is secreted by the polar cells, it is easy to imagine that levels of JAK activity within the follicular epithelium would be greater near the poles. Consequently, the sum of the endogenous and ectopic Upd activities would be higher near the termini and could better repress mirr-lacZ. An alternative explanation is that the level of JAK signaling or another required signaling pathway is limited near the central region of the epithelium. It is concluded that JAK activation is necessary and sufficient for terminal identity in the follicular epithelium (Xi, 2003).

These results do not distinguish whether JAK signaling induces general terminal cell identity or is instructive for specific terminal fates. JAK activity could make the termini competent to respond to another signal that determines specific terminal fates or, alternatively, JAK activity could directly specify terminal fates, perhaps through varying levels of activation. To determine whether JAK signaling is essential for determining specific fates within the terminal domains, previously characterized markers for anterior subpopulations were examined. Work by others has already shown that JAK signaling can recruit the most terminal anterior fate, border cells. Here it is demonstrated that high levels of JAK activity are necessary and sufficient for border cell identity, at least within the anterior terminal domain. Consistent with the previous studies, the loss of JAK activity in clones of presumptive border cells invariably leads to failure of those cells to differentiate as border cells. However, this does not address whether JAK signaling is instructive for specific anterior fates. To address this question, the JAK pathway was activated to high levels in cells that are not at the terminus. Ectopic expression of either hop or upd stimulates additional cells to adopt border cell fate, again, in a cell-autonomous manner for hop and in a nonautonomous manner for upd. The location of the ectopic border cells suggests that they would normally have become stretched or centripetal cells. The ability of increased JAK signaling to alter fate within the anterior domain supports the hypothesis that levels of JAK signaling instruct specific fates within that domain. Increased JAK activity in the posterior terminal domain failed to induce ectopic border cells, presumably because of EGFR-mediated specification of posterior identity within this domain that would preclude expression of any anterior markers (Xi, 2003).

Involvement of JAK signaling in the specification of the more central anterior fates was examined with dpp-lacZ and MA33, which mark both stretched and centripetal cells, and BB127, which is specific for centripetal cells. JAK activity is essential for both fates. Loss of JAK signaling in either population results in a failure to express the population-specific markers. In addition to loss of the stretched cell marker, hop mutant cells also fail to migrate, spread out, and adopt the squamous morphology distinctive for stretched cells. On the basis of the expression of mirr-lacZ in JAK mutant clones above, it is presumed that these cells adopt a default main body fate. Moreover, effects of a weak general reduction of JAK signaling in the egg can be examined in ovaries of females transheterozygous for one severe and one weak allele of upd. In such eggs, the number of cells expressing a border cell marker is reduced. But, in addition, a marker for the stretched/centripetal fates is prominently expressed in the remaining border cells. Comparable expression of stretched cell markers in border cells is never observed in wild-type. Furthermore, the defective 'border cells' of upd mutant chambers also show aberrant migration. It is concluded that high levels of JAK activity are required for border cell fate, while lower levels direct stretched cell fate. Consistent with this, the border cells of upd mutant chambers did not express the centripetal cell-specific marker. Moreover, aberrant border cell specification in the Upd mutants indicates that Upd must normally activate JAK signaling during this process (Xi, 2003).

As with loss-of-function, clones of JAK-activated cells fail to express stretched and centripetal markers appropriate for their positions within the egg. On the basis of the morphology of the misexpressing cells and the complementary evidence with border cell markers, the presumptive stretched or centripetal cells with increased JAK activation are converted to the terminal-most border cell fate. Furthermore, JAK activation in the presumptive main body can induce the adoption of centripetal cell fate. However, it is somewhat surprising that JAK activation is unable to induce the most terminal (border cell) fate. One possible reason is that the endogenous JAK activity is likely highest near the poles and lowest in the central region, making it easier to convert cells closer to the terminus to border cell fate. This model assumes that the levels of ectopic activity must be lower than the highest levels of endogenous JAK activity, though evidence below suggests that this may not be true. A second possibility suggests that downstream components or cofactors required for high-level activation of JAK or for another required pathway may be in limited supply in the central region. Despite limited response in the central region, all the epithelial follicle cells are responsive to changes in levels of JAK activation. This indicates that the JAK pathway plays an active role, not a permissive role, in assigning specific terminal fates within the follicular epithelium (Xi, 2003).

The transformation of hop mutant cells in the posterior terminal domain into main body cells, as marked by mirr-lacZ, suggests that JAK signaling is essential for posterior terminal identity, as well as anterior fates. To address this hypothesis, two enhancer trap markers for posterior cells were analyzed in hop mutant clones. The pnt-lacZ marker is normally expressed strongly at the posterior, with a graded reduction in posterior cells farther from the pole. Cells mutant for hop show complete loss of pnt-lacZ expression in a cell-autonomous fashion. Moreover, because the cells at the posterior do not undergo the dramatic migrations seen at the anterior, it is possible to analyze the mutant and wild-type cells in their relative positions to one another. Significantly, it can be seen that wild-type cells express normal levels of pnt-lacZ, even when mutant cells intervene between them and the polar cells. Similar results were seen for a second posterior marker, blot⁰¹⁶⁵⁸. This suggests that the wild-type cells are receiving a signal directly from the polar cells and not via a local signal relay or 'bucket brigade' mechanism from neighboring cells (Xi, 2003).

To confirm that JAK activity influences posterior terminal fate, the function of those terminal cells was examined. At approximately stage 7, the posterior terminal cells send a signal to the underlying oocyte that stimulates microtubule reorganization in the oocyte. This microtubule reorganization is important for the migration of the oocyte nucleus to the dorsal anterior and for the proper sequestration of A/P determinants that direct development of the resulting embryo. After reorganization, one of these determinants, Staufen, is tightly associated with the posterior end of the oocyte. However, in egg chambers that lack JAK activity at the posterior terminus, Staufen fails to localize and is dispersed in the cytoplasm. Furthermore, in eggs with JAK mutant clones that cover only a portion of the presumptive posterior terminal cells, Staufen becomes localized directly underneath the wild-type cells at the posterior. This suggests that high JAK activity in the posterior follicle cells very precisely stimulates aggregation of a Staufen-bound complex in the underlying membrane. Despite the consistent mislocalization of Staufen in eggs with hop mutant clones at the posterior, the oocyte nucleus rarely fails to migrate to the dorsal anterior. This may indicate either that global microtubule reorganization is separable from Staufen localization or that Staufen is more sensitive to perturbations in microtubule reorganization (Xi, 2003).

Conversely, cell clones that express either hop or upd are able to activate pnt-lacZ, but only near the posterior of the chamber. Once again, the activation is autonomous for hop and nonautonomous for upd, supporting a direct role for JAK signaling in determining cell fates. Moreover, as with the mirr-lacZ reporter, the nonautonomous activity of Upd results in a graded response in the marker, such that the level of pnt-lacZ decreases as the distance from the ectopic Upd source increases. Again, this points to a gradient of JAK activity being established around the upd-expressing cells. Interestingly, activation of the posterior marker in cells neighboring upd-expressing clones was stronger on the posterior side of the clone. Again, this may be due to additive influences of the endogenous Upd signal coming from the polar cells and the ectopically expressing cells (Xi, 2003).

The initial A/P pattern in the follicular epithelium has a mirror image symmetry, such that cells at either end that are equidistant from the polar cells have equivalent identities. Subsequently, Grk from the oocyte, which always lies at the posterior of the egg, breaks the symmetry by stimulating EGFR in the follicle cells, inducing posterior terminal fate. Loss of EGFR activation in the posterior cells causes adoption of the underlying anterior fates. The requirement for both EGFR and JAK activation explains the failure of ectopic JAK activation to induce posterior identity at the anterior. But clones of cells that express activated EGFR can induce pnt-lacZ at the anterior. However, as in the posterior, induction of the marker at the anterior is graded, with highest levels closest to the pole. Furthermore, in the main body, activated EGFR is unable to induce posterior fate. This suggests that another factor essential for posterior identity is normally present, but limiting, in the anterior region. These domains that are competent to respond to activated EGFR coincide with the JAK activation. So, is EGFR limited in specifying posterior fate by the underlying activity of the JAK pathway? Consistent with this supposition, the coexpression of activated EGFR and JAK is capable of inducing posterior fate in all follicular epithelial cells. Thus, the coordinated activities of the two pathways are necessary and sufficient for induction of posterior identity (Xi, 2003).

Graded response of the mirr-lacZ marker and the ability of altered levels of JAK activity to change anterior fates are consistent with a model in which graded levels of JAK activity specify different follicular fates along the A/P axis. This model predicts that an overall increase or decrease of JAK activity would alter the number of cells adopting fates for each of the anterior subpopulations. Specifically, an overall reduction of JAK activity should reduce the number of border cells while shifting and/or reducing the number of cells adopting the more central stretched and centripetal fates and expanding the main body domain. To test this hypothesis, egg chambers from reduced function mutants of upd and hop were examined for the number and distribution of cells within each of the anterior subpopulations. Reduction of JAK activity dramatically reduces the number of border cells. Combination of one weak and one strong mutant allele of upd reduces the number of border cells by nearly half. Furthermore, combination of two weak hop alleles completely eliminates all border cells, despite producing morphologically normal eggs. Moreover, stretched cells are somewhat reduced in the hop mutant, while centripetal cells are only slightly affected. Similar results were seen at the posterior, where reduced hop activity results in marker expression that is only detectable to about four cell diameters from the posterior, rather than the normal eight cell diameters. However, graded marker expression is maintained, just shifted toward the posterior. A more substantial reduction of the most terminal fates strongly supports existence of graded JAK activity that is highest at the termini (Xi, 2003).

A model is presented for anterior-posterior follicular patterning. Patterning of the follicular epithelium requires the coordination of several signaling pathways. In the A/P axis, prominent roles for the EGFR and Notch pathways have been established. By incorporating the functions of the JAK pathway, an integrated model of A/P patterning in the follicular epithelium is proposed. The activation of the JAK pathway in the follicular epithelium is graded, with highest levels at the anterior and posterior poles. This is consistent with the production of the secreted ligand, Upd, from the polar cells, which is then received by cells of the follicular epithelium. The expression of Upd in the polar cells begins even within the germarium, so it is established as a potential graded signal from the earliest stages of follicular epithelial development. The polar cells have an organizer function in the establishment of A/P pattern. This organizer activity is consistent with the functions and behaviors described for JAK signaling in the surrounding epithelium. It is proposed that the gradient of JAK activity from both termini determines the presumptive border, stretched, and centripetal cells, on the basis of thresholds of JAK activity that define each fate, establishing a symmetrical prepattern. However, JAK signaling may not be the only patterning element in this process. Ectopic JAK activation in the main body domain is insufficient to induce the most terminal fate, the border cells. Though this could arise because of an inherent limitation to JAK signaling in the main body, the induction of Stat92E and Dome to high levels in similar activation clones argues against this. Alternatively, the main body may have low levels of some downstream coactivator for JAK signaling or of an independent patterning element, perhaps another signaling pathway. With the exception of this reduced response in the main body, adoption of each epithelial follicular fate can be simply ascribed to varying thresholds of JAK pathway activity (Xi, 2003).

The symmetrical prepattern established by JAK signaling is broken by EGFR activation in the posterior follicle cells stimulated by the secreted Grk ligand from the oocyte. The combined activation of JAK and EGFR signaling at the posterior defines posterior terminal follicle cell identity, overriding the default anterior fates specified by JAK activity alone. By the end of stage 6, when proliferation ceases, the cell fates of the follicular epithelium must already be determined. At that time, Notch pathway activation in all epithelial follicle cells triggers the transition from active division to an endocycle. By stage 9, the epithelial cells express markers for the various fates, begin migrations toward the posterior, and undergo morphological changes appropriate for ultimate function of that fate. Thus, the combined and sequential functions of the JAK, EGFR, and Notch pathways establish a series of anterior and posterior fates in the follicular epithelium (Xi, 2003).

The essential nature of morphogens, signals that have the ability to induce cell fates on the basis of levels of activity, is a central theme in animal development. Yet, despite this centrality, very few proteins have been demonstrated to have morphogenic function. Interestingly, most of the known morphogens have retained that activity throughout animal evolution. In both vertebrates and invertebrates, well-known signaling proteins of the Wnt, Hedgehog, and TGF-ß families act as morphogens. Though not all of the criteria have been explored, it is suggested that the properties of Upd and its stimulation of the JAK pathway in follicular epithelial cells are consistent with function as a morphogen. While the JAK intracellular cascade is highly conserved from flies to man, no proteins with significant homology to the Upd ligand have been found in other organisms. Therefore, Upd may be an unusual example of a morphogen that has rapidly diverged evolutionarily (Xi, 2003).

Morphogens are generally regarded to have four defining characteristics. (1) They are released from a localized source. In the ovary, Upd is secreted by the polar cells. (2) Morphogens form a concentration gradient from nearby to distant cells that respond directly to the signal, not through a relay mechanism. Although a gradient of Upd has not been directly visualized, the underlying gradient of JAK activation is apparent. Moreover, the response of cells to Upd activity requires downstream components of the JAK pathway in a cell-autonomous manner, demonstrating that the response to Upd is direct and not relayed. (3) Cells within the region of the gradient must show at least two different responses in addition to the default. In the follicular epithelium, the region that corresponds to the presumed JAK gradient gives rise to the border cells, stretched cells, and centripetal cells, in addition to the default main body cells. (4) Over- and underexpression should change cell fates in opposite directions. Clonal analysis clearly demonstrates that the anterior terminal and main body cell fates can be influenced by gain or loss of JAK pathway activity in an opposite and predictable manner. Thus, despite no direct visualization of a Upd gradient, the characteristics of the JAK pathway are consistent with a system that transduces a morphogenic signal (Xi, 2003).

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).

Apoptosis-mediated cell death within the ovarian polar cell lineage of Drosophila

Polar cells are pairs of specific follicular cells present at each pole of Drosophila egg chambers. They are required at different stages of oogenesis for egg chamber formation and establishment of both the anteroposterior and planar polarities of the follicular epithelium. Definition of polar cell pairs is a progressive process since early stage egg chambers contain a cluster of several polar cell marker-expressing cells at each pole, while as of stage 5, they contain invariantly two pairs of such cells. Using cell lineage analysis, it has been demonstrated that these pre-polar cell clusters have a polyclonal origin and derive specifically from the polar cell lineage, rather than from that giving rise to follicular cells. In addition, selection of two polar cells from groups of pre-polar cells occurs via an apoptosis-dependent mechanism and is required for correct patterning of the anterior follicular epithelium of vitellogenic egg chambers. Prevention of pre-polar cell death and subsequent generation of supernumerary polar cells may lead to production of an excess of signaling molecules, such as Unpaired, and alteration of endogenous morphogen gradients which could explain why both squamous cells and border cells exhibit aberrant behavior when pre-polar cell death is blocked (Besse, 2003).

Thus, each pair of mature polar cells derives from a pool of precursor pre-polar cells within which supernumerary cells are eliminated via an apoptosis-dependent mechanism. This mechanism probably requires both caspase activity and the 'death' gene reaper, since death is inhibited by ectopic expression of the bacculoviral p35 protein and is associated with specific induction of reaper expression. However, whereas the self-death machinery appears to be evolutionary conserved, a wide range of distinct signaling mechanisms can be used to elicit apoptosis. Cellular interactions within or without the pre-polar cell cluster may also be crucial for regulation of the selective pre-polar cell loss. In the present study, no correlation could be made between pre-polar cell position and cell removal, at least for apoptosis events occurring after egg chamber budding. It would be interesting nonetheless to examine Notch signaling as a survival factor in this system. Indeed, induction of Notch loss-of-function clones in prefollicular cells is associated with absence of polar cells. Conversely, egg chambers with terminal clones expressing an activated form of Notch contain up to 6 polar cell marker-positive cells. Such phenotypes, interpreted as reflecting a role for Notch signaling in polar cell specification, could also correspond to a Notch-dependent control of apoptosis within the pre-polar cell lineage (Besse, 2003).

Considering both the embryonic and oogenesis systems, the question can be asked as to the biological significance of creating supernumerary cells to remove them afterwards. One proposed explanation for what is observed in the embryonic CNS is that a differential number of glial cells may be required depending on the developmental stage. By analogy, at least 6 pre-polar cells may be required in the ovary to assume early germarial functions, whereas only 2 polar cells may be needed during later egg chamber maturation. Another possibility is that the process of polar cell production corresponds to an evolutionarily conserved mechanism to which removal of deleterious cells would have been added later. Further studies are now needed to determine whether the pre-polar cell clusters have a functional role in early oogenesis (Besse, 2003).

Polar cells are involved in several important signaling processes during oogenesis, including posterior positioning of the oocyte, induction of stalk cells in the germarium, organization of follicular cell epithelial planar polarity during mid-oogenesis and anteroposterior patterning of follicular epithelial cells at stage 9 (Besse, 2003 and references therein).

Strikingly, polarization of basal actin filament bundles in the follicular epithelium arises progressively, starting at stage 5 and proceeding from the poles, suggesting the existence of a diffusible signal produced by polar cells. Consistent with this, ectopic polar cells generated upon hedgehog overexpression have the capacity to reorganize actin bundles locally. However, orientation of planar actin filaments probably does not require a precise level of polarizing signal since it is shown here that an increase in polar cell number does not affect the establishment or maintenance of planar polarity (Besse, 2003).

In contrast, it has been found that the restriction in the number of polar cells seems to be required for correct anterior patterning of follicular epithelial cells. Indeed, preventing the elimination of supernumerary pre-polar cells results in morphogenetic defects affecting both stretching of anterior squamous cells and migration of border cell clusters. The latter is also accompanied by an increase in the number of recruited border cells. Production of ectopic polar cells has been described to induce ectopic and poorly migrating border cells at stage 9. Yet, this phenomenon was observed upon ectopic activation of hedgehog signal transduction (patched and Costal 2 mutant contexts). Therefore it is not clear in these cases whether migration defects are a direct consequence of extra border cell number or whether they reflect an additional effect of the Hedgehog signaling pathway on border cell migration and/or specification (Besse, 2003).

The fused gene encodes a serine/threonine kinase identified as a positive effector of the Hedgehog signal transduction pathway. In the ovary, Hedgehog signal transduction controls somatic stem cell (SSC) proliferation. Indeed, SSC self-renewing properties are not maintained in the absence of Hh signaling, whereas excessive Hh signaling produces supernumerary stem cells and leads to the accumulation of poorly differentiated somatic cells between egg chambers. Analysis of fu mutations had indicated that fu function is not involved in this process. Rather, fu-dependent Hedgehog signal transduction is necessary for somatic prefollicular cell differentiation and morphogenesis. In particular, fu function seems to be required for correct timing of the polar cell differentiation program. Indeed, fu mutant females exhibit a global shift in the dynamics of A101 staining, as visualized after anti-ß-galactosidase staining of fu^JB3/fu^JB3; A101/+ females. (1) The appearance of A101 staining is delayed, since 28% of stage 2 fu egg chambers do not exhibit any marked anterior cells compared to 19% in heterozygous sisters. (2) Restriction of A101 staining to 2 polar cells is also delayed, since 60% of stage 3 and 19% of stage 4 fu egg chambers contained 3 or more stained anterior cells compared to 33% and 4%, respectively, for fu⁺ egg chambers. Strikingly, 100% of stage 5 fu mutant egg chambers exhibit only 2 A101⁺ cells, indicating that restriction in the number of polar cells does eventually occur as in wild-type ovarioles. Altogether, these results suggest that fu mutations lead to a delay in the polar cell differentiation program (Besse, 2003).

Close examination of fu ovarioles has revealed that a higher proportion of groups containing 4 A101⁺ cells, 5 A101⁺ cells (8/156), and even 6 A101⁺ cells (1/156) can be found in stage 2 as well as stage 3 fu mutant egg chambers compared to the wild-type situation. It was reasoned that the presence of such groups of cells could result either from abnormally slow apoptosis-dependent elimination of pre-polar cells, or from an overproduction of pre-polar cells or their precursors. The first hypothesis could not be tested directly because the relatively low number of TUNEL-positive cells found in both wild-type and fused females (possibly due to rapid elimination of apoptotic cells) made it impossible to compare quantitatively the dynamics of polar cell apoptotic cell death between these two contexts. Therefore the second hypothesis was tested; defects were sought in polar and pre-polar cell proliferation, or in the number of polar cell precursor cells. First, polar cell proliferative properties do not seem to be altered in the vitellarium of fu ovarioles since (1) no increase in the size of A101⁺ terminal clusters is observed with increasing age of fu egg chambers from stage 2 to 5, and (2) no prolongation beyond stage 6 of somatic cell mitotic activity is observed in fu ovarioles. Second, using a dominantly marked clone approach, it has been shown that early clusters of 4-6 A101⁺ cells found in fu females never contain more than 2 GFP⁺ cells, and therefore that they do not result from extra divisions of precursor cells within the germarium. Third, it was reasoned that preventing apoptosis in the polar cell lineage in a fu mutant context should give an indication about the number of polar cell precursors present in these flies. If the number of such precursors is greater in fu females than in wild-type females, then blocking apoptosis should result in a greater number of 'rescued' cells in polar cell clusters than in a wild-type context (that is more than 6 cells). Therefore, the flp-out/Gal4 system was used to generate large somatic clones of p35 overexpressing cells in a fu mutant context. Although an increase in the average size of the terminal Fas III⁺ cell cluster was observed after p35 overexpression, only groups containing 2 to 6 cells were recovered. This indicates that fused females contain the same number of polar cell precursors as wild-type females (Besse, 2003).

Therefore, the supernumerary polar cells in both wild-type and fused mutant contexts is interpreted to represent pre-polar cells, and it is proposed that slower apoptosis-mediated reduction in the number of these cells in a fused context allows easier visualization of these cells. Thus, fused mutations, by delaying the somatic cell differentiation program, confirm the existence of pre-polar cell clusters and allow detection of up to 6 pre-polar cells. However, restriction in the final number of polar cells is achieved by stage 5 and is probably also mediated by apoptosis since TUNEL-positive A101⁺ cells are found in fused females (Besse, 2003).

In this study, defective border cell migration was detected after having prevented cell death specifically within neuralized-expressing pre-polar and polar cells. This suggests, first, that rescued pre-polar cells are not inert and, second, that final polar cell number per se is critical for both border cell recruitment and migration. Interestingly, polar cells show specific expression of Unpaired (Upd), an extracellular ligand that activates the conserved JAK/Stat signaling pathway. In the absence of positive effectors of this pathway, such as Unpaired, Hopscotch or STAT92E, defects in both recruitment and migration of border cells and sometimes also in stretching of squamous cells are observed. In addition, ectopic expression of Upd in a subset of anterior somatic cells is sufficient to induce expression of border cell markers in adjacent squamous cells. Interestingly, high levels of Upd result in the formation of egg chambers in which both normal and supernumerary border cells frequently fail to migrate. Altogether, this suggests that Upd could act as a morphogen produced by polar cells and necessary for establishing anteroposterior patterning of the follicular epithelium. In the present study, prevention of pre-polar cell death and subsequent generation of supernumerary polar cells may lead to production of an excess of signaling molecules, such as Upd, and alteration of endogenous morphogen gradients, which could explain why both squamous cells and border cells exhibit aberrant behavior. These results therefore provide further evidence for a non cell-autonomous role for anterior polar cells in patterning of the follicular epithelium (Besse, 2003).

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 (hop^TumL) 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(os^1a]. 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(os^1a) 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 (dsx^D/ dsx¹) proliferate, while XY germ cells in a feminized soma (UAS-tra^F; 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(os^1a)) 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).

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

During development, specific cells are eliminated by apoptosis to ensure that the correct number of cells is integrated in a given tissue or structure. How the apoptosis machinery is activated selectively in vivo in the context of a developing tissue is still poorly understood. In the Drosophila ovary, specialised follicle cells [polar cells (PCs)] are produced in excess during early oogenesis and reduced by apoptosis to exactly two cells per follicle extremity. PCs act as an organising centre during follicle maturation as they are the only source of the JAK/STAT pathway ligand Unpaired (Upd), the morphogen activity of which instructs distinct follicle cell fates. This study shows that reduction of Upd levels leads to prolonged survival of supernumerary PCs, downregulation of the pro-apoptotic factor Hid, upregulation of the anti-apoptotic factor Diap1 and inhibition of caspase activity. Upd-mediated activation of the JAK/STAT pathway occurs in PCs themselves, as well as in adjacent terminal follicle and interfollicular stalk cells, and inhibition of JAK/STAT signalling in any one of these cell populations protects PCs from apoptosis. Thus, a Stat-dependent unidentified relay signal is necessary for inducing supernumerary PC death. Finally, blocking apoptosis of PCs leads to specification of excess adjacent border cells via excessive Upd signalling. These results therefore show that Upd and JAK/STAT signalling induce apoptosis of supernumerary PCs to control the size of the PC organising centre and thereby produce appropriate levels of Upd. This is the first example linking this highly conserved signalling pathway with developmental apoptosis in Drosophila (Borensztejn, 2013).

A role for STAT in cell death and survival has been clearly documented in mammals, and depending on which of the seven mammalian Stat genes is considered and on the cellular context, both pro- and anti-apoptotic functions have been characterised. In the Drosophila developing wing, phosphorylated Stat92E has been shown to be necessary for protection against stress-induced apoptosis, but not for wing developmental apoptosis. This study provides evidence that Upd and the JAK/STAT pathway control developmental apoptosis during Drosophila oogenesis (Borensztejn, 2013).

This study demonstrated that the JAK/STAT pathway ligand, Upd, and all components of the JAK/STAT transduction cascade (the receptor Dome, JAK/Hop and Stat92E) are involved in promoting apoptosis of supernumerary PCs produced during early oogenesis. It is argued that The JAK/STAT pathway is essential for this event for several reasons. Indeed, in the strongest mutant context tested, follicle poles containing large TFC and PC clones homozygous for Stat92E amorphic alleles, almost all of these (95%) maintained more than two PCs through oogenesis. Also, RNAi-mediated reduction of upd, dome and hop blocked PC number reduction and deregulated several apoptosis markers, inhibiting Hid accumulation, Diap1 downregulation and caspase activation in supernumerary PCs. Altogether, these data, along with what has already been shown for JAK/STAT signalling in this system, fit the following model. Upd is secreted from PCs and diffuses in the local environment. Signal transduction via Dome/Hop/Stat92E occurs in nearby TFCs, interfollicular stalks and PCs themselves, leading to specific target gene transcription in these cells, as revealed by a number of pathway reporters. An as-yet-unidentified Stat92E-dependent pro-apoptotic relay signal (X) is produced in TFCs, interfollicular stalks and possibly PCs, which promotes supernumerary PC elimination via specific expression of hid in these cells, consequent downregulation of Diap1 and finally caspase activation. An additional cell-autonomous role for JAK/STAT signal transduction in supernumerary PC apoptosis of these cells is also consistent with, though not demonstrated by, the results (Borensztejn, 2013).

Relay signalling allows for spatial and temporal positioning of multiple signals in a tissue and thus exquisite control of differentiation and morphogenetic programmes. In the Drosophila developing eye, the role of Upd and the JAK/STAT pathway in instructing planar polarity has been shown to require an as-yet-uncharacterised secondary signal. In the ovary, the fact that JAK/STAT-mediated PC apoptosis depends on a relay signal may provide a mechanism by which PC apoptosis and earlier JAK/STAT-dependent stalk-cell specification can be separated temporally (Borensztejn, 2013).

Although neither the identity, nor the nature, of the relay signal are known, it is possible to propose that the signal is not likely to be contact-dependent, and could be diffusible at only a short range. Indeed, Stat92E homozygous mutant TFC clones in contact with PCs, as well as those positioned up to three cell diameters away from PCs, are both associated with prolonged survival of supernumerary PCs, whereas clones further than three cell diameters away from PCs are not. In addition, fully efficient apoptosis of supernumerary PCs may require participation of all surrounding TFCs, stalk cells and possibly PCs, for production of a threshold level of relay signal. In support of this, large stat mutant TFC clones are more frequently associated with prolonged survival of supernumerary PCs, and the effects of removing JAK/STAT signal transduction in several cell populations at the same time are additive. Interestingly, the characterisation of two other Drosophila models of developmental apoptosis, interommatidial cells of the eye and glial cells at the midline of the embryonic central nervous system, also indicates that the level and relative position of signals (EGFR and Notch pathways) is determinant in selection of specific cells to be eliminated by apoptosis (Borensztejn, 2013).

The results indicate that only the supernumerary PCs respond to the JAK/STAT-mediated pro-apoptotic relay signal, whereas two PCs per pole are always protected. Indeed, this study found that overexpression of Upd did not lead to apoptosis of the mature PC pairs and delayed rather than accelerated elimination of supernumerary PCs. Recently, it was reported that selection of the two surviving PCs requires high Notch activation in one of the two cells and an as-yet-unknown Notch-independent mechanism for the second cell. Intriguingly, expression of both Notch and Stat reporters is dynamic in PC clusters and PC survival and death fates are associated with respective activation of the Notch and JAK/STAT pathways. However, this study found that RNAi-mediated downregulation of upd did not affect either expression of Notch or that of two Notch activity reporters. Therefore, JAK/STAT does not promote supernumerary PC apoptosis by downregulating Notch activity in these cells. Identification of the relay signal and/or of Stat target genes should help further elucidate the mechanism underlying the induction of apoptosis in selected PCs (Borensztejn, 2013).

Interfollicular stalk formation during early oogenesis has been shown to depend on activation of the JAK/STAT pathway. The presence of more than two PCs during these stages may be important to produce the appropriate level of Upd ligand to induce specification of the correct number of stalk cells. Later, at stages 7-8 of oogenesis, correct specification of anterior follicle cell fates (border, stretch and centripetal cells) depends on a decreasing gradient of Upd signal emanating from two PCs positioned centrally in this field of cells. Attaining the correct number of PCs per follicle pole has been shown to be relevant to this process and border cells (BC) specification seems to be particularly sensitive to the number of PCs present. Previously work has shown apoptosis of supernumerary PCs is physiological necessary for PC organiser function, as blocking caspase activity in PCs such that more than two PCs are present from stage 7 leads to defects in PC/BC migration and stretch cell morphogenesis. This study now shows that the excess PCs produced by blocking apoptosis lead to increased levels of secreted Upd and induce specification of excess BCs compared with the control, and these exhibit inefficient migration. These results indicate that reduction of PC number to two is necessary to limit the amount of Upd signal such that the correct numbers of BCs are specified for efficient migration to occur. Taken together with the role shown for Upd and JAK/STAT signalling in promoting PC apoptosis, it is possible to propose a model whereby Upd itself controls the size of the Upd-producing organising centre composed of PCs by inducing apoptosis of supernumerary PCs. Interestingly, in the polarising region in the vertebrate limb bud, which secretes the morphogen Sonic Hedgehog (Shh), Shh-induced apoptosis counteracts Fgf4-stimulated proliferation to maintain the size of the polarising region and thus stabilise levels of Shh. It is likely that signal autocontrol via apoptosis of signal-producing cells will prove to be a more widespread mechanism as knowledge of apoptosis control during development advances (Borensztejn, 2013).

Tissue landscape alters adjacent cell fates during Drosophila egg development

Extracellular signalling molecules control many biological processes, but the influence of tissue architecture on the local concentrations of these factors is unclear. This study examines this issue in the Drosophila egg chamber, where two anterior cells secrete Unpaired (Upd) to activate Signal transducer and activator of transcription (STAT) signalling in the epithelium. High STAT signalling promotes cell motility. Genetic analysis shows that all cells near the Upd source can respond. However, using upright imaging, surprising asymmetries in STAT activation patterns were shown, suggesting that some cells experience different Upd levels than predicted by their location. A three-dimensional mathematical model was developed to characterize the spatio-temporal distribution of the activator. Simulations show that irregular tissue domains can produce asymmetric distributions of Upd, consistent with results in vivo. Mutant analysis substantiates this idea. The study concludes that cellular landscape can heavily influence the effect of diffusible activators and should be more widely considered (Manning, 2015).

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

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

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

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

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

upd^sisC1, a loss-of-function mutation that appears to specifically affect sex determination was examined, because it has no observable effect on later upd functions. Consistent with the fact that upd has a modest effect on SxlPe, it was found that two-thirds of homozygous upd^sisC embryos expressed SxlPe in a manner indistinguishable from that of the wild-type. A small proportion of embryos, 15%, had within their middle sections several clusters of 5-15 nuclei that did not express SxlPe, whereas the remaining 18% had severe defects, with SxlPe expression being absent from most of the central regions of the embryos. Despite early aberrations in SxlPe activity, immunostaining revealed no lasting defect in the expression of SXL, because upd^sisC1 embryos that reached germband extension stained in a 1:1 male:female ratio. To determine the effects of a complete loss of zygotic upd activity, upd^YC43, a probable null mutation, and the deficiency Df(1)ue69, which deletes upd and the upd-like gene, upd3, were examined. With respect to SxlPe, it was found that upd-null-mutant females were more severely affected than were upd^sisC1 embryos. At cellularization, the defects ranged from embryos containing large clusters of nuclei that did not express SxlPe in the central part of embryo to those in which the entire central region failed to express the promoter. The poles, however, expressed SxlPe normally. Immunostainings of upd^YC43 and Df(1)ue69 embryo collections revealed that these alleles had strong but incompletely penetrant effects on the later distribution of SXL. The fact that an estimated 40% of mutant female embryos stage 6 and older failed to express SXL in their central regions is consistent with the observed defects in SxlPe activity. The remainder eventually expressed normal levels of SXL in all their tissues, indicating that most upd mutant females were able to compensate for reduced SxlPe activity and ultimately engaged autoregulatory Sxl mRNA splicing. Two upd-like genes, upd2 and upd3, map adjacent to upd. Loss of zygotic upd2 had no effect on SxlPe, and the effects of Df(1)ue69 (upd3^-,upd^-) appeared identical to those of upd^YC43 when analyzed in a common genetic background. This shows that XSE activity in this region of the X is due to upd alone (Avila, 2007).

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

When Sxl expression was examined in cycle 14 embryos derived from hop^C111 germline clones, it was found that SxlPe was active in the anterior and posterior regions of the embryos but almost completely inactive in the central region of the embryos. In contrast to the results with upd mutants and deficiencies, all of which exhibited considerable embryo-to-embryo variation, loss of maternal hop had nearly identical effects on SxlPe in every embryo. This more potent effect of maternal hop^C111 as compared to upd mutants suggests that zygotic Upd might not be the only activator of JAK in the precellular embryo (Avila, 2007).

The findings with hop^C111 were confirmed by using the Stat92E⁰⁶³⁴⁶ mutation. Cycle 14 embryos derived from Stat92E⁰⁶³⁴⁶ germline clones also lacked nearly all SxlPe expression in their central regions, but they were even more strongly affected than hop^C111 females because SxlPe activity was also reduced in the termini. These findings are contrary to predictions of a linear JAK/STAT pathway going from zygotic Upd through receptor and kinase to activated STAT. Instead, the progressive weakening of SxlPe by removal of upd and Stat92E suggests that there is hop-independent control of Stat92E function in sex determination. The possibility of cross-talk between signaling systems is supported by the finding that the torso receptor-tyrosine-kinase pathway activates STAT92E in the embryo termini (Avila, 2007).

Although the hop^C111 and Stat92E⁰⁶³⁴⁶ mutations had large effects on SxlPe during cycle 14, the period of maximum SxlPe expression, it was found that these mutations had little effect on SxlPe at earlier stages. In wild-type females, SxlPe is first activated in cycle 12. Expression increases throughout cycle 13 and reaches a peak in the first minutes of cycle 14. In embryos from hop^C111 mothers, SxlPe was expressed as in the wild-type during cycles 12 and 13. However, upon entry into cycle 14, SxlPe activity ceased in the middle sections of the embryos. A similar phenomenon was observed in embryos carrying strong upd mutants and in those derived from Stat92E⁰⁶³⁴⁶ germline clones. These results show that JAK/STAT, and thus upd XSE function, is not needed for the initial activation of SxlPe. Instead, upd must function as a different kind of XSE: one dispensable for the initial assessment of X-chromosome dose, but needed to maintain SxlPe activity in the final stage of the X-counting process (Avila, 2007).

When the progeny of hop^C111 mutant mothers were examined for Sxl protein, it was found that defects in SxlPe expression led to a permanent failure to express SXL in the central regions in 35% of female embryos. This suggests that the loss of SxlPe activity in cycle 14 can reduce the level of early Sxl to below the threshold normally required to activate autoregulatory mRNA splicing. Although 35% of female embryos were defective for later Sxl expression, most females that completed gastrulation expressed Sxl uniformly. This striking discordance between the effects of hop (and upd and Stat92E) mutants on SxlPe activity and ultimate Sxl levels suggests that stable Sxl autoregulation can be established even when SxlPe function has been seriously compromised. Although some rescuing Sxl mRNA or protein may have diffused from the poles, an alternative explanation is that expression of SxlPe during cycles 12 and 13 might often have provided sufficient Sxl to trigger autoregulation once the maintenance promoter, SxlPm, had been activated (Avila, 2007).

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

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

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

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

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

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

The establishment of sexual identity in the Drosophila germline

The establishment of sexual identity is a crucial step of germ cell development in sexually reproducing organisms. Sex determination in the germline is controlled differently than in the soma, and often depends on communication from the soma. To investigate how sexual identity is established in the Drosophila germline, a molecular screen was conducted for genes expressed in a sex-specific manner in embryonic germ cells. Sex-specific expression of these genes is initiated at the time of gonad formation (stage 15), indicating that sexual identity in the germline is established by this time. Experiments where the sex of the soma was altered relative to that of the germline (by manipulating transformer) reveal a dominant role for the soma in regulating initial germline sexual identity. Germ cells largely take on the sex of the surrounding soma, although the sex chromosome constitution of the germ cells still plays some role at this time. The male soma signals to the germline through the JAK/STAT pathway, while the nature of the signal from the female soma remains unknown. The genes ovo and ovarian tumor (otu) are expressed in a female-specific manner in embryonic germ cells, consistent with their role in promoting female germline identity. However, removing the function of ovo and otu, or reducing germline function of Sex lethal, had little effect on establishment of germline sexual identity. This is consistent with findings that signals from the soma are dominant over germline autonomous cues at the initial stage of germline sex determination (Casper, 2009).

This analysis demonstrates that a sex-specific program of gene expression is present in the germline soon after the time of gonad formation [stage 15, ~12 hours after egg laying (AEL)]. Examples were identified of both male-specific germline genes and female-specific germline genes, with other genes being expressed equally in the two sexes. Therefore, it is likely that the sex-specific pattern of gene expression in the germline represents the establishment of true sexual identity in the germline, as opposed to other differences that might be observed in the germline between the sexes, such as proliferation status or transcriptional competence. It is concluded that sexual identity in the germline is established at least as early as stage 15. The observation that many genes examined initiate sex-specific germline expression at this time might indicate that this is when germline sexual identity is first established. However, it was also found that the soma signals to the germline in a sex-specific manner just prior to gonad formation (stage 13, ~10 hours AEL), indicating that germ cells could determine their sex even earlier. Because the zygotic genome is only robustly activated in the germline during gastrulation (stage 9, ~4 hours AEL), this sets a narrow time window during which sexual identity is established in the germline (Casper, 2009).

It is significant that germline sexual identity appears to be first manifested as the germ cells contact the somatic gonad. This is also the time that sexual dimorphism is first observed in the somatic gonad and that it exhibits sex-specific patterns of gene expression, Jheh2 and CG5149). The finding that germ cells might establish their sexual identity only as they contact the somatic gonad is consistent with the strong role of the soma in determining germline sexual identity. In addition, signaling from the germline back to the soma also occurs at this time (Kitadate, 2007; Casper, 2009).

The establishment of a sex-specific pattern of gene expression in the germline requires these cells to acquire germ cell identity, in addition to sexual identity. Previous work has established that germ cell-specific transcription is independent of the somatic environment, and is autonomously regulated in the germ cells. Indeed, it was seen that germ cell expression of the genes reported in this study can be independent of the somatic gonad. Germ cell-specific gene expression is likely to be regulated by the germ plasm, and several maternally expressed germline transcription factors have recently been identified (Yatsu, 2008; Casper, 2009).

Previously, it was known that sexual identity in the germline requires autonomous cues, along with non-autonomous cues from the surrounding soma, but little was known about how the signals work together to establish sexual identity. XX germ cells cannot develop normally in a male soma, and XY germ cells cannot develop normally in a female soma. Although evidence was found for both autonomous and non-autonomous regulation of germline sexual identity in the embryo, it appears that non-autonomous signals from the soma are dominant over germline autonomous cues. When XX germ cells are present in a male soma (tra mutants), they exhibit a clear increase in expression of male germ cell genes, and decreased expression of the female-specific gene otu. Similarly, XY germ cells present in a female soma (UAS-tra) exhibit a strong repression of male germ cell genes, while otu expression is increased. Thus, in each case the germ cells largely take on the sexual identity of the surrounding soma, independent of their own sex chromosome constitution. However, subtle differences in gene expression between XX and XY germ cells remain in these situations, indicating some germ cell autonomous control of sexual identity. In addition, when germ cells are present outside of the gonad (srp mutants), some differences are also observed between XX and XY germ cells. When outside of the gonad, XY germ cells are more likely than XX germ cells to express male-specific genes, and XX germ cells maintain some otu expression, while otu is largely off in XY germ cells. Thus, there is at least some autonomous contribution of the germ cell sex chromosome genotype to germline sexual identity at this early stage (Casper, 2009).

The genes of the ovarian tumor loci, ovo, otu, stil and Sxl, are thought to contribute to autonomous germline sexual identity by promoting female identity in XX germ cells. At this early stage, no change was observed in sex-specific germ cell gene expression in mutants for ovo, otu or stil, or in embryos with mutations that reduce the function of Sxl in the germline. Although this could indicate that these genes do not play a role in the initial establishment of germline sexual identity, these observations could also be due to the dominant effects of the soma on sex-specific germ cell gene expression at this time. These mutations would be expected to masculinize XX germ cells, causing them to exhibit a more male-like pattern of gene expression. However, even fully male (XY) germ cells exhibit a female pattern of gene expression when in a female soma. Thus, the dominant effect of the female soma might mask any masculinizing effects from the removal of ovarian tumor locus genes at this time (Casper, 2009).

Evidence is seen for at least two types of non-autonomous regulation of germline sexual identity by the soma, one coming from the male soma and the other from the female soma. Both XY and XX germ cells can activate male-specific gene expression when not in contact with the somatic gonad, but expression of these genes does not appear to be as robust as when germ cells are in contact with a male soma. Furthermore, when XX germ cells are in contact with a male soma, expression of the female germ cell gene otu is partially repressed. Thus, the male soma is required for full levels of male gene expression in the germ cells, and for the repression of female genes (Casper, 2009).

The signal from the male soma to the germline appears to be primarily, and perhaps exclusively, acting through the JAK/STAT pathway. Previously, it was shown that the male soma activates the JAK/STAT pathway in the germ cells, and that this is required for proper male germ cell behavior (Wawersik, 2005). This pathway is necessary for the proper male-specific proliferation of embryonic germ cells, and for the maintenance, but not the initiation, of male-specific mgm-1 expression (male germline marker-1, which appears to be an enhancer trap in the escargot locus). The JAK/STAT ligand UPD is also sufficient for the induction of germ cell proliferation and mgm-1 expression, and for partial induction of mcm5 and dpa expression. This study found that, when the JAK/STAT pathway is inactivated, sex-specific gene expression in the germline resembles that of germ cells that are not in contact with the somatic gonad. Ectopic expression of upd in females is able to induce some aspects of male-specific gene expression in XX germ cells, but not as robustly as when XX germ cells are present in a male soma. This is likely to be due to the fact that, when upd is expressed in an otherwise female soma, it is in competition with female signals that repress male gene expression. It is concluded that the JAK/STAT pathway is important for the regulation of germline sex determination by the male soma, and may be the primary or only signal from the male soma to the embryonic germ cells. It is essential for the male-specific pattern of germ cell proliferation, and for the robust initiation and maintenance of male-specific gene expression (Casper, 2009).

It is clear that the female soma also plays a key role in regulating the sexual identity of the germline. Both XX and XY germ cells exhibit some level of male-specific gene expression when outside of the gonad, but this expression is dramatically repressed when in contact with a female soma. This repression has also been observed with mgm-1. In addition, XY germ cells exhibit some female-specific otu expression when in contact with a female soma. Therefore, the female soma is essential for the proper sex-specific regulation of germline gene expression, although the nature of the signal from the female soma to the germ cells remains unknown (Casper, 2009).

The source of both the male and female somatic signals to the germline is likely to be the somatic gonad. The germ cells show signs of receiving the JAK/STAT signal only when in contact with male somatic gonad, and germ cells outside of the gonad do not appear to receive the proper sex-specific signals (e.g. srp mutants). Furthermore, it is known that somatic regulation of germline sex is controlled by the sex determination cascade, acting through tra and dsx. However, the only cells to express DSX within the embryo are part of the somatic gonad. Thus, the somatic control over germline sex determination is likely to represent local signaling within the gonad environment, rather than long-range signaling from other somatic cell types (Casper, 2009).

Somatic control over germline sex determination is a common feature of germ cell sexual development. In the mouse, germ cells first manifest sex-specific behaviors as they contact the somatic gonad in the genital ridge. The initial behavior of the germ cells is dependent on the sex of the soma, as male germ cells exhibit female behavior (early meiosis) when contacting a female soma, and female germ cells exhibit male behavior when in contact with a male soma. At least some aspects of this sex-specific behavior are regulated by retinoic acid (RA) levels controlled by the soma; female germ cells receive a high level of RA signal, but the male soma degrades RA so that male germ cells receive less of this signal. Interestingly, this study found that the somatic gonad in Drosophila expresses Jheh2 in a male-specific manner. JHEH2 hydrolyzes Juvenile Hormone (JH), which is structurally related to RA. JH analogs have also been observed to influence sex determination in crustaceans, which further supports this interesting parallel between vertebrates and invertebrates (Casper, 2009).

In mammals, the germline sex chromosome constitution is also important for germ cell development. As in Drosophila, mouse and human Y chromosome genes are crucial for spermatogenesis. However, the number of X chromosomes also appears to play an autonomous role in germline sexual development in mammals. XX germ cells in a male soma [e.g. in Sex reversed (Sxr) mice appear initially male and do not enter meiosis, but eventually die. XO germ cells in a male soma survive and progress further in spermatogenesis. Similarly, having two X chromosomes promotes female germ cell identity. XX germ cells are predisposed to enter meiosis on the female timetable compared with XO germ cells under the same conditions, and are biased towards a female pattern of imprinting. Thus, as in Drosophila, the germ cell genotype contributes to germ cell sex determination in the mouse and depends on the number of X chromosomes. Similarly, humans with altered sex chromosome constitutions, such as those with Turner's Syndrome (XO females) or Klinefelter's Syndrome (XXY males) have relatively normal somatic development but exhibit severe germline defects, indicating that the proper number of X chromosomes in the germline is essential for germline development (Casper, 2009).

However, in other species, the sex chromosome constitution of germ cells does not play an important role in germline sex determination. In the housefly, Musca domestica, transplanted germ cells develop normally according to the somatic sex of their host and produce fertile gametes. Some species also exhibit dramatic sexual plasticity in the germline, with the same individual being able to produce both sperm and egg, such as in hermaphrodites (e.g., C. elegans or in species that exhibit natural sex reversal. i.e., wrasses and gobies). Given the diversity of mechanisms animals use for establishing sex determination in the soma, it is not surprising that there might be considerable diversity in the germline as well. However, it appears that an important role for the soma in controlling sexual identity in the germline is a common theme in germ cell development. Whether or not the sex chromosome constitution of the germline is also important for fertility in an organism places additional constraints on the evolution of sex determination mechanisms, sexual plasticity and the sex chromosomes (Casper, 2009).

Drosophila RalA is essential for the maintenance of Jak/Stat signalling in ovarian follicles

Small GTPases of the Ras-like (Ral) family are crucial for signalling functions in both normal and cancer cells; however, their role in a developing organism is poorly understood. This study identified the Drosophila Ral homologue RalA as a new key regulator of polar-cell differentiation during oogenesis. Polar cells have a crucial role in patterning the egg chamber and in recruiting border cells, which undergo collective and guided migration. RalA function is essential for the maintenance of anterior and posterior polar-cell fate and survival. RalA is required cell autonomously to control the expression of polar-cell-specific markers, including the Jak/Stat ligand Unpaired. The loss of RalA also causes a cell non-autonomous phenotype owing to reduced Jak/Stat signalling in neighbouring follicle cells. As a result, border-cell assembly and migration as well as the polarization of the oocyte are defective. Thus, RalA is required in organizing centres to control proper patterning and migration in vivo (Ghiglione, 2008).

RalA shows a cell non-autonomous phenotype originating from the PC. PCs are essential anteriorly for recruiting a ring of around six outer border cells (oBCs) that make a mature BC cluster, which depends on the secretion of the Unpaired (Upd) ligand from the PC and subsequent Jak/Stat activation in the oBC. Secretion of Upd and binding to Domeless (Dome), the Drosophila Jak/Stat receptor, induces ligand-dependent internalization of Dome in cells surrounding PCs, both in BCs and posterior follicle cells. When PCs were mutant for RalA, Dome-containing endocytic vesicles were no longer observed, both anteriorly and posteriorly, suggesting that Jak/Stat signalling was not activated. In wild-type egg chambers, Stat is localized in the nucleus as a gradient, with higher levels of nuclear Stat close to the PC, thus reflecting Jak/Stat pathway activation. The nuclear localization of Stat was normal when PCs were wild type with adjacent follicle cells mutant for RalA, indicating that RalA does not have a role in the function of oBCs and posterior follicle cells in controlling Jak/Stat signalling. By contrast, in egg chambers with mutant PCs, Stat nuclear localization was completely abolished. To discriminate between a role of RalA in Upd expression or activity, egg chambers were stained using a Upd antibody, which shows a gradient of this ligand in egg chambers. Using this assay, it was shown that RalA mutations in PCs strongly affect the expression of the Upd protein (Ghiglione, 2008).

Does the reduction of Upd lead to non-autonomous defects posteriorly? It was shown previously that the Jak/Stat pathway is essential for specifying posterior follicle cells, which then signal back to the oocyte for anterior–posterior polarization. When polarity is normal, the Staufen protein forms a posterior crescent in the oocyte. In egg chambers containing posterior RalA mutant PCs, the localization of Staufen was not normal and it was found centrally in strongly affected oocytes, similar to mutants that fail to reorganize the microtubules (Ghiglione, 2008).

Among follicle cells, PCs have been shown to be important in patterning the egg chamber and in establishing BCs. This study identified RalA as a new key regulator of PC fate. RalA is essential both cell autonomously for maintaining PC differentiation and cell non-autonomously for patterning terminal follicle cells through Jak/Stat signalling. However, RalA mutations do not reproduce the full range of Jak/Stat mutations, consistent with the fact that some Upd is still produced by RalA mutant PCs. For example, the follicle cell markers MA33 and dpp-lacZ, the expression of which in stretched cells is controlled by Jak/Stat signalling, are expressed normally when PCs are mutant for RalA. Altogether, these data indicate that the function of RalA is essential for maintaining the PC fate and for ensuring high levels of Upd expression, which are required for patterning the most terminal follicle cells, including BCs and posterior follicle cells. The RalA phenotype suggests the existence of a maintenance signal taking place around stage 6-7 -- that is, following egg chamber proliferation phase -- which would be necessary to complete egg chamber patterning by providing sustained Jak/Stat activation (Ghiglione, 2008).

Previous studies have shown that Ral proteins interact with Sec5 and Exo84 to regulate the exocyst function during proliferation and tumorigenesis. The current in vivo study suggests a role for RalA in cell differentiation and patterning, independent of secretion. Not only Upd but also several non-secreted PC markers lose expression following RalA loss of function, reminiscent of a more general differentiation phenotype. Contrary to what would be expected of a secretion phenotype, the Upd protein does not accumulate within PCs that are mutant for RalA. The analysis of sec5 mutations in the follicle cells showed that this gene is required for the positioning of the oocyte and for follicle cell morphology, two phenotypes that were never observed in RalA mutant egg chambers. Finally, expression in BCs of RNA-mediated interference against the sec5, sec6, sec8 or sec15 genes did not show any phenotype (Ghiglione, 2008).

Thus, instead of showing a ubiquitous activity, the data indicate a cell-type-specific function for RalA in PCs, independent of secretion. Controlling the differentiation of PCs, which have a central organizing role through Jak/Stat ligand production, might represent a way to monitor the number of invasive cells during both normal development and tumour cell invasion. Interestingly, in mouse M1 myeloid leukaemia cells, Stat3 can activate Ral by controlling the expression of its exchange factor. These data suggest a conserved functional link between Ral proteins and Stat activity and provide a basis for the maintenance of Jak/Stat activity in PCs through a positive feedback loop involving RalA and Stat (Ghiglione, 2008).

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).

Niche appropriation by Drosophila intestinal stem cell tumours

Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. This study investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch(N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis (Patel, 2015).

This paper described a step-wise series of events during the earliest stage of tumour development in a stem cell niche. First, the combination of environmentally triggered mitogenic signalling and a mutation that compromises differentiation generates small clusters of differentiation-defective stem-like cells. Autocrine (Spi/EGFR) signalling between these cells then promotes their expansion into clusters, which quickly reach a size capable of physically disrupting the surrounding epithelium and driving the detachment and apical extrusion of surrounding epithelial cells (that is, ECs). This loss of normal cells seems to involve tumour cell/epithelial cell competition through integrin-mediated adhesion. Subsequently, the loss of epithelial integrity (specifically, EC detachment) triggers stress signalling (JNK, Yki/YAP) in the surrounding epithelium and underlying VM, and these stressed tissues respond by producing cytokines (Upd2,3) and growth factors (Vn, Pvf, Wg, dILP3). These signals are normally used within the niche to activate stem cells for epithelial repair, but in this context they further stimulate tumour growth in a positive feedback loop. It is noteworthy that in this example a single mutation that blocks differentiation is sufficient to drive early tumour development, even without secondary mutations in growth signalling pathways that might make the tumour-initiating cells growth factor- and niche-independent (for example, Ras, PTEN). Thus, tumour cell-niche interactions can be sufficient to allow tumour-initiating cells to rapidly expand, increasing their chance to acquire secondary mutations that might enhance their growth or allow them to survive outside their normal niche. This study highlights the importance of investigating the factors that control paracrine stem cell mitogens and survival signals in the niche environment. Tumour-niche interactions may be important to acquire a sizable tumour mass before the recruitment of a tumour-specific microenvironment that supports further tumour progression. A careful analysis of similar interactions in other epithelia, such as in the lung, skin or intestine could yield insights relevant to the early detection, treatment and prevention of cancers in such tissues (Patel, 2015).

Growth control in the Drosophila eye disc by the cytokine Unpaired

A fundamental question in developmental biology is how organ size is controlled. Previous studies have shown that the area growth rate in the Drosophila eye primordium declines inversely proportionally to the increase in its area. How the observed reduction in the growth rate is achieved is unknown. This study explored the dilution of the cytokine Unpaired (Upd) as a possible candidate mechanism. In the developing eye, upd expression is transient, ceasing at the time when the morphogenetic furrow first emerges. It was confirmed experimentally that the diffusion and stability of the JAK/STAT ligand Upd are sufficient to control eye disc growth via a dilution mechanism. It was further shown that sequestration of Upd by ectopic expression of an inactive form of the receptor Domeless (Dome) results in a substantially lower growth rate, but the area growth rate still declines inversely proportionally to the area increase. This growth rate-to-area relationship is no longer observed when Upd dilution is prevented by the continuous, ectopic expression of Upd. It is concluded that a mechanism based on the dilution of the growth modulator Upd can explain how growth termination is controlled in the eye disc (Vollmer, 2017).

EFFECTS OF MUTATION

Loss of zygotic os activity causes segmentation defects in the Drosophila embryo that resemble the phenotype of hopscotch and stat92E mutant embryos (Wieschaus, 1984). These defects always include loss of the fifth abdominal denticle band and the posterior mid-ventral portion of the fourth band. Defects in other segments are variable, but often include reduction of the second thoracic and eighth abdominal denticle bands and fusion of the sixth and seventh bands. In contrast to hop or stat92E (Perrimon and Mahowald 1986; Hou et al. 1996), zygotic os activity is essential but maternal activity is not, as evidenced by the lack of a maternal effect phenotype for os mutants (Eberl et al. 1992). The similarity between embryos that lack zygotic os and those that lack maternal hop or stat92E suggests that os is a component of the JAK signaling pathway. This hypothesis is further supported by genetic interactions between these genes. It has been observed previously (Perrimon, 1986) that certain allelic combinations of hop are viable, but have adult defects. The partial loss of hop activity in such animals causes reduced viability, held-down wings, reduced production of mature eggs, and/or defects in eggs produced. Each of the heteroallelic combinations results in a consistent and predictable degree of severity with respect to these phenotypes. To test whether the hop and os genes interact genetically, one copy of os was removed from animals carrying allelic combinations of hop. Altering the dose of os activity exacerbates the defects observed for these hop mutant combinations. Such enhancement is likely to occur if the two gene products are active in the same pathway (Harrison, 1998).

Strong alleles of unpaired are embryonic lethal, but weaker alleles show an outstretched (os) phenotype, resulting in adult flies with wings held out away from the body. Allelism of upd and os is based on the failure of zygotic lethal upd alleles to complement the wing phenotype of os alleles (Harrison, 1998 and Eberl, 1992). For example, combination of the embryonic lethal allele upd^YC43 with the viable allele os^o results in viable adult flies with outstretched wings (Harrison, 1998).

The JAK/STAT pathway is central to the establishment of planar polarity during Drosophila eye development. A localized source of the pathway ligand, Unpaired/Outstretched, present at the midline of the developing eye, is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror (Zeidler, 1999).

Given the known function of Upd as a JAK/STAT pathway ligand during Drosophila embryonic development, and the results showing that expressing Upd during eye development can mimic activation of the JAK/STAT pathway, it was decided to investigate whether Upd was likely to be acting as the endogenous JAK/STAT pathway ligand during eye patterning. Therefore, the time course of Upd expression during eye development was examined, using a polyclonal anti-Upd antibody. In first instar discs, only very weak expression is observed, in a faint horseshoe-shaped pattern around the poles and posterior of the disc. By second instar, expression is seen localized to the posterior margin of the disc, lying on the DV midline adjacent to the optic stalk. This pattern of expression persists into the third instar stage, however, by late third instar, an additional patch of staining is seen at the anteroventral margin of the eye disc adjacent to the junction with the antennal disc. Higher magnification views of an early third instar disc reveal that the Upd protein is highly expressed in only a small group of cells on the DV midline, where it can be seen cytoplasmically localized, but consistent with it being a secreted ligand, the protein is seen around the periphery of cells away from the site of expression in a concentration gradient toward the poles of the disc (Zeidler, 1999).

Considering the expression domain of endogenous Upd in the eye and the negative regulation of stat92E-lacZ by ectopically expressed Upd, it seems likely that the wild-type pattern of stat92E-lacZ is at least partly a consequence of endogenous Upd expression. The expression pattern of stat92E-lacZ in the third instar disc does not seem to be the exact inverse of the Upd expression pattern. However, it must be borne in mind that it is difficult to predict the exact stat92E-lacZ pattern that would be expected, because stat92E-lacZ is probably a lagging indicator of Upd repression (due to the perdurance of the beta-galactosidase gene product) and the cells in the posterior of the disc (behind the furrow) undergo fewer cell divisions, which would tend to distort the pattern seen. On the basis of this evidence, it has been proposed that the Upd expression at the optic stalk is sufficient to set up a gradient of JAK/STAT activation across the DV axis of the developing eye disc (Zeidler, 1999).

Further experiments were carried out to test the hypothesis that localized expression of Upd at the DV midline and the consequent gradient of JAK/STAT expression is important for normal eye patterning. The normal pattern of Upd expression is altered by ectopically expressing Upd in the developing eye imaginal disc using the GAL4/UAS system and the 30A-GAL4 line, which drives expression at the dorsal and ventral poles of the developing eye disc. In wild-type eyes, the array of facets appears externally regular, however, misexpression of Upd at the poles of the eye gives rise to adults in which the normal regular array of ommatidial facets is externally disrupted at both the dorsal and ventral poles of the eye. Sections through either the dorsal or ventral regions of such eyes show that although equatorially ommatidial polarity is normal, at the poles ommatidia have an inverted orientation. The inversions generated by misexpressing Upd in this manner do not form a regular and straight equator but rather appear to define a field of inversion with no clear boundary between dorsal and ventral ommatidial fates. This confused region may well represent the area in which ommatidia are responding to competing polarity signals, produced either by endogenous Upd expression at the optic stalk and Upd at the poles or possibly by other independent signaling mechanisms (Zeidler, 1999).

Next, the effect of completely removing Upd expression from the developing eye was investigated. Clones homozygous mutant for two independent Upd alleles were generated. These were recovered at high frequency and none of the clones analyzed (those which lay within the eye field) displayed any visible phenotype, either in imaginal discs or adult eyes. However, clones that overlapped the region of Upd expression adjacent to the optic stalk produced almost complete dorsalization of the eye field as assayed by ommatidial polarity. In an effort to better understand the ommatidial dorsalization observed following removal of Upd, the expression of mirr was examined. Wild-type mirr expression is restricted to the dorsal hemisphere of the eye, and via repression of fng results in activation of the Notch pathway at the DV midline, thereby defining the position of the endogenous equator. To investigate mirr regulation, P-element insertions in mirr were used that function as enhancer detectors and express both the lacZ gene and the P-element mini-white⁺ marker gene specifically in the dorsal half of the adult eye, henceforth referred to as mirr-lacZ. When mirr-lacZ is present in a background in which clones removing upd are generated, a low frequency of flies are recovered in which all or almost all of the eye field expresses the dorsal fate-specific white⁺ mirr reporter. When the eyes of flies showing this external dorsalization phenotype are sectioned, the dorsal fate of the ommatidia within the region ectopically expressing mirr is confirmed, although intriguingly, such eyes show occasional ommatidia still exhibiting ventral fate in the mirr-expressing region. Therefore, it is concluded that Upd expression at the optic stalk during normal eye patterning is required for restriction of mirr expression to the dorsal hemisphere of the eye. This restriction then determines the position of the equator along the DV midline of the eye disc via activation of Notch (Zeidler, 1999).

Given the role of Upd in restricting mirr expression, one possible mechanism by which JAK/STAT LOF clones might induce ectopic axes of mirror-image symmetry would be through the generation of ectopic boundaries of mirr expression. The expression of mirr-lacZ was examined in hop clones. Many clones lying both dorsally and ventrally were examined in eye discs, and in no case was an alteration in mirr-lacZ expression observed. Additionally, hundreds of adults carrying mirr-lacZ were examined, in which hop clones had been induced, and, again, in no case was a change in mirr-regulated white⁺ expression observed (Zeidler, 1999).

Thus, ommatidial polarity inversions generated by hop clones are mirr independent. It is therefore concluded that the process of midline equator definition by dorsally restricted mirr expression and the regulation of ommatidial polarity by the JAK/STAT pathway are separable processes. It is also noted that these results suggest that Upd might act independently of Hop to regulate mirr expression (Zeidler, 1999).

These results show that LOF JAK/STAT clones give nonautonomous inversions of ommatidial polarity on the polar clonal boundary, and that JAK/STAT activity is highest at the DV midline as a result of localized Upd expression. This situation is reciprocal to that seen for LOF Wg pathway clones that cause nonautonomous inversions of ommatidial polarity on the equatorial clonal boundary. An important question is whether the Wg and JAK/STAT pathways are acting independently to regulate ommatidial polarity decisions, or whether one acts through the other. Experiments were carried out to test directly whether Upd and Wg might regulate each other's expression. Ectopic Upd expression has no effect on Wg expression in the developing eye disc and also ectopic expression of Wg adjacent to the optic stalk does not alter Upd expression. Thus, Upd cannot be producing its phenotype via negative regulation of Wg and, similarly, Wg does not act via regulation of Upd. These results indicate that Wg and Upd do not regulate each other's expression, and, thus, that one of these pathways is not likely to be downstream of the other. Instead, it is surmised that Wg and Upd act in parallel to one another in the regulation of ommatidial polarity (Zeidler, 1999).

The ommatidial polarity phenotype produced by removal of JAK activity in mosaic clones has a number of important features: (1) the phenotype observed is an inversion of ommatidial polarity in which either the dorsal rotational form is seen in the ventral hemisphere of the eye or vice versa; (2) the phenotype is only observed on the polar boundary of the mosaic tissue; (3) the strength of the phenotype (in terms of the number of inverted ommatidia seen) is dependent on the size and shape of the clone; (4) the phenotype is cell nonautonomous as either fully mutant, fully wild-type, or as mosaic clusters that can manifest the phenotype (Zeidler, 1999).

From these characteristics, the following can be deduced: the nonautonomy of the phenotype produced by removal of the autonomously acting pathway component JAK, and its dependence on clone size and shape, suggests that JAK/STAT affects ommatidial polarity via a secreted downstream signal (which subsequently will be referred to as a second signal, most likely detected by Frizzled). The direction of the nonautonomy (only in a polar direction) and the strict DV nature of the polarity inversions indicates that this second signal must be graded in its activity along the DV axis, with a change in direction of the gradient at the equator. The direction of this gradient would then be the instructive cue to which ommatidia respond when rotating to establish their mature polarity (Zeidler, 1999).

Since Upd expression does not regulate Wg and vice versa, the possibility that these two pathways act sequentially can be excluded, and so it is proposed that they must act in parallel. An attractive possibility is that the Upd and Wg pathways might act in parallel to regulate the concentration of a single second signal. In this case, Upd expression at the DV midline would activate the signal and Wg expression at the poles would repress the signal. Adding together the effect of two such opposing signals produces a predicted second signal concentration that has a fairly even slope from the DV midline to the poles. In contrast, a single signal that is high at the DV midline and decays to zero at the poles has a very shallow gradient in the polar regions. Since reading the slope of a steep gradient is presumably easier than reading the slope of a shallow gradient, the use of two opposing gradients to set up second signal concentration is thus highly advantageous. Therefore, this presents a possible biological explanation for the proposed redundant use of both an Upd and a Wg concentration gradient in determining ommatidial polarity (Zeidler, 1999).

The most likely candidate for a receptor of the second signal is the seven pass transmembrane protein encoded by the frizzled (fz) locus. fz function is required in the presumptive R3/4 cells of the pre-ommatidium, and clonal analysis suggests that it interprets a gradient of positional information that is high at the equator and low at the poles. Recent results suggest that differential activation of Fz signaling in R3/4 results in asymmetric Notch activation in this photoreceptor pair, which ultimately leads to a binary cell-fate decision such that the cell closest to the equator takes on the R3 fate and the ommatidial unit as a whole adopts the correct rotational fate (Zeidler, 1999 and references).

The simplest model would be that there is a single second signal secreted from the equator, which is downstream of mirr/fng/Notch, and that Wg and Upd/JAK/STAT feed into this pathway upstream of Notch. This is consistent with the roles of Wg and Upd as regulators of mirr expression and, thus, in positioning the endogenous equator. However, it is not consistent with the observed ommatidial polarity inversions produced in the eye field both dorsally and ventrally by Wg-pathway and JAK/STAT-pathway LOF and GOF clones. These phenotypes indicate that second-signal concentration is dependent on Wg pathway and JAK/STAT pathway activity across the whole of the eye field, and thus the second signal cannot be only secreted from the DV midline as a consequence of localized Notch activation. It is conceivable that Notch is activated on the polar boundary of JAK/STAT LOF clones, but in this context the only known mechanism of Notch activation is via mirr/fng interactions, and this possibility has been ruled out (Zeidler, 1999).

Instead, the data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localize activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, becausefng LOF clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity (Zeidler, 1999).

Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal. A number of observations provide a great deal of support for such a model. (1) It is consistent with the known timing of the events involved. The requirement for fng function has been shown to lie between late first instar and mid second instar, which coincides with the first appearance of high levels of Upd expression at the optic stalk. However, the ommatidia are not formed (and thus do not respond to the polarity signal) until the start of the third instar, a stage when localized Upd expression still persists. Furthermore, extracellular Upd protein can be seen in a concentration gradient many cell diameters from the optic stalk at the early third instar stage, consistent with Upd being at least partly responsible for setting up the long-range gradient of JAK/STAT activity across the DV axis of the eye disc that is revealed by the stat92E-lacZ reporter. (2) This model does not require that a single source of second signal secreted by a narrow band of cells at the equator should be capable of determining ommatidial polarity across the whole of the DV axis of the disc during the third instar stage of development. Instead, the band of activated Notch at the equator would serve to draw a straight line between the fields of dorsally and ventrally polarized ommatidia, and need only secrete a localized source of second signal to polarize ommatidia in this region. Further from the equator, the opposing gradients of Upd and Wg signaling would provide a robust mechanism for maintenance of correct ommatidial polarity across the DV axis. Conversely, without the mirr/fng/Notch mechanism to draw a straight line, it would be impossible to imagine how Upd at the posterior margin and Wg at the poles alone could provide the perfectly straight equator that is ultimately formed. (3) The phenotypes that are observed are consistent with multiple competing mechanisms responsible for determining ommatidial polarity. When inversions of ommatidial polarity are induced by generating hop clones or ectopically expressing Upd, straight equators are not produced, such that two cleanly abutting fields of dorsal and ventral ommatidia are produced. Instead, there is usually some confusion of ommatidial identities as if they might be receiving conflicting signals. Additionally, when upd activity is removed from the optic stalk, an equator still forms (albeit at the ventral edge of the disc), but some ommatidia dorsal to the equator still adopt a ventral fate as if the determination of ommatidial polarity is less robust in the absence of Upd (Zeidler, 1999).

It is concluded that Upd is required to position the future equator via dorsal repression of mirr and localized activation of Notch (as a consequence of the restricted expression of the fringe (fng) gene product in the ventral half of the disc and Mirror in the dorsal half of the disc) at the DV midline. Upd also appears to regulate ommatidial polarity via activation of a gradient of JAK/STAT pathway activity and secretion of an unidentified second signal. Intriguingly, in both of these contexts, Wg secreted from the poles of the disc appears to cooperate with Upd. It will be interesting to see whether this represents a general mechanism for cooperation of these signaling pathways in pattern formation (Zeidler, 1999).

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

The two copies of XSEs present in XX individuals in Drosophila specify female development by transiently activating Sxl_Pe in the young embryo. A positive autoregulatory feedback loop acting on RNA splicing keeps Sxl active in females thereafter. Male development ensues in XY individuals because their single set of XSEs is insufficient to activate Sxl_Pe. Because Sxl controls the vital process of X-chromosome dosage compensation as well as sex determination, sexually inappropriate expression of Sxl is lethal. For example, simultaneous duplication of sisA and sisB kills males, as a female dose of these two XSEs in males causes Sxl to be expressed in its female mode, thereby reducing X-linked gene expression (Sefton, 2000).

Because XSEs act additively, males that would be killed by an excess dose of one group of XSEs can be rescued by compensating mutations that reduce the dose of other XSEs. A genetic screen based on this principle of additivity has generated five new mutations that define a new XSE, sisC. The first four sisC alleles recovered, including an apparent null, sisC¹, have no phenotype by themselves, even in trans to deficiencies of the region. In contrast, sisC ⁵, which is also null for sex-determination, exhibits phenotypes unrelated to sex: variably reduced viability (females more than males), female sterility and tergite defects. All mutations were mapped by recombination and deficiency analysis close to os based on their interactions with mutations in other XSEs (Sefton, 2000).

sisC and os are now shown to be the same gene, but this possibility seemed to have been excluded by the initial characterization of the putative sisC region. Df(1)os^1a seemsto be wild-type for sex-determination function. Moreover, construction of an os^ssisC¹ chromosome placed sisC centromere-proximal to os and the phenotype of the double mutant adults gives no hint that both lesions affect the same gene. Using DNA centromere-proximal to os, a DNA breakpoint has been identified for the atypical allele sisC⁵ precisely where the genetics had predicted sisC to be -- outside the region deleted by Df(1)os^1a; however, complications were suspected when no candidate sisC RNAs or other sisC lesions near this breakpoint could be found (Sefton, 2000).

The subsequent discovery that embryonic lethal os alleles (os^upd by convention) fail to complement sisC⁵ female sterility and tergite defects indicates that the non-sex-determination defects of this atypical sisC allele must be due to a different lesion that disrupts os slightly. The finding that embryonic lethal os alleles are deficient for XSE function indicates that this second lesion in sisC⁵ might also be responsible for the sex-determination defect. This finding coincides with the recovery of a candidate os complementary DNA (lambdaKZ-GR) (Sefton, 2000).

With this cDNA as a probe, the anticipated second change was found in sisC⁵, 100 kilobases (kb) centromere-distal to the first and within Df(1)os^1a, just upstream of the 5' end of lambdaKZ-GR. DNA sequencing has showen sisC⁵ to be a simple inversion, and allows the design of polymerase chain reaction (PCR) primers that will amplify only os ^upd DNA from sisC⁵/os^upd females. The discovery that os^upd-3 and os ^upd-4 are a 2-base-pair (bp) insertion at codon 143 and a C-to-T change causing a stop at codon 60, respectively, shows that lambdaKZ-GR corresponds to os (Sefton, 2000).

Characterization of os DNA for the other sisC mutants shows that sisC and os are the same gene and that the first sisC⁵ breakpoint and the Df(1)os^1a XSE test results were misleading. sisC ¹ is a 1.6-kb DNA insertion and a ~100-bp duplication just upstream of lambdaKZ-GR. All weaker sisC lesions also fall within os: sisC³ deletes 7 bp upstream of the translation start; sisC⁴ inserts tyrosine after alanine 128, and sisC² substitutes CGG for GTT 5 bp downstream of a 5' splice site (decreasing RNA splicing efficiency as determined by reverse transcription [RT]-PCR). The contradiction that Df(1)os^1a is sisC⁺ was resolved by showing that the Df chromosome carries a closely linked, maternal-effect suppressor. Mutations that only disrupt sex determination are now designated as os^sisC, but the locus is referred to as sisC when discussing os as an XSE (Sefton, 2000).

The 5' end of os transcripts was defined to guide construction of transgenes essential for showing the role of os in sex determination and for understanding sex-specific mutations in a non-sex-specific gene. 5' rapid amplification of cDNA ends (RACE) indicates two potential transcription start sites 1,316 and 1,439 bp upstream of the 5' end of lambdaKZ-GR, with the product for the +1,316 species terminating in a non-coding G that could correspond to the 5' methyl cap of a bona fide messenger RNA end. RNase protection shows that the two RACE products correspond to the major and minor os mRNA species present during the first half of embryonic development. Sequencing the RACE products, other RT-PCR products and genomic DNA reveal the true first exon and redefine the start of the exon designated II. The 5' end of lambdaKZ-GR may be artifactual, as it was not found in mRNA from embryos (Sefton, 2000).

Identification of these transcription start sites shows that sisC fits a pattern established for all other XSEs that control Sxl throughout the embryo: sisC has three copies of the sequence CAGGTAG less than 0.5 kb upstream of its first transcription start site (-242, -424 and -450 bp). Several conserved copies of this sequence or its complement have been found previously upstream of the transcription start sites for sisA, sisB and Sxl, leading to speculation that this sequence might be involved in the unusually early onset of XSE transcription (Sefton, 2000).

Three genomic transgenes were constructed with different amounts of 5' sequence but the same 3' end. Lines with 6.5 kb or 0.6 kb of genomic sequence upstream of the transcription start sites (P{sisC} 10 and P{sisC}5.8, respectively) provide high XSE function but only partially complemente os^upd lethals. In contrast, transgenes (P{sisC}4.8) lacking the transcription start sites and most of exon 1, but still containing 43 bp more uninterrupted 5' sequence than In(1)os^sisC-5, have no XSE and os activity. Hence, the significant level of os⁺ activity provided by In(1)os^sisC-5 despite its disrupted transcription start sites is unlikely to be due to an undiscovered endogenous os promoter, but rather to introduction of a foreign promoter providing activity sufficient only for non-sex-specific functions (Sefton, 2000).

The data show that os is an XSE. To merit XSE status, lowering the zygotic dose of the gene must decrease the probability of Sxl⁺ activation in females, but increasing the dose of the same gene must also increase that probability in males. The two larger sisC transgenes can compensate for mutations that lower perceived X-chromosome dose in females. Females simultaneously homozygous for a sisC null and heterozygous for a mutation in sisA would die but are rescued by the two larger transgenes. Line-to-line variation in rescue probably reflects the effects of different insertion-site positions. os⁺ transgenes can kill males by increasing perceived X-chromosome dose. Males were sensitized to increased sisC ⁺ by an increase in sisA⁺ dose. A double dose of sisC⁺ is more lethal to males than a single dose, reflecting the additive behaviour expected of XSEs. That this expected lethality is Sxl dependent is shown by the fact that Sxl ^- males are fully viable regardless of XSE dose. The effect of sisC⁺ transgenes is comparable to that of a chromosomal duplication of sisC⁺, Dp(1;3)JC153, but is smaller than seen for extra doses of sisA⁺ or sisB⁺ (Sefton, 2000).

As is true for other Drosophila XSEs, eliminating sisC activity reduces expression of Sxl_Pe, but less than eliminating sisA or sisB. Like sisA and sisB mutations, but unlike runt mutations, sisC mutations affect Sxl_Pe throughout the embryo. Mutations in the Drosophila JAK/STAT signaling pathway reduce expression of the Sxl 'establishment' promoter Sxl _Pe throughout the embryo. In non-mutant situations, most Sxl_Pe:lacZ females stain darkly and comprise the expected 50% of the progeny. The other 50% are males whose light staining matches that of embryos lacking P {Sxl_Pe:lacZ}. Most Df(1)os/os^- females stain lighter than os ⁺ female controls but darker than males, showing that os ^- generally reduces but does not eliminate Sxl_Pe expression. The reduction is not always uniform across the embryo. Any region could be affected, but anterior expression seemed to be reduced the least. The range of effects is considerable: fewer than 50% of the mutant embryos stained above background; some mutant females may not have expressed Sxl_Pe at all, but Sxl _Pe expression in a few others matched that of their os ⁺ sisters. In contrast to the Df(1)os/os^upd females, Df(1)os/os^sisC-1 females are fully viable, but they show a comparable reduction in Sxl_Pe expression. This observation confirms that os^sisC-1 is near null with respect to sex determination, but still supports normal development (Sefton, 2000).

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

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

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 hop^Tum, 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, Hop^Tum1, 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).

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).

Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and are specified by the activation of Notch. Their role in follicle patterning has been investigated by creating clones of cells mutant for the Notch modulator fringe. In the absence of fng or Notch function, polar cells do not form, and the requirement for these genes in polar cell fate is strictly cell autonomous. This genetic ablation of polar cells results in cell fate defects within surrounding follicle cells. At the anterior, the border cells, the immediately adjacent follicle cell fate, are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. These observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis. Only one gene, upd, is known that encodes for a signaling molecule that is expressed by polar cells. Although loss of upd, or other components of the JAK-STAT pathway, reduces the number of border cells, this contrasts markedly with the complete elimination of border cells observed in the absence of polar cells. Moreover, loss of upd does not have obvious effects on any of the other terminal cell fates that are polar-cell dependent. Thus, the existence of additional signaling molecules must be invoked to account for the organizing activity of the polar cells (Grammont, 2002).

domeless was identified using a screen for suppressors of an eye phenotype caused by overexpression of unpaired. Overexpression of upd using a UAS-upd and GMR-Gal4 driver causes compound eye dramatic overgrowth in the adult eye because of an increase in the number of ommatidia. The average number of ommatidia in the compound eye of UAS-upd/GMR-Gal4 female flies is 978 ± 10 compared with 745 ± 7 in wild-type flies. Histological sections through the overgrown eyes reveal that most ommatidia have normal photoreceptor cells and regular cell size, indicating that Upd activity mainly regulates cell proliferation in the compound eye. However, the ommatidia look more crowded and have irregular space and arrangement, and several big vacuoles are integrated into the ommatidia lattice. The severity of eye morphology appears proportional to the strength of the Hop/Stat92E-mediated signaling, because removing one copy of hop partially suppresses the big eye phenotype; the average number of ommatidia is 854 ± 9). The advantage of this sensitized system lies in the possibility of conducting a screen for mutations that reduce (suppressors) or increase (enhancers) the degree of eye size. It was reasoned that a twofold reduction in the dose of a gene (by mutating one of its two copies) that functions downstream of Upd should dominantly alter signaling strength, which, in turn, should visibly modify the eye size. Based on this assumption, available X-chromosome P-element insertion mutations were screened and one complementation group of suppressors with four alleles was identified at the cytological location 18E. Based on its presumed role in the Hop/Stat92E signal transduction pathway, this novel gene was named master of marelle (mom). The relative strength of four mom alleles in suppressing the UAS-Upd/GMR-Gal4 fly big eye phenotype is mom¹ > mom² >  mom³ = mom⁴, and mom¹ is the strongest allele. mom is indeed the same gene as domeless (Chen, 2002).

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

unpaired (upd) encodes a ligand for the Jak/STAT signaling pathway in Drosophila. In the second instar and early third larval eye disc, upd is expressed in the center of the posterior margin. upd loss-of-function mutations causes eye size reduction and upd overexpression causes eye enlargement. Upd regulates eye size through the Dome/Jak(Hop)/STAT92 signaling pathway to promote cell proliferation. Interestingly, the effect of Upd is only on cells located anterior to the morphogenetic furrow (MF), but has no effect on the second mitotic wave, which is posterior to MF. Overexpression of upd behind the MF can nonautonomously induce cell proliferation up to 20 rows of cells anterior to MF. The G1 cyclin, cycD transcript level is also enhanced anterior to MF. Consistent with the long-range effect, it was found that the extracellular Upd protein can be detected over a comparable long range, suggesting that Upd acts directly over a long distance as a signaling molecule (Tsai, 2004).

Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury

To characterize the features of JAK/STAT signaling in Drosophila immune response, totA was identified as a gene that is regulated by the JAK/STAT pathway in response to septic injury. Septic injury triggers the hemocyte-specific expression of upd3, a gene encoding a novel Upd-like cytokine that is necessary for the JAK/STAT-dependent activation of totA in the Drosophila counterpart of the mammalian liver, the fat body. In addition, totA activation is shown to require the NF-KB-like Relish pathway, indicating that fat body cells integrate the activity of NF-KB and JAK/STAT signaling pathways upon immune response. This study reveals that, in addition to the pattern recognition receptor-mediated NF-kappaB-dependent immune response, Drosophila undergoes a complex systemic response that is mediated by the production of cytokines in blood cells, a process that is similar to the acute phase response in mammals (Agaisse, 2003).

In order to identify genes that are regulated by the JAK/STAT pathway in response to septic injury in adult flies, a screen was performed for candidates that display an inducible expression upon immune challenge and that are constitutively expressed in flies carrying a gain-of-function mutation in the JAK/STAT pathway. To this end, custom-made cDNA microarrays were used to compare gene expression profiles of nonchallenged wild-type flies to gene expression profiles of challenged wild-type flies and to gene expression profiles of nonchallenged TumL flies displaying a gain-of-function mutation in the Drosophila JAK kinase Hopscotch. MP1 was identified as a gene that fulfilled both criteria for induction upon challenge and constitutive expression in a JAK/STAT gain-of-function mutation. MP1 expression was not induced in challenged flies displaying loss-of-function mutation in hop (hop^M38/hop^msv1), confirming the involvement of Drosophila JAK in MP1 expression (Agaisse, 2003).

Sequence analysis of MP1 cDNA reveals that MP1 codes for Turandot A (TotA), a polypeptide that is produced by the larval fat body and accumulates in hemolymph in response to various stress conditions in flies. totA expression is mainly fat body specific in adult flies. totA was weakly expressed in the fat body of unchallenged flies and strongly induced after septic injury (Agaisse, 2003).

Activation of the JAK/STAT pathway culminates in translocation of phosphorylated STAT dimers from the cytoplasm to the nucleus. In mosquitoes, it has been shown that AgSTAT, the homolog of Drosophila STAT, translocates into the nucleus of fat body cells in response to bacterial infection. To further confirm the activation of the JAK/STAT pathway in Drosophila fat body in response to septic injury, the subcellular location of STAT protein was examined in fat body cells by immunostaining. In unchallenged flies, STAT protein is located both in the cytoplasm and nucleus. In challenged flies, STAT substantially clears the cytoplasm and accumulates in the nucleus. In contrast, there was no STAT nuclear translocation in Drosophila JAK mutant adult flies. Conversely, a very strong staining was detected both in the cytoplasm and nucleus of flies carrying a Drosophila JAK gain-of-function mutation. These results demonstrate that totA activation correlates with the JAK-dependent activation of STAT in Drosophila fat body cells in response to septic injury (Agaisse, 2003).

A Drosophila homolog of the vertebrate cytokine class I receptor, Dome (a.k.a. Mom), has been identified. Mutations in dome result in embryonic defects similar to the embryonic phenotype associated with mutation in the JAK/STAT pathway components. A truncated version of Dome, DomeΔCYT, has been generated by deletion of the intracellular region that is involved in signal transduction. This mutated receptor still contains the extracellular cytokine binding module and acts as a signaling antagonist, probably by titrating the ligand. Accordingly, DomeΔCYT overexpression during embryogenesis mimics the loss-of-function phenotype of dome mutants. To test whether Dome plays a role in totA expression, the GAL4/UAS system was used to express the dominant-negative form of Dome, DomeΔCYT, in adult fat body. Northern blot analysis has revealed that totA expression upon immune challenge is totally abolished in the corresponding animals. It is concluded that totA activation in response to bacterial infection is the result of a signaling event that is transduced by the Drosophila homolog of the vertebrate cytokine receptor, Dome, in the fat body (Agaisse, 2003).

The involvement of dome in totA expression strongly suggests the existence of a cytokine-like molecule involved in the control of totA expression. upd has been characterized as a gene encoding the cytokine that activates the JAK/STAT pathway during Drosophila embryogenesis. Strong alleles of upd are embryonic lethal, but weaker alleles, such as outstrechted (os), give rise to adult flies that hold their wings at right angles and have small eyes. totA activation was found to be strongly decreased in os flies, suggesting that upd might be involved in totA expression. However, totA activation is nearly wild-type in transheterozygous flies displaying the os mutation over a null mutation in upd (updYM55), suggesting that a defect in upd expression is not responsible for the lack of totA activation in the os genetic background. Interestingly, totA activation is abolished in transheterozygous flies displaying the os mutation over a large deficiency (os1A) of the upd locus. This suggests that the os mutation affects the expression of a gene involved in totA activation that maps to the os/upd locus, but that is not upd. Blast search analysis reveals the presence of two other upd-like cytokine-encoding genes at the upd/os locus. upd2 corresponds to CG5988 and maps 50 kb downstream from upd. upd3 corresponds to CG15062 (for the first and second exon) and CG5963 (for the third exon) and maps 25 kb downstream from upd. These observations prompted a hypothesis that upd2 and/or upd3 might be involved in totA expression (Agaisse, 2003).

To further investigate the potential role of upd2 and/or upd3 in totA expression, whether the expression of the upd-like genes was inducible upon septic injury was examined. No upd2 expression was detected in adult flies by using RT-PCR analysis. In contrast, the level of upd3 expression, which was very low in control animals, was found to be significantly increased after septic injury. The pattern of GFP expression was examined in flies harboring a upd3 promoter region-GAL4 fusion and a UAS-GFP reporter. No GFP production was detected in fat body cells of control or challenged animals. However, GFP production was strongly increased in blood cells after challenge, indicating that hemocytes might be the main site of upd3 expression. To investigate the functional importance of upd3 hemocyte-specific expression in totA expression, an in vivo RNAi strategy was designed to silence upd3 expression in a tissue-specific manner. The hemolectin-GAL4 and the yolk-GAL4 constructs were used to drive the expression of the UAS-iupd3 hairpin construct in hemocytes and fat body, respectively. While the production of upd3 dsRNA in fat body did not interfere with totA expression, upd3 dsRNA expression in hemocytes led to a strong decrease in totA activation upon septic injury. Altogether, these experiments suggest that upd3 activation in hemocytes subsequently leads to totA activation in the fat body (Agaisse, 2003).

To further analyze the regulation of totA expression in fat body cells, totA expression was monitored in response to clean injury, septic injury with gram-negative bacteria (E. coli), or septic injury with gram-positive bacteria (M. luteus). Clean injury and septic injury with M. luteus resulted in a modest but significant induction of totA expression: 4-fold induction 6 hr after challenge and 7-fold induction 18 hr after challenge. In sharp contrast, septic injury with E. coli resulted in a robust induction of totA expression: 25-fold induction at 6 hr and 35-fold induction at 18 hr. Gram-negative bacteria therefore constitute the best inducer for totA expression. It is well established in flies that immune response to gram-negative bacteria is mediated by the Imd pathway through activation of TAK1 and the NF-KB-like transcription factor Relish. Therefore totA expression was analyzed in TAK1 and in relish mutant flies. totA activation after challenge was totally abolished in these mutants, indicating that, in addition to being JAK/STAT dependent, totA expression also requires the activity of the Relish pathway. Whether the activity of the Relish pathway is specifically required in the fat body was analyzed. To this end, relish dsRNA was overexpressed in fat body using the UAS-irel construct and the yolk-GAL4 driver. dsRNA-mediated silencing of relish expression leads to a failure in totA activation, indicating that Relish activity is specifically required in the fat body. Finally, whether Relish activation in the fat body is sufficient to activate totA expression was analyzed. Overexpression of Imd in fat body cells has been shown to lead to activation of Relish and therefore constitutive expression of the antimicrobial peptide genes, such as diptericin, in the absence of immune challenge. totA is not constitutively expressed in the corresponding flies, indicating that Relish activation is required in fat body but is not sufficient to activate totA expression (Agaisse, 2003).

Altogether, these results demonstrate that TotA qualifies as a bona fide acute phase protein. In addition, totC and totM are also controlled by the JAK/STAT pathway upon septic injury, a characteristic that is probably shared by all the members of the tot family. Moreover, the tot family members are not the sole target of the JAK/STAT pathway upon septic injury. Expression of CG11501, a cysteine-rich polypeptide related to scorpion toxin, is also controlled by the JAK/STAT pathway. These data indicate that the JAK/STAT pathway contributes to a global response upon immune challenge by controlling the expression of several acute phase proteins. As for most of their mammalian counterparts, the function of these Drosophila acute phase proteins is unclear. totA overexpression does not appear to protect NF-kappaB mutants, such as kenny, from gram-negative bacteria infection, indicating that TotA, unlike antimicrobial peptides such as Diptericin and Drosomycin, does not prevent bacterial growth. Altogether, these observations suggest that TotA is probably a general stress response factor involved in homeostasis of (damaged) tissues. Accordingly, it has been shown that TotA overexpression confers extended survival to flies subjected to heat stress, a treatment that certainly leads to disturbances of physiological homeostasis. Further in vivo characterization of acute phase protein function, such as TotA, using Drosophila as a model system will help gain an understanding of the overall physiology of the acute phase response in insects and mammals (Agaisse, 2003).

Upd was first identified as a secreted molecule that activates the JAK/STAT pathway during Drosophila embryogenesis. Evidence is provided for the existence of a component of the JAK/STAT pathway: Upd3 that is produced in hemocytes in response to immune challenge. Although cytokine-like activities, such as IL1 and TNFα, have been previously reported as being produced by hemocytes from Lepidopteran larvae in response to LPS stimulation, none of these activities have been shown to have a physiological function in vivo. upd3 is thus the first example of a gene coding for a cytokine that is expressed in hemocytes and is required for signaling in fat body. This study therefore constitutes the first demonstration that sentinel cells, such as hemocytes, play a signaling role in the Drosophila immune response. The nature of the signals that are detected by hemocytes and the signaling pathway(s) that trigger upd3 activation in response to septic injury remain to be determined. Preliminary experiments indicate that upd3 expression is severely impaired in TAK1 flies after septic injury, suggesting that components of the Relish pathway (as defined in fat body cells) might be involved in upd3 activation in hemocytes in response to bacterial infection. However, further analysis in PGRP-LC and relish mutant backgrounds was not consistent with this hypothesis. Clearly, the mechanisms involved in upd3 regulation potentially constitute a new paradigm for studying the signals and the transduction machinery involved in the control of gene expression in activated hemocytes (Agaisse, 2003).

Differing characteristics of the two Upd-like molecules of Drosophila

The characterisation of ligands that activate the JAK/STAT pathway has the potential to throw light onto a comparatively poorly understood aspect of this important signal transduction cascade. This study describes an analysis of the only invertebrate JAK/STAT pathway ligands identified to date, the Drosophila unpaired-like family. upd2 is expressed in a pattern essentially identical to that of upd and the proteins encoded by this region activate JAK/STAT pathway signalling. Mutational analysis demonstrates a mutual semi-redundancy that can be visualised in multiple tissues known to require JAK/STAT signalling. In order to better characterise the in vivo function of these ligands, a reporter based on a natural JAK/STAT pathway responsive enhancer was developed, and ectopic upd2 expression was shown to effectively activate the JAK/STAT pathway. While both Upd and Upd2 are secreted JAK/STAT pathway agonists, tissue culture assays show that the signal-sequences of Upd and Upd2 confer distinct properties, with Upd associated primarily with the extracellular matrix and Upd2 secreted into the media. The differing biophysical characteristics identified for Upd-like molecules have implications for their function in vivo and adds another aspect to understanding of cytokine signalling in Drosophila (Hombria, 2005).

Three unpaired-like genes have been identified by sequence homology searches within the 17A interval of the Drosophila X-chromosome. The founding family member upd has been molecularly characterised and its activation of the JAK/STAT signal transduction pathway is required for multiple developmental processes. In addition, a recent report has identified Upd3 as an infection specific cytokine produced by haemocytes in response to septic injury (Agaisse, 2003). However, no function has been proposed for upd2 and no analysis of the upd locus as a whole has been undertaken (Hombria, 2005).

To investigate the potential developmental roles of upd2 and upd3, their embryonic expression was analyzed. Although adult haemocytes have been shown to express Upd3 in response to bacterial challenge the only detectable expression of upd3 during embryogenesis is observed in the gonads from embryonic stages. By contrast, upd2 is expressed in the central region of the blastoderm, segmentally repeated stripes at stage 9, in the tracheal placodes at stage 10, and in a region within the hindgut and posterior spiracles from stage 11. Given the almost complete overlap between the upd2 expression pattern and that previously described for upd these results suggest that both upd2 and upd could be regulating embryonic development (Hombria, 2005).

Conceptual translation of the upd-like genes present in Drosophila melanogaster identifies three related proteins. Each of these proteins contains an N-terminal region representing a predicted signal- or anchor-sequence and multiple potential N-linked glycosylation sites. Glycosylation of these sites has been suggested to mediate the binding of Upd to the extracellular matrix (ECM). Analysis of Upd2 shows that it contains a strongly hydrophobic region from amino acid 30 to 55. However, using the SignalP 3.0 server, a hidden Markov model predicts the likelihood of Upd2 containing a signal sequence at only 28%, with a 25% confidence level of cleavage between positions 55 and 56. The same model also predicts an anchor sequence (at a 71% confidence level), a motif required to insert Type II, III and IV trans-membrane proteins into the endoplasmic reticulum (ER). By contrast, when Upd is analysed using the same hidden Markov model, a signal peptide is predicted (at 100% confidence level) with an 89% probability of cleavage between position 27 and 28. Strikingly, the Upd2-like molecules present in D. melanogaster, simulans and yakuba contain predicted anchor-sequences while more distantly related Upd2-like molecules include N-termini predicted to act as signal-sequences. This prediction therefore suggests that D. melanogaster Upd2 cannot be secreted by the 'classical' Golgi/ER-based secretion machinery and may be trapped as a trans-membrane protein within the ER (Hombria, 2005).

All related Drosophilid species for which genome sequence is available, encode clear homologues of all three upd-like genes. However, no unambiguous upd-like homologues are identifiable in more distantly related species including the fellow dipteran Anopheles gambiae. It therefore appears that the upd gene family is evolving rapidly in the Drosophilids (Hombria, 2005).

Although the N-terminus of D. melanogaster Upd2 is predicted to function as an anchor-sequence, the true nature of this signal ultimately requires experimental validation. Therefore C-terminal fusions of both Upd and Upd2 to enhanced GFP were generated to allow the direct visualisation of the resulting proteins. When expressed in S2 Drosophila tissue culture cells, UpdGFP appears to be present extracellularly around transfected cells (identified by their co-expression of nuclear localised mRFP). This extracellular GFP is only detectable in the most basal confocal sections and appears to be associated with the substrate on which the cells grow. This is consistent with previous results that identify Upd as an ECM-associated protein. By contrast, fluorescence associated with Upd2GFP expressing cells appears to be intracellular with particular accumulations surrounding the nucleus in structures that may represent the endoplasmic reticulum. Although no pattern comparable to the surrounding ECM-associated halo of UpdGFP protein is detected, very low levels of extracellular Upd2GFP are occasionally detected adjacent to Upd2GFP transfected cells. It therefore appears that Upd2 cannot associate with the ECM in a manner comparable to Upd (Hombria, 2005).

Whether the dissimilar signal-/anchor-sequences could explain the observed differences of Upd and Upd2 secretion was examined. Domain swap experiments were undertaken to determine the individual contributions of the signal-/anchor-sequences for the activity of Upd and Upd2. GFP-tagged fusion molecules consisting of the secreted portion of Upd2 joined to the signal sequence of Upd (termed Upd2SS1) and another comprising the Upd2 anchor sequence attached to the secreted portion of Upd (called Upd1SS2) were constructed. Expression in S2 cells showed that Upd2SS1 can now be visualised as an ECM-associated halo surrounding transfected cells while Upd1SS2 appears to be located exclusively intracellularly. These results appear to indicate that the nature of the signal-/anchor-sequences present is responsible for the differential ECM interactions of UpdGFP and the low levels of extracellular Upd2GFP detected. In addition, the extracellular halo of Upd2SS1 indicates that the secreted region of Upd2 is capable of associating with the ECM under these tissue culture conditions (Hombria, 2005).

Upd and Upd2 induced JAK/STAT activation can be assayed using transcriptional reporter systems in tissue culture cells. To undertake such experiments, a 6x2xDrafLuc reporter transgene containing twelve STAT92E binding sites located upstream of the gene encoding firefly Luciferase was used in the haemocyte-like Kc₁₆₇ cell line. Stimulation of endogenous JAK/STAT pathway activity by ectopic expression of Upd has been shown to result in a strong 6x2xDrafLuc response dependent on endogenously expressed pathway components. This study showed that Upd2 can act as a potent activator of JAK/STAT signalling. Additional studies showed that Upd2 is unlikely to be ECM associated and is probably a freely diffusible pathway ligand (Hombria, 2005).

To test if upd2 can activate the JAK/STAT pathway in vivo, reporters of JAK/STAT activation were sought. The only rigorously analysed target of STAT, the even-skipped stripe 3 enhancer, is not useful to test induced STAT activation, as it is under negative regulation by gap genes. While searching for an alternative reporter it was observed that the dome gene might contain enhancers regulated by STAT. (1) dome mRNA expression increases after stage 11 in areas where upd is expressed: the pharynx, the hindgut, the tracheae and the posterior spiracles. (2) lacZ expressed by heterozygous P-element enhancer traps inserted in the dome 5'UTR show the same areas of elevated expression. (3) When hemizygous (and therefore causing a dome mutation), these same P-elements no longer up-regulate expression in these areas indicating that JAK/STAT activation through dome is required for dome up-regulation. Consistent with these observations, potential STAT binding sites were found in the 5'UTR and first intron of dome. Reporter constructs made using a 2.8-kb genomic fragment containing part of the first exon and most of the first intron activate lacZ expression in the pharynx and hindgut mesoderm, two of the areas expressing increased dome mRNA levels. Double RNA in situ/antibody staining shows that upd RNA is expressed in the ectoderm of the pharynx and in the hindgut, in a region adjacent to the mesoderm cells expressing the reporter construct. Given its mesoderm specific expression, the 2.8-kb construct will be referred to as dome-MESO (Hombria, 2005).

To determine if dome-MESO is regulated by JAK/STAT signalling, its expression was studied in different JAK/STAT mutant backgrounds. In dome⁹ mutant embryos, dome-MESO is not activated in either the pharynx or the hindgut, showing that dome-MESO regulation is comparable to that of the endogenous dome. The same result is observed in Df(1)os1A embryos lacking all Upd-like ligands. Whether pathway activation is sufficient to drive dome-MESO expression was tested. Gal4 mediated expression of upd or the activated form of the JAK kinase Hop^Tuml using the 24B-Gal4 or the twi-Gal4 mesodermal drivers results in ectopic dome-MESO expression both in the visceral and somatic mesoderm. Also, whether signalling from the ectoderm could non-autonomously activate mesoderm specific expression of dome-MESO was tested by using the ectodermal specific line 69B-Gal4 to express upd. Under these conditions, dome-MESO was activated in the mesoderm, with the salivary glands being the only non-mesodermal tissue induced. As a control Hop^Tuml was also expressed with 69B-Gal4. In these conditions dome-MESO was not ectopically activated in the mesoderm, confirming the ectodermal specific expression of this Gal4 line. These results show that the dome-MESO enhancer is a mesodermal specific reporter for JAK/STAT activation that mimics the behaviour of endogenous dome. These results also provide evidence of JAK/STAT signalling across germ layers and demonstrates the existence of a positive feed back loop in dome regulation (Hombria, 2005).

Having established the dome-MESO reporter as a useful tool to show the status of JAK/STAT pathway activity in mesodermal cells, whether both Upd and Upd2 have similar functions was tested in vivo. As would be expected of a bona fide pathway ligand, Upd2 expression in the mesoderm using 24B-Gal4 or in the ectoderm using 69B-Gal4 results in strong ectopic mesodermal dome-MESO activation. Upd2 is therefore able to non-autonomously activate JAK/STAT signalling in vivo (Hombria, 2005).

The expression of vvl, a gene whose expression in the anterior part of the hindgut ectoderm has been shown to depend on JAK/STAT pathway activity, was tested. Pathway induction driven by expression of either Upd or Upd2 with the 69B-Gal4 line, is sufficient to expand the domain of vvl within the hindgut. The morphological effect of Upd2 activation on germ band movements was studied; as reported for Upd, Upd2 is also sufficient to block germ band retraction (Hombria, 2005).

Finally, whether upd and upd2 expression can exert the same effects when expressed in the imaginal discs was tested. JAK/STAT pathway activation is sufficient to modulate cellular proliferation during the development of the wing imaginal disc. Using the Bx-Gal4 (also known as 1096-Gal4) driver line, either upd or upd2 was expressed in wing imaginal discs. In both cases, the resulting adults developed reduced size wings (Hombria, 2005).

All these studies taken together indicate that ectopic expression of Upd2 is sufficient to reproduce all effects caused by misexpression of Upd and suggest that both molecules are likely to activate the same pathway (Hombria, 2005).

To analyse if both ligands activate the JAK/STAT pathway through the Dome receptor, whether the wing defects caused by the ectopic misexpression of Upd and Upd2 could be rescued by simultaneous expression of dominant negative versions of the Dome receptor was tested. Having established that misexpression of dominant negative Dome alone has only mild effects, Upd or Upd2 and the dominant negative receptors were coexpressed. Expression of both transgenes resulted in an almost complete rescue of the Upd/Upd2 mediated wing defects, indicating that both Upd and Upd2 induce their effect via Dome. A similar blockage of Upd2-induced signalling is also observed in tissue culture-based assays by RNAi-induced knock down of dome mRNA (Hombria, 2005).

Since both the loss-of-function analysis and the ectopic expression tests indicate that Upd2 and Upd can act as partially redundant JAK/STAT ligands, the capacity of each ligand to rescue the absence of all other upd-like ligands was tested. For that purpose, focus was placed on the requirement of the JAK/STAT pathway for spiracle morphogenesis. Mutations in any of the components of the JAK/STAT pathway result in a highly abnormal posterior spiracle. The Klu-Gal4 spiracle driver line was used to test rescue of the Df(1)os1A spiracle defect. As a positive control, the expression of the activated JAK kinase was induced; under these conditions, UAS-hop^Tuml results in a partial rescue of the spiracle phenotype. Very similar rescues where obtained when expressing either UAS-upd or UAS-upd2 thus confirming that Upd and Upd2 represent redundant ligands in vivo (Hombria, 2005).

Given the sequence similarity of the upd-like genes and their clustering within the genome, it appears that this region has undergone two genomic duplication events; a similar scenario has been proposed to explain the functional redundancy of the proneural ac and sc genes. Since all available Drosophilid genomes encode all three upd-like genes, it is likely that the duplication events occurred before the radiation of the Drosophila family. However, no plausible upd-like genes were identified in the genome of the fellow dipteran Anopheles gambiae, suggesting that the Upd ligand is undergoing a phase of rapid evolutionary change. This conjecture is supported by the restriction of the upd3 expression pattern and its apparent specialisation to roles in immune signalling as well as the apparent divergence of the N-terminal signal-/anchor-sequences of Upd2 molecules in the closely related D. melanogaster, simulans and yakuba (Hombria, 2005).

Although negative autoregulation of dome in the follicle cells has been suggested to occur through two STAT binding sites 12 kb upstream of dome, this study shows that positive dome autoregulation in the mesoderm is driven by intronic regulatory sequences used to generate the dome-MESO reporter gene. Expression of dome-MESO requires the function of the JAK/STAT pathway and can be induced by its ectopic activation. The reporter includes several putative Drosophila STAT binding sites and future experiments should confirm molecularly if the autoregulation is direct. In any case, these results show that dome-MESO is a useful tool to test the state of activity of the pathway in embryos and that, in the mesoderm, JAK/STAT induces positive autoregulation of dome. In addition to the mesodermal up-regulation, dome mRNA expression also increases in the posterior spiracles and in the ectoderm of the pharynx and hindgut close to where upd is expressed, suggesting the existence of another ectoderm-specific positive autoregulatory element. It is interesting to speculate that several dome tissue-specific enhancers exist to modulate the strength of JAK/STAT signalling by increasing or decreasing the amount of receptor. These changes in Dome levels would either amplify the reception of ligands in the embryonic mesoderm and ectoderm (pharynx and hindgut), or act to down-regulate the signal in cases where only transient pathway activity is desired (such as in the follicle cells) (Hombria, 2005).

Although tissue culture-based secretion assays indicate that Upd2 is only weakly associated with the ECM immediately surrounding expressing cells, the ability of Upd2 to condition media indicates that the molecule is secreted and active under these conditions. This result is further supported by the mesodermal induction of the dome-MESO reporter following ectoderm specific expression of Upd2 (Hombria, 2005).

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

Striking similarities continue to emerge between the mammalian and Drosophila JAK/STAT signaling pathway. However, until now there has not been the ability to monitor global pathway activity during development. A transgenic animal was generated with a JAK/STAT responsive reporter gene that can be used to monitor pathway activation in whole Drosophila embryos. Expression of the lacZ reporter regulated by STAT92E binding sites can be detected throughout embryogenesis, and is responsive to the Janus Kinase hopscotch and the ligand Upd. The system has enabled identification of the effect of a predicted gene related to upd, designated upd2, whose expression initiates during germ band extension. The stimulatory effect of upd2 on the JAK/STAT reporter can also be demonstrated in Drosophila tissue culture cells. This reporter system will benefit future investigations of JAK/STAT signaling modulators both in whole animals and tissue culture (Gilbert, 2005; full text of article).

To visualize global JAK/STAT pathway activation in the whole animal a transgenic Drosophila line containing the lacZ gene regulated by STAT DNA binding sequences. This construct included three STAT target sites (GAS) upstream of a minimal Drosophila heat shock promoter in the pCaSpeR.hsp.bas vector. This reporter gene and the Drosophila line is referred to as (GAS)₃-lacZ. Expression during embryogenesis is strikingly dynamic (Gilbert, 2005).

To evaluate expression of the reporter gene, in situ hybridization was performed to detect the lacZ mRNA transcript in homozygous (GAS)₃-lacZ embryos during various stages of development. Expression of the lacZ gene is not detectable in syncytial blastoderm embryos. However, at the onset of cellularization, lacZ mRNA is detected throughout the embryo with strongest expression in the ventral region. Just prior to gastrulation, expression becomes more spatially restricted. At the onset of gastrulation, an intense lacZ signal is detected in a broad region anterior to the presumptive cephalic furrow and invaginating presumptive mesoderm. As germ band extension proceeds, expression is reduced, but reappears by early stage 9 in the head region and as a weak 14 stripe pattern. By stage 10 this pattern resolves into strong expression in 14 parasegments. The lacZ signal then recedes and is detected in small clusters of segmentally repeated cells. These data indicate that STAT binding sites within a promoter context can drive expression of a reporter gene in the Drosophila embryo. Homozygous Drosophila were also generated that contain a lacZ transgene regulated by a single GAS element. The (GAS)₁-lacZ embryos exhibited a similar pattern of gene expression but with significantly weaker intensity (Gilbert, 2005).

To ensure that expression of the reporter system was in fact responsive to JAK/STAT activity, (GAS)₃-lacZ expression was evaluated in embryos lacking maternal hop. These hop embryos have also been shown to exhibit reduced STAT92E protein levels. Maternal hop was removed using the FLP-DFS technique to generate females with hop^C111 homozygous germ cells. These females were crossed to (GAS)₃-lacZ males to generate embryos that lack maternal hop and carry a single copy of the (GAS)₃-lacZ transgene. Embryos were evaluated for expression of lacZ transcript by in situ hybridization. Expression was found to be dramatically reduced in all stages examined. During early cellularization only a weak anterior signal is detected with little or no signal in the remainder of the embryo. At the onset of gastrulation, the embryos exhibited reduced expression throughout, particularly in the area corresponding to mesoderm. During germ-band extension, the expression in 14 parasegments was reduced with residual signals in small clusters of cells at the midline of the original 14 stripes. The residual activity detected may be hop independent. stat92E null alleles were also tested and showed residual activity. These results indicate that the (GAS)₃-lacZ reporter system is responsive to a reduction of in vivo levels of JAK/STAT pathway components, and it was confirmed that early JAK/STAT signaling is established in cellularizing embryos in a ubiquitous manner (Gilbert, 2005).

The result suggests that the presence of Upd ligand alone is not sufficient to activate the pathway. In addition, when Upd was ectopically expressed with the paired-Gal4 driver, (GAS)₃-lacZ was hyperactivated, but only in a subset of cells. These data support the finding of the inability of ectopic ligand to induce Domeless dimerization (Brown, 2003), and hence pathway activation in early embryos (Gilbert, 2005).

The removal of maternal hop by the production of germline clones had a clear inhibitory effect on the establishment of (GAS)₃-lacZ expression in early cellularized embryos, gastrulation, and the maintenance of reporter expression during germ-band extension. Since loss of maternal hop has been shown to negatively regulate STAT92E protein levels, it was expected that this loss of function allele would cause the strongest loss of (GAS)₃-lacZ expression. However, all three stages displayed some residual expression. Given that the hop^C111 mutation in the germ line clone corresponds to a small internal deletion, it is possible that the residual reporter expression is due to low activity of a mutant Hop protein. The possiblility that residual expression is due to the activity of an unknown DNA binding factor that can interact with the reporter gene cannot be ruled out. It is also possible that there is minimal but constitutive activity of the promoter used in the construction of the (GAS)₃-lacZ gene. The presence of a CAAT box and a TATA box could facilitate a low level of expression by the basal transcriptional machinery (Gilbert, 2005).

The contribution of upd, upd3 and upd2 to JAK/STAT signaling during embryo development was also evaluated. The removal of upd and upd3 or upd, upd3 and upd2 significantly decreased pathway activity. The effect was similar to the removal of maternal hop during the establishment of (GAS)₃-lacZ expression in cellularized embryos. The earliest developmental stage examined for both deficiencies is slightly later than that examined for hop embryos. Given the slight difference in staging, the residual expression is similar, consisting of a weak head stripe and 2 weak stripes in the trunk. The maintenance of (GAS)₃-lacZ expression in germ-band extended embryos was also analyzed. The removal of both upd, upd3 and upd2 had a more severe effect on reporter gene expression than removal of upd and upd3 alone. This increase in severity was manifest as a reduction in the number of expressing cells within the segmentally repeated cell clusters (Gilbert, 2005).

These studies performed both in vivo and in Drosophila tissue culture cells provide evidence that Upd2 can act to stimulate activation of the JAK/STAT pathway. Since the expression pattern of both upd and upd2 is similar during germ-band extension, it is possible that they serve certain biologically redundant functions similar to the manner in which IL-6 cytokines function in mammalian systems. These are pleiotropic cytokines that share structural similarity and functional redundancies in part due to the fact that they share a common receptor subunit. Alternatively, signaling by Upd and Upd2 may serve specific functions either in the embryo or during other stages of larval, pupal, or adult development. In monitoring upd2 expression by Western blot analysis, multiple isoforms were detected that may indicate post-translational modifications. The nature of the Upd2 proteins remains to be characterized and could provide insight on additional levels of signaling specificity and receptor binding. Upd2 may not be associated with the extracellular matrix like Upd and thereby able to act at a greater distance from its production to influence gene expression. The in vitro studies in Drosophila S2 cells clearly demonstrated the ability of Upd2 to stimulate specific expression of (GAS)₃-lacZ (Gilbert, 2005).

This report provides evidence that JAK/STAT signaling can be monitored in vivo using the lacZ reporter gene regulated by STAT DNA binding elements. A complementary assay has recently been developed to monitor the pattern of STAT92E phosphorylation in the embryo, however this method of detecting lacZ by in situ hybridization provides a highly sensitive assay with little background to detect pathway activation from the cell surface receptor to gene expression in the nucleus. In addition, dynamic expression of the reporter can be visualized by a simple X-gal staining of whole embryos and remains sensitive enough to allow detection of changes in reporter activity in response to the removal and ectopic expression of pathway components. This capability could facilitate a genetic screen for enhancers or suppressors of JAK/STAT pathway activity during specific developmental stages. In addition, since (GAS)₃-lacZ can be used to monitor JAK/STAT activation in tissue culture cells, this could facilitate a screen in tissue culture cells as well as providing a method of verifying screen-based genetic interactions. The reporter line should also be useful to characterize JAK/STAT function during later developmental stages. Preliminary experiments with X-gal staining of (GAS)₃-lacZ third instar larval structures revealed β-galactosidase activity in a subset of structures known to require or possess competence for JAK/STAT signaling (Gilbert, 2005).

Until now direct monitoring of JAK/STAT pathway activation has only been possible in tissue culture cells. The establishment of an in vivo monitor of JAK/STAT pathway activation will provide an indispensable tool for the discovery of interacting proteins and tissue-specific requirements during Drosophila development. This assay can be used to visualize pathway activation and identify novel regulators of JAK/STAT signaling during embryogenesis and the isolation of a novel gene, upd2, which bears sequence homology to upd and encodes a functional ligand of the JAK/STAT pathway is specifically described. These data add to the mounting evidence that suggests the Drosophila JAK/STAT pathway is not simple, but contains multiple ligands that may act to elicit tissue and gene-specific responses (Gilbert, 2005).

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

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

Analysis of phenotypes associated with mutations in Drosophila JAK/STAT pathway components have identified a wide variety of requirements for the pathway during embryonic development and in adults. What is less clear is how the repeated stimulation of a single pathway is able to generate this pleiotropy of developmental functions. In order to identify modulators of JAK/STAT signaling that may be involved in this process, a genetic screen was undertaken for modifiers of the dominant phenotype caused by the ectopic expression of the pathway ligand Unpaired (Upd) in the developing eye imaginal disc. Such misexpression by GMR-updΔ3′ results in overgrowth of the adult eye, a phenotype sensitive to the strength of pathway signaling activity. With this assay, one genomic region, defined by Df(2R)Chi^g320, was found to enhance the GMR-updΔ3′-induced eye overgrowth phenotype. Of the genes deleted by Df(2R)Chi^g320, only mutations in ken showed consistent and reproducible enhancement of the phenotype. In addition, other dominant phenotypes induced by transgene expression from the GMR promoter are not modulated by ken mutations, indicating that Ken is unlikely to interact with the misexpression construct used (Arbouzova, 2006).

The enhancement of the GMR-updΔ3′ phenotype after removal of one copy of ken implies that Ken normally functions antagonistically to JAK/STAT signaling. Therefore phenotypes associated with mutations in other pathway components were tested to establish the reliability of this initial observation. Consistent with this, genetic interaction assays between ken mutations and the hypomorphic loss-of-function allele stat92E^HJ show a reduction in the frequency of wing vein defects normally associated with this stat92E allele. Moreover, the degree of suppression is consistent with the strength of ken alleles tested. Similarly, the frequency of “strong” posterior spiracle phenotypes caused by the dome³⁶⁷ allele of the pathway receptor is also reduced when crossed to ken alleles or the Df(2R)Chi^g320 deficiency, with a concomitant increase in “weak” phenotypes (Arbouzova, 2006).

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

The ken locus contains three exons encoding a 601 aa protein. Ken possesses an N-terminal BTB/POZ domain between aa 17 and 131 and three C-terminal C2H2 zinc finger motifs from aa 502 to 590. Strikingly, a number of Zn finger-containing proteins that also contain BTB/POZ domains have also been shown to function as transcriptional repressors—often via the recruitment of corepressors such as SMRT, mSIN3A (see Drosophila Sin3A), N-CoR, and HDAC-1 (Arbouzova, 2006).

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

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

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

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

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

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

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

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

Having established that Ken functions at the level of DNA binding in cell culture, it was asked whether Ken also acts as a transcriptional repressor of JAK/STAT pathway target genes in vivo. For this, the effect of ectopically expressed Ken on the expression of putative JAK/STAT pathway target genes was examined and, given the high levels of maternally loaded STAT92E present at blastoderm stage, focus was placed on targets expressed later in embryogenesis. These include the hindgut-specific expression of vvl, the expression of trachealess (trh) and knirps (kni) in the tracheal placodes, and the dynamic expression of socs36E throughout the embryo (Arbouzova, 2006).

First, the effect of Ken was addressed on trh, whose expression precedes the formation of the tracheal pits in the embryonic segments T2 to A8. Levels of trh are greatly reduced in embryos uniformly misexpressing Ken driven by the daughterless-GAL4 (da-GAL4) line. Many tracheal placodes express little or no trh, and tracheal pits fail to form even in the presence of residual trh. Similar effects are seen in upd^OS1A mutant embryos lacking all pathway activity. Likewise, downregulation of Kni expression is also observed in embryos misexpressing ken. These results show that both endogenous trh and kni are downregulated by ectopically expressed Ken (Arbouzova, 2006).

Whether Ken can modulate the expression of socs36E, a Drosophila homolog of mouse SOCS-5, was tested. socs36E expression closely mirrors that of upd, showing JAK/STAT pathway-dependent upregulation in segmentally repeated stripes, tracheal pits, and the hindgut. By contrast to trh and kni, ectopically expressed Ken does not affect any aspect of socs36E transcription. However, controls expressing a dominant-negative form of the pathway receptor DomeΔCyt, using the same Gal4 driver line, show a strong downregulation of socs36E, an effect reproduced by the complete removal of all JAK/STAT pathway activity by the upd^OS1A allele. Taken together, these results illustrate that ectopic expression of Ken during Drosophila development is sufficient to downregulate the expression of only a subset of putative JAK/STAT pathway target genes (Arbouzova, 2006).

As part of this analysis, modulation of vvl by Ken was tested. In wild-type embryos, vvl is expressed in the developing trachea and lateral ectoderm (in a JAK/STAT-independent manner) and in the hindgut of stage 12–14 embryos, where it requires JAK/STAT signaling. In upd^OS1A mutants, no vvl expression in the hindgut can be detected, indicating that this locus is a target of pathway activation. When Ken is uniformly misexpressed throughout the embryo, vvl expression is no longer detectable in the hindgut. Thus vvl, like trh and kni, can be a target of Ken-mediated repression (Arbouzova, 2006).

Having established that ectopic Ken is sufficient to downregulate vvl in the hindgut, whether endogenous Ken performs a similar role was determined. One overlap between ken expression and regions known to require JAK/STAT signaling are the developing posterior spiracles, structures in which both the pathway ligand upd and ken are simultaneously expressed. However, vvl is never detected in the posterior spiracle primordia in wild-type embryos, despite JAK/STAT pathway activity induced by upd expression in these tissues. Intriguingly, in a heteroallelic combination of the strongest ken^k11035 allele and Df(2R)Chi^g320, vvl transcript was detected not only in its normal expression domain within the hindgut but also in the posterior spiracles. This ectopic expression is initially detected from late stage 13 and rapidly strengthens during stage 14–15. When ken^k11035/Df(2R)Chi^g320 embryos simultaneously mutant for the amorphic upd^OS1A allele were analyzed, upregulation of vvl in the presumptive posterior spiracles was never observed at the stage by which ectopic vvl expression was first detected in the ken mutant embryos. At later stages, JAK/STAT pathway activity is required for posterior spiracle morphogenesis, posterior spiracles do not form, and upregulated vvl is not present (Arbouzova, 2006).

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

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

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

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

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 hop^M13 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).

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).

Decline in self-renewal factors contributes to aging of the stem cell niche in the Drosophila testis

Aging is characterized by compromised organ and tissue function. A decrease in stem cell number and/or activity could lead to the aging-related decline in tissue homeostasis. This study analyzed how the process of aging affects germ line stem cell (GSC) behavior in the Drosophila testis; significant changes within the stem cell microenvironment, or niche, occur that contribute to a decline in stem cell number over time. Specifically, somatic niche cells in testes from older males display reduced expression of the cell adhesion molecule DE-cadherin and a key self-renewal signal unpaired (upd). Loss of upd correlates with an overall decrease in stem cells residing within the niche. Conversely, forced expression of upd within niche cells maintains GSCs in older males. Therefore, these data indicate that age-related changes within stem cell niches may be a significant contributing factor to reduced tissue homeostasis and regeneration in older individuals (Boyle, 2007. Full text of article).

Repression of Wasp by JAK/STAT signalling inhibits medial actomyosin network assembly and apical cell constriction in intercalating epithelial cells

Tissue morphogenesis requires stereotyped cell shape changes, such as apical cell constriction in the mesoderm and cell intercalation in the ventrolateral ectoderm of Drosophila. Both processes require force generation by an actomyosin network. The subcellular localization of Myosin-II (Myo-II) dictates these different morphogenetic processes. In the intercalating ectoderm Myo-II is mostly cortical, but in the mesoderm Myo-II is concentrated in a medial meshwork. Spacial constriction is repressed by JAK/STAT signalling in the lateral ectoderm independently of Twist. Inactivation of the JAK/STAT pathway causes germband extension defects because of apical constriction ventrolaterally. This is associated with ectopic recruitment of Myo-II in a medial web, which causes apical cell constriction as shown by laser nanosurgery. Reducing Myo-II levels rescues the JAK/STAT mutant phenotype, whereas overexpression of the Myo-II heavy chain (also known as Zipper), or constitutive activation of its regulatory light chain, does not cause medial accumulation of Myo-II nor apical constriction. Thus, JAK/STAT controls Myo-II localization by additional mechanisms. Regulation of actin polymerization by Wasp, but not by Dia, is important in this process. Constitutive activation of Wasp, a branched actin regulator, causes apical cell constriction and promotes medial 'web' formation. Wasp is inactivated at the cell cortex in the germband by JAK/STAT signalling. Lastly, wasp mutants rescue the normal cortical enrichment of Myo-II and inhibit apical constriction in JAK/STAT mutants, indicating that Wasp is an effector of JAK/STAT signalling in the germband. Possible models are discussed for the role of Wasp activity in the regulation of Myo-II distribution (Bertet, 2009).

Myo-II subcellular localization controls different cell shape changes such as cell constriction or intercalation. The data shed new light on the mechanisms of the subcellular localization of actomyosin networks in the early Drosophila ectoderm. VL ectodermal cells intercalate via the cortical recruitment of Myo-II at AJs, which drives polarized junction remodelling. This contrasts with the behaviour of immediately adjacent cells in the mesoderm, which undergo apical constriction and recruit Myo-II into a medial apical web. The data indicate that the cortical enrichment of Myo-II in ectodermal intercalating cells is not a 'default pathway', and requires at least activity of the JAK/STAT pathway. Indeed, in JAK/STAT pathway mutants, Myo-II is aberrantly recruited in a medial apical meshwork and cells consequently undergo apical constriction. This is surprising, as apical constriction is normally only observed in mesodermal ventral cells and is considered to be a unique attribute owing to their selective expression of Twist and Snail. Twist and Snail induce expression of the ligand Fog in the ventral cells only, which activates RhoGEF2, Rok and Myo-II. It also regulates expression of the transmembrane protein T48, which participates in the apical recruitment of RhoGEF2 and contributes to apical constriction. However it is not clear whether activation of the RhoGEF2 pathway is sufficient to drive the apical medial recruitment of Myo-II. This study shows that apical constriction is not simply induced in mesodermal cells by Fog, but is also prevented in ectodermal cells by activity of the JAK/STAT pathway and that this is essential for germ-band extension (GBE). In JAK/STAT pathway mutants, ectodermal cells undergo apical constriction despite the absence of ectopic Twist expression. Note, however, that apical constriction is not as rapid in these mutants as in mesodermal cells, so Twist and Snail accelerate or render more efficient the capacity to apically constrict. Moreover, the fact that Wasp mediates JAK/STAT function in the ectoderm but is not required in the mesoderm indicates that the mechanisms promoting medial Myo-II in mesoderm cells are likely to be different (Bertet, 2009).

These findings provide a novel opportunity to investigate the regulation of cortical or medial Myo-II localization in the ectoderm. The data document two novel features of this regulation (Bertet, 2009).

MRLC (Sqh) phosphorylation by the RhoGEF2 and the Rok pathway are both necessary for apical constriction; lowering the dose of RhoGEF2, Rho or Rok suppress the apical constriction observed in upd mutants. However, neither constitutive activation of this pathway by expression of a phosphomimetic form of Sqh, ShqE20E21, which rescues Rok inhibition, nor overexpression of MHC (Zip) is sufficient to promote medial accumulation of Myo-II. The medial accumulation of Myo-II requires additional regulation apart from the activation of Myo-II. Since RhoGEF2 and Rok are key regulators of Myo-II, this suggests that activation of the RhoGEF2/Rok pathway is necessary but not sufficient to explain medial Myo-II accumulation and apical constriction (Bertet, 2009).

This analysis of the JAK/STAT mutant phenotypes indicates a key role of Wasp in this process. Wasp is shown to be necessary for medial Myo-II accumulation, at least in ectodermal cells, and very strong activation of Wasp at the cortex (myrWasp) also causes medial Myo-II accumulation. Moreover, although Wasp is normally downregulated in VL ectodermal cells, in JAK/STAT pathway mutants Wasp is strongly recruited and hence activated at the plasma membrane, which suggests that JAK/STAT signalling represses the membrane activation of Wasp. Importantly, lowering the dose of Wasp maternally suppresses medial accumulation of Myo-II in upd mutants, and restores prominent accumulation at the cortex, as in wild-type embryos. Consistent with this, ectopic apical constriction is completely rescued in these double mutant embryos (Bertet, 2009).

Dia and Wasp play different roles in the regulation of Myo-II localization. Consistent with previous data, Dia controls the amount of apical Myo-II, but the specific localization of Myo-II at the cortex or in the medial network is not affected by loss of Dia. Dia promotes polymerization of non-branched filaments, and might control the formation of a good substrate for the stable association of Myo-II minifilaments. The fact that in dia heterozygotes the amount of apical Myo-II is reduced indicates that the amount of actin filaments might be limiting and controlled. Indeed, more F-actin is detected at the cortex of intercalating cells, preceding by a few minutes the enrichment of Myo-II. The role of Wasp is more surprising and unique; it is shown to mediate specifically repression of medial Myo-II accumulation and, hence, cell constriction in the germband. Because activation of Wasp leads to activation of medial web formation and reduction of Wasp dosage rescues cortical Myo-II in JAK/STAT mutants, it is concluded that Wasp controls an essential feature of Myo-II subcellular localization that is essential for the regulation of apical constriction. How does Wasp control Myo-II localization? Two non-exclusive models. In the first model, Wasp controls actin branching through activation of the Arp2/3 complex. Because Wasp has been implicated in endocytosis via Arp2/3 in Drosophila, Wasp could promote Myo-II web formation indirectly by regulating endocytosis of a surface protein required to anchor the medial actomyosin network at the membrane, such as E-cadherin. Consistent with this, downregulation of E-cadherin by RNAi disrupts the faint medial Myo-II pool. In mesodermal cells, E-cadherin appears to anchor the strong medial Myo-II pool. In the second model, Wasp might act more directly via the regulation of actin network architecture and its impact on the dynamic interactions between the medial web and the cortex, and thereby might affect the steady-state distribution of Myo-II exchanging between these two pools. Although Wasp uniquely mediates Myo-II regulation via JAK/STAT in the ectoderm and not in the mesoderm, regulation of Arp2/3 might be more generally implicated in the control of Myo-II regulation (Bertet, 2009).

Although wasp is an important mediator of JAK/STAT function in the ectoderm, it is unlikely to be the only one. Indeed wasp mutants rescue the cortical accumulation of Myo-II and apical constriction in upd mutants, but GBE is still strongly affected; it was noticed that cortical Myo-II distribution was not properly polarized in the plane of the epithelium. This suggests that other subcellular processes are also perturbed in the mutant. The fact that a reduction of Myo-II levels suppresses the upd defects indicates that the overall dosage of Myo-II is important as well. Identifying the transcriptional targets of JAK/STAT might shed light on its complex regulatory role during embryonic morphogenesis (Bertet, 2009).

Finally, although this work identifies an important regulator of Myo-II network subcellular distribution in epithelial cells, it is still not clear what regulates the polarized distribution of Myo-II at the cortex (Bertet, 2009).

JAK/STAT signalling controls a number of developmental processes. Importantly, this pathway has been implicated in diverse morphogenetic processes, such as convergent extension movements in the zebrafish embryo, hindgut elongation in Drosophila embryos, which probably involves intercalation movements as well, and posterior spiracle morphogenesis in Drosophila embryos. JAK/STAT signalling also controls border cell migration. The data indicate that JAK/STAT signalling plays an important and hitherto unappreciated morphogenetic function in gastrulating embryos. These data document evidence that JAK/STAT controls, via Wasp, a morphogenetic switch based on the regulation of medial or cortical Myo-II distribution. Interestingly, dorsal cells do not undergo apical constriction in JAK/STAT mutants. Indeed, dorsal cells exhibit neither cortical nor medial web Myo-II and are thus unable to participate in profound tissue remodelling. It appears that DV patterning provides a first general subdivision within the embryonic epithelium whereby Myo-II is globally repressed dorsally, and activated laterally and ventrally. Cortical or medial web distribution then results from the combinatorial input of Fog and JAK/STAT (Bertet, 2009).

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, 2009).

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, 2009).

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, 2009).

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, 2009).

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, 2009).

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, 2009).

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, 2009).

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, 2009).

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, 2009).

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, 2009).

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, 2009).

Loss of the Polycomb group gene polyhomeotic induces non-autonomous cell overproliferation

Polycomb group (PcG) proteins are conserved epigenetic regulators that are linked to cancer in humans. However, little is known about how they control cell proliferation. This study reports that mutant clones of the PcG gene polyhomeotic (ph) form unique single-cell-layer cavities that secrete three JAK/STAT pathway ligands, which in turn act redundantly to stimulate overproliferation of surrounding wild-type cells. Notably, different ph alleles cause different phenotypes at the cellular level. Although the ph-null allele induces non-autonomous overgrowth, an allele encoding truncated Ph induces both autonomous and non-autonomous overgrowth. It is proposed that PcG misregulation promotes tumorigenesis through several cellular mechanisms (Feng, 2010). <>In summary, mosaic clones homozygous for the ph-null allele induce overproliferation of surrounding wild-type cells through Notch-Upd-JAK/STAT signalling, whereas mosaic clones homozygous for a ph hypomorphic allele that encodes truncated Ph proteins induce both autonomous and non-autonomous cell overproliferation. These results highlight an important but largely overlooked phenomenon: different mutations in the same gene might induce tumours and cancers through distinct cellular mechanisms, depending on the nature of the mutations and/or genetic backgrounds. This fact adds another layer of complexity to cancer pathology (Feng, 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).

Diverse tumor pathology due to distinctive patterns of JAK/STAT pathway activation caused by different Drosophila polyhomeotic alleles

Drosophila polyhomeotic (ph) is one of the important polycomb group genes that is linked to human cancer. In the mosaic eye imaginal discs, while ph^del, a null allele, causes only non-autonomous overgrowth, ph⁵⁰⁵, a hypomorphic allele, causes both autonomous and non-autonomous overgrowth. These allele-specific phenotypes stem from the different sensitivities of ph mutant cells to the Upd homologs that they secrete (Feng, 2012).

Different ph alleles cause tissue overgrowth in different ways. While a ph null allele, ph^del , causes only non-autonomous cell over-proliferation, a ph hypomorphic allele, ph⁵⁰⁵ , causes both autonomous and non-autonomous cell overproliferation. In mosaic tissues, overproliferation of mutant cells was defined as autonomous, whereas over-proliferation of genotypically wild type cells induced by mutant cells was defined as non-autonomous. The signaling pathway involved in ph^del induced non-autonomous cell over-proliferation. In summary, elevated Notch activity in ph cells up-regulates the expression of JAK/STAT pathway ligands Upd homologs, which in turn activate the JAK/STAT pathway in neighboring wild type cells and cause their over-proliferation. This study addressed why a ph null allele and a ph hypomorphic allele both cause tumors but in such different ways (Feng, 2012).

First whether the same signaling pathway underlay non-autonomous overproliferation induced by both ph^del and ph⁵⁰⁵ was tested. The functions of Notch and Upd homologs in the ph⁵⁰⁵ mosaic eyes were examined with the same strategy used for ph^del. A ph⁵⁰⁵ -Notch double mutant line was generated, and eyes mosaic for this line were essentially of the same size as wild type eyes. The mosaic eye discs had normal size and normal cell proliferation level, as shown by PH3 staining, which marks mitotic cells. Moreover, the size of ph⁵⁰⁵ -Notch clones was significantly reduced when compared to that of ph⁵⁰⁵ clones. These results indicated that Notch was required for both autonomous and non-autonomous overproliferation induced by ph⁵⁰⁵ (Feng, 2012).

Next ph⁵⁰⁵ was recombined with upd^Δ1-3, a deficiency line that lacks all three upd homologs in the Drosophila genome Mosaic analyses were then performed using this double mutant line. ph⁵⁰⁵ -upd^Δ1-3 mosaic eyes were significantly smaller than ph⁵⁰⁵ mosaic eyes and were comparable to wild type eyes, indicating that tissue overgrowth was largely suppressed. PH3 staining of the double mutant mosaic eye discs showed that these discs had relatively normal size and cell proliferation level. Importantly, ph⁵⁰⁵ -updd1-3 clones were also drastically reduced in size compared to ph⁵⁰⁵ clones. These results indicated that Upd homologs are required for not only non-autonomous but also autonomous cell over-proliferations induced by ph⁵⁰⁵ (Feng, 2012).

It is not surprising that the same signaling pathway is responsible for non-autonomous over-proliferation induced by both ph^del and ph⁵⁰⁵ , and it is not completely unexpected that Notch is also required for ph⁵⁰⁵ induced autonomous over-proliferation, as Notch is a transcription factor that has been shown to autonomously regulate cell proliferation. However, the three Upd proteins are secreted and are not expected to have any direct effect on autonomous cell proliferation. To interpret these observations, it was hypothesized that ph⁵⁰⁵ cells still respond to Upd ligands secreted by themselves in an autocrine or paracrine manner, and therefore over-proliferate. However, ph^del cells were thought to be no longer responsive to Upd ligands (Feng, 2012).

To functionally test this hypothesis, the double mutant strategy was applied, taking advantage of the fact that the genes domeless (dome, encoding the only transmembrane receptor of the Drosophila JAK/STAT pathway) and hopscotch (hop, encoding the only Drosophila JAK kinase) are also on X chromosome as is ph. First ph⁵⁰⁵ was recombined with two dome alleles to generate ph⁵⁰⁵ -dome double mutant lines. Eye discs mosaic for these lines were still significantly larger than wild type, but the size of double mutant clones was dramatically reduced, so that only a tiny portion of the disc was composed of mutant cells. PH3 staining indicated that non-autonomous proliferation level was still high, but autonomous proliferation largely disappeared. The adult eyes mosaic for such double mutant lines were further examinedm and these eyes were found to be still much larger than wild type and similar to ph⁵⁰⁵ mosaic eyes in size, but they generally were not folded as seen in ph⁵⁰⁵ mosaic eyes (Feng, 2012).

Next a ph⁵⁰⁵ -hop double mutant line was generated. Autonomous proliferation was found in mosaic eye discs of this double mutant that was also significantly suppressed, with mutant cells only accounted for a small portion of the whole disc. In contrast, non-autonomous cell over-proliferation was not affected and the overall size of these discs was still significantly larger than wild type. Adult eyes mosaic for this double mutant showed similar phenotypes as those of ph⁵⁰⁵ -dome mosaic eyes. These eyes were still significantly larger than wild type but they were generally not folded. Therefore, the removal of either dome or hop from ph⁵⁰⁵ cells only suppressed autonomous over-proliferation but did not affect non-autonomous overproliferation, making such double mutant mosaic discs phenotypically similar to ph^del mosaic discs (Feng, 2012).

As controls, ph^del -dome and ph^del -hop double mutant lines were also generated using the same dome and hop alleles. Mosaic analyses on eye discs showed that the removal of dome or hop from ph^del cells did not affect non-autonomous cell over proliferation. It did, however, mildly reduce the mutant clone size, suggesting that ph^del cells might still have a weak response to Upd ligands. Adult eyes mosaic for these double mutant lines were phenotypically indistinguishable from ph^del mosaic eyes, consistent with the above observations in mosaic eye discs (Feng, 2012).

Finally it was asked why ph^del and ph⁵⁰⁵ cells responds differently to the Upd ligands secreted by themselves. It was hypothesized that some of the JAK/STAT pathway modulators might be differentially expressed in ph^del and ph⁵⁰⁵ cells. To test this hypothesis, TU-Tagging, a technique that enables the purification of RNA from mutant cells without having to physically isolate such cells, was chosen. Briefly, Drosophila is unable to synthesize uridine from uracil due to the lack of phosphoribosyltransferase (UPRT). When exogenous UPRT is expressed in mutant cells by MARCM, such cells would acquire the ability to utilize uracil. If these larvae are fed with 4-thiouracil (4-TU), a uracil derivative that contains a thio group, only mutant cells would be able to use 4-TU and eventually incorporate thio- containing uridine into newly synthesized RNA. This treatment has little toxicity, and the thio-labeled RNA can be purified from total RNA using conventional biochemical methods (Feng, 2012).

TU-tagging was performed to isolate RNA from ph^del cells and ph⁵⁰⁵ cells, and qRTPCR was used to examine candidate gene expression. The expression of the JAK/STAT pathway receptor dome was significantly higher in ph⁵⁰⁵ cells than in ph^del cells. A higher receptor expression might sensitize ph⁵⁰⁵ cells to the Upd ligands. The levels of enok and socs42a, both negative regulators of the JAK/STAT pathway, were also significantly higher in ph⁵⁰⁵ cells compared to ph^del cells. This might represent feedback loops that negatively regulate the pathway activity. In fact, several such negative feedback loops, in which elevated pathway activity upregulates a negative pathway regulator, have been reported in JAK/STAT pathway (Feng, 2012).

Together, it is concluded that ph^del and ph⁵⁰⁵ both cause autonomous over-expression of Upd homologs in mutant cells, which represents the only driving force of cell overproliferation in ph^del and ph⁵⁰⁵ mosaic tissues and in essence acts non-autonomously to activate JAK/STAT pathway. The different phenotypes of these two types of mosaics are due to different sensitivity of mutant cells to Upd homologs. ph⁵⁰⁵ mutant cells robustly respond to Upd ligands that they secreted. Therefore, Upd ligands secreted by ph⁵⁰⁵ cells simultaneously induce over-proliferation in both mutant and wild type cells. In contrast, ph^del cells are largely insensitive to Upd ligands, so that Upd ligands secreted by ph^del cells only induce over-proliferation in wild type but not mutant cells. Furthermore, differential expression of the JAK/STAT pathway receptor dome might underlie the different sensitivity of ph^del and ph⁵⁰⁵ cells to Upd ligands (Feng, 2012).

Heart- and muscle-derived signaling system dependent on MED13 and Wingless controls obesity in Drosophila

Obesity develops in response to an imbalance of energy homeostasis and whole-body metabolism. Muscle plays a central role in the control of energy homeostasis through consumption of energy and signaling to adipose tissue. MED13, a subunit of the Mediator complex, acts in the heart to control obesity in mice. To further explore the generality and mechanistic basis of this observation, this study investigated the potential influence of MED13 expression in heart and muscle on the susceptibility of Drosophila to obesity. This study shows that heart/muscle-specific knockdown of MED13 or MED12, another Mediator subunit, increases susceptibility to obesity in adult flies. To identify possible muscle-secreted obesity regulators, an RNAi-based genetic screen of 150 genes was performed that encode secreted proteins; Wingless inhibition was also found to cause obesity. Consistent with these findings, muscle-specific inhibition of Armadillo, the downstream transcriptional effector of the Wingless pathway, also evoked an obese phenotype in flies. Epistasis experiments further demonstrated that Wingless functions downstream of MED13 within a muscle-regulatory pathway. Together, these findings reveal an intertissue signaling system in which Wingless acts as an effector of MED13 in heart and muscle and suggest that Wingless-mediated cross-talk between striated muscle and adipose tissue controls obesity in Drosophila. This signaling system appears to represent an ancestral mechanism for the control of systemic energy homeostasis (Lee, 2014).

The results reveal a role of muscle in systemic regulation of obesity via the function of MED13 in Drosophila. A genetic screen identified muscle-secreted obesity-regulating factors, including Wg, and demonstrated that Wg signaling in muscle is necessary and sufficient to suppress obesity. Furthermore, it was shown that a skd-null mutation dominantly enhances the arm phenotype in muscle and that wg is epistatic to skd, suggesting that Wg is a downstream effector of MED13 in muscle (Lee, 2014).

The results reveal that MED13 in Drosophila muscle functions to suppress obesity based on several criteria, such as histology, measurement of whole-body triglycerides, tolerance to starvation stress, and susceptibility to high-fat diet. Similarly, muscle-specific knockdown of MED12 also increases fat accumulation, suggesting that MED12 and MED13 function similarly in the control of fat deposition in Drosophila. The finding that MED12 and MED13 modulate energy homeostasis adds to a growing number of examples in which components of the kinase module of the Mediator complex influence metabolic signaling on an organismal level. For example, the other two components of the kinase module, Cyclin-dependent kinase 8 and Cyclin C, have also been reported as negative regulators of fat accumulation in flies and mice. The finding that the activity of MED13 in cardiac muscle regulates fat accumulation in Drosophila is consistent with earlier observation with mice and suggests that the function of cardiac MED13 in systemic regulation of fat storage represents an ancestral mechanism conserved in metazoans. Although it seems most likely that the effect of MED13 on obesity is mediated by overall changes in metabolism, it is also conceivable that changes in feeding behavior contribute to the obesity phenotypes that were observed. Knockdown of MED12 and MED13 using drivers that are active specifically in the heart using Tin-Gal4 or generally in all muscles using Mef2-Gal4 or Mhc-Gal4 commonly evoked obesity and MED13 can control metabolic signaling from the heart, consistent with prior conclusions regarding the functions of MED13 in the mouse heart. However, these Gal4 drivers do not enable reaching of conclusions regarding the specific role of somatic or visceral muscle in this signaling process because Mhc-Gal4 and Mef2-Gal4 are active in diverse muscle-cell types. Given that MED12 and MED13 are ubiquitously expressed, it is possible that they also act in nonmuscle tissues to regulate metabolic homeostasis (Lee, 2014).

It is hypothesized that muscle-secreted factors mediate the function of MED13 in Drosophila muscle to suppress systemic fat accumulation. To identify such factors, a screen was carried out for muscle-secreted obesity-regulating proteins using two different muscle drivers, Mef2-Gal4 and Mhc-Gal4. Six genes were identified that increased fat accumulation of flies in both screens by >60%, including the genes encoding (1) an antimicrobial peptide, Diptericin B; (2) a Drosophila homolog of Angiotensin converting enzyme; (3) a G protein-coupled receptor ligand SIFamide; (4) one of seven Drosophila Insulin/IGF homologs, Insulin-like peptide 4; (5) a JAK/STAT signaling ligand, Unpaired 3; and (6) Wg. Interestingly, it has been shown recently that MED13 and MED12 are required for the expression of Diptericin B in response to Immune Deficiency (IMD) pathway activation, suggestive of additional regulatory functions of MED13 and the genes identified from the current screens beyond obesity control (Lee, 2014).

This study demonstrated that Wg and its autonomous signaling activity, controlled by Arm, in muscle are necessary and sufficient for systemic regulation of obesity in vivo. Previously, the correlation between obesity and the expression of genes involved in the Wnt signaling pathway in heart has been raised from transcriptome analyses using heart biopsies from obese patients. Similarly, correlations between obesity and differential expression of genes for Wnt signaling, as well as genes for insulin sensitivity and myogenic capacity, were also found in skeletal-muscle samples from obese rats. These findings suggest that Wg signaling activity in muscle serves as an intrinsic rheostat for obesity control (Lee, 2014).

Muscle-specific arm knockdown caused partial-patterning defects in the embryonic musculature, and a skd-null allele dominantly enhanced this phenotype to complete lethality. Given the central role of Arm in Wg target gene expression, the findings are consistent with the established function of wg in the development of mesoderm and the embryonic musculature. The findings reveal a close functional connection between MED13 and Arm, suggestive of the role of MED13 in Wg target gene expression. In fact, in the developing Drosophila eye and wing, MED13 and MED12 are essential for Wg target gene expression, and the MED13/MED12 complex physically interacts with Pygopus, a component of the Wg transcriptional complex. Furthermore, MED12 hypomorphic mutant mice are embryonic lethal with impaired expression of Wnt targets. Therefore, the genetic interaction data along with these previous reports suggest that MED13 is a general component of the canonical Wg/Wnt pathway (Lee, 2014).

epistasis experiments indicate that muscle-secreted Wg functions downstream of MED13 in muscle to suppress obesity. Because both wg and arm in muscle are crucial for obesity regulation, one function of muscle-secreted Wg might be to act on muscle. Accordingly, the nonautonomous function of Wg to suppress obesity may occur through autonomous Wg signal activity in muscle. However, if MED13 functions at the level of transcriptional control of Wg target genes and the sole function of muscle-secreted Wg ligand is to activate the Wg signal 'in' muscle, Wg should be upstream of MED13, which is contrary to the epistasis studies. Based on the data, it stands to reason that muscle-secreted Wg should also act directly on a tissue other than muscle for its nonautonomous effect. If so, which tissue may be the target? Ectopic expression of Wg using a fat body-specific Dcg-Gal4 decreased larval abdominal fat body mass, which demonstrates the role of Wg signaling in the fat body for fat-mass regulation. Similarly, in mammals, autonomous activation of the Wnt pathway in adipose tissue decreases fat mass. Wnt signaling blocks mammalian adipogenesis in vitro, and, in mice, activation of the canonical Wnt pathway in adipocytes by ectopic expression of Wnt10b, a Wnt ligand, inhibits obesity. Furthermore, autonomous activation of the Wnt pathway in adipose progenitors with constitutively active β-catenin expression decreases fat mass. Therefore, the reduced fat mass in Dcg > wg larvae indicates that autonomous Wg signaling activity in the fat body serves as a regulator of fat mass. Considered together with the data showing that muscle-secreted Wg contributes to nonautonomous regulation of adiposity in vivo, it is concluded that muscle serves as a source of Wg to regulate adiposity by modulating Wg signaling activity in fat body. However, the possibility cannot be ruled out that the systemic effect of Wg from muscle is mediated through an alternative tissue, such as nervous system (Lee, 2014).

Wg acts on short- and long-range targets. Wg is highly hydrophobic and has been shown to diffuse through the extracellular space and act on long-range targets by associating with solubilizing molecules such as lipoprotein particles and Secreted Wg-interacting molecule. Furthermore, Wnt-1 has been identified in serum, and decreased Wnt-1 levels in serum correlate with premature myocardial infarction and metabolic syndrome, suggesting that Wg may act on remote organs as an endocrine factor. Therefore, this study proposes a model in which muscle-secreted Wg is a downstream effector of MED13 and acts both to activate the signal in muscle and to act on the fat body ultimately to achieve systemic inhibition of obesity (Lee, 2014).

JAK/STAT signaling in Drosophila muscles controls the cellular immune response against parasitoid infection

The role of JAK/STAT signaling in the cellular immune response of Drosophila is not well understood. This study shows that parasitoid wasp infection activates JAK/STAT signaling in somatic muscles of the Drosophila larva, triggered by secretion of the cytokines Upd2 and Upd3 from circulating hemocytes. Deletion of upd2 or upd3, but not the related os (upd1) gene, reduces the cellular immune response, and suppression of the JAK/STAT pathway in muscle cells reduces the encapsulation of wasp eggs and the number of circulating lamellocyte effector cells. These results suggest that JAK/STAT signaling in muscles participates in a systemic immune defense against wasp infection (Yang, 2015).

Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity

A relatively small number of signaling pathways drive a wide range of developmental decisions, but how this versatility in signaling outcome is generated is not clear. In the Drosophila follicular epithelium, localized epidermal growth factor receptor (EGFR) activation induces distinct cell fates depending on its location. Posterior follicle cells respond to EGFR activity by expressing the T-box transcription factors Midline and H15, while anterior cells respond by expressing the homeodomain transcription factor Mirror. This study shows that the choice between these alternative outputs of EGFR signaling is regulated by antiparallel gradients of JAK/STAT and BMP pathway activity and that mutual repression between Midline/H15 and Mirror generates a bistable switch that toggles between alternative EGFR signaling outcomes. JAK/STAT and BMP pathway input is integrated through their joint and opposing regulation of both sides of this switch. By converting this positional information into a binary decision between EGFR signaling outcomes, this regulatory network ultimately allows the same ligand-receptor pair to establish both the anterior-posterior (AP) and dorsal-ventral (DV) axes of the issue (Fregoso Lomas, 2016).

This study shows that the choice between two alternative Grk/EGFR signaling outcomes in the follicular epithelium depends on positional input provided by Upd and Dpp. At the posterior, the presence of Upd allows Grk to induce Mid/H15 while, at the anterior, Grk together with Dpp positively regulates Mirr. In this context, Upd and Dpp serve to define the response to Grk/EGFR signaling, since they are not sufficient to induce Mid/H15 and Mirr, respectively, in the absence of Grk (Fregoso Lomas, 2016).

Mutual repression is demonstrated between Mid/H15 and Mirr that is proposed to generate a double-negative feedback circuit that toggles the system between anterior and posterior outcomes. Moreover, in addition to their mutual regulation, analysis of double-mutant clones reveals that Upd and Dpp each regulate both Mid/H15 and Mirr and, thus, each provides input to both sides of this circuit. Upd is required for the expression of Mid and H15 even in the absence of a functional mirr gene, demonstrating that Upd is required for Mid/H15 expression independent of its ability to repress Mirr. Similarly, Dpp signaling can repress Mid independently of its positive effect on Mirr. The choice of Grk/EGFR signaling outcome in this context thus depends not only on mutual repression between these alternate targets but also on their opposing regulation by Upd and Dpp (Fregoso Lomas, 2016).

It is proposed that these elements define a bistable network that controls the choice between two alternative outcomes of Grk/EGFR signaling. These outcomes are irreversible -- e.g., posterior EGFR signaling in later stages cannot induce Mirr unless Mid and H15 are absent - and mutually exclusive, and the factors described in this study include two key elements found in bistable networks: feedback and non-linearity. The feedback in this case is provided by the reciprocal repression between Mirr and Mid/H15, generating a double-negative feedback loop that reinforces the choice of signaling outcome. In addition, bistability requires non-linearity in the response of the circuit to its upstream regulators, which makes the switch more sensitive to graded inputs. It is proposed that, in the follicular epithelium, this is achieved by the joint opposing regulation of the feedback circuit by both Dpp and Upd; each activates one side of the switch while repressing the other, biasing the outcome in the same direction (Fregoso Lomas, 2016).

These alternative responses to Grk are separated in time, as the source of Grk moves from posterior to anterior during the course of development. An important element that determines the choice between them is the early pattern of Mirr expression. In early stages of oogenesis, Mirr is Grk independent and is restricted to the main body follicle cells, due to its repression in the terminal regions by Upd. These main-body follicle cells correspond to the future anterior region of the columnar epithelium, and it is proposed that this early expression of Mirr predisposes them to express Mirr instead of Mid/H15 when Grk adopts its final dorsal anterior localization. Such a role for the early phase of Mirr expression is also consistent with the DV asymmetry of the Mid expression domain; as Grk moves anteriorly, leaving the range of posterior Upd and entering this domain of early Mirr expression and Dpp pathway activity, only the peak dorsal levels of Grk are capable of inducing Mid (Fregoso Lomas, 2016).

These observations also provide an example of how antiparallel signaling gradients can be integrated during epithelial patterning. Tissue patterning by opposing morphogen gradients is observed in developmental contexts as diverse as the Drosophila blastoderm and vertebrate neural tube, where they engage downstream transcriptional networks whose dynamic properties generate reproducible gene expression boundaries. Mutual repression between downstream transcription factors helps define the position and boundaries of cell fate domains, but how the opposing gradients are integrated is not well understood. This study shows that, in the follicular epithelium, the opposing Upd and Dpp gradients are integrated at the level of the Mirr-Mid/H15 feedback circuit. This integration occurs not only at the level of the mutual repression between Mid/H15 and Mirr but also by the ability of each gradient to regulate both sides of this circuit (Fregoso Lomas, 2016).

Together, these elements define the framework of a regulatory network that integrates localized positional information to regulate a binary choice of EGFR signaling outcome. The results allow construction of a model that both accounts for how an individual cell responds to Grk/EGFR signaling and explains how these spatial inputs are integrated across the epithelium to generate a defined pattern of Mid/H15 and Mirr expression, ultimately defining the pattern of the eggshell. Mirr is required for the generation of the high- and low-Broad domains, and Mid/H15 expression is required to define the posterior limit of these domains. The ability of Dpp and Upd to influence the outcome of EGFR signaling allows a single signaling input, namely localized secretion of Grk by the oocyte, to generate multiple distinct outputs that are localized in space and time, thus establishing both the AP and DV polarity of the epithelium and generating a complex and reproducible pattern of cell fates (Fregoso Lomas, 2016).

Remote control of intestinal stem cell activity by haemocytes in Drosophila

Only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. This study used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. It was shown that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. It was also found that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Also, intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. The release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival (Chakrabarti, 2016).

A leptin analog locally produced in the brain acts via a conserved neural circuit to modulate obesity-linked behaviors in Drosophila

Leptin, a typically adipose-derived "satiety hormone," has a well-established role in weight regulation. This study describes a functionally conserved model of genetically induced obesity in Drosophila by manipulating the fly leptin analog unpaired 1 (upd1). Unexpectedly, cell-type-specific knockdown reveals upd1 in the brain, not the adipose tissue, mediates obesity-related traits. Disrupting brain-derived upd1 in flies leads to all the hallmarks of mammalian obesity: increased attraction to food cues, increased food intake, and increased weight. These effects are mediated by domeless receptors on neurons expressing Drosophila neuropeptide F, the orexigenic mammalian neuropeptide Y homolog. In vivo two-photon imaging reveals upd1 and domeless inhibit this hedonic signal in fed animals. Manipulations along this central circuit also create hypersensitivity to obesogenic conditions, emphasizing the critical interplay between biological predisposition and environment in overweight and obesity prevalence. The study proposes that adipose- and brain-derived upd/leptin may control differing features of weight regulation through distinct neural circuits (Beshel, 2017).

Spatiotemporal regulation of cell fusion by JNK and JAK/STAT signaling during Drosophila wound healing

Cell-cell fusion is widely observed during development and disease and imposes a dramatic change on participating cells. Cell fusion should be tightly controlled, but the underlying mechanism is poorly understood. This study found that the JAK/STAT pathway suppresses cell fusion during wound healing and delimits the event to the vicinity of the wound in the Drosophila larval epidermis. In the absence of JAK/STAT signaling, a large syncytium containing 3-fold the number of nuclei observed in wild-type tissue forms in wounded epidermis. upd2 and upd3 are transcriptionally induced by wounding and are required for suppressing excess cell fusion. JNK is activated in the wound vicinity and activity peaks at approximately 8 h after injury, whereas JAK/STAT signaling is activated in an adjoining concentric ring and activity peaks at a later stage. Cell fusion occurrs primarily in the wound vicinity, where JAK/STAT activation is suppressed by fusion-inducing JNK signaling. JAK/STAT signaling is both necessary and sufficient for the induction of βPS integrin expression, suggesting that the suppression of cell fusion is mediated at least in part by integrin protein (Lee, 2017).

EVOLUTIONARY HOMOLOGS

Optic morphology (Om) mutations in Drosophila ananassae are a group of retrotransposon (tom)-induced gain-of-function mutations that map to at least 22 independent loci and exclusively affect the compound eye morphology. In marked contrast to other Om mutations, which are characterized by fewer-than-normal and disorganized ommatidia, the Om(1E) mutation exhibits a peculiar phenotype as enlarged eyes with regularly arrayed normal ommatidia. To characterize the Om(1E) mutation, molecular analyses was carried out. A putative Om(1E) locus cloned by tom tagging and chromosome walking contains two transcribed regions in the vicinity of tom insertion sites of the Om(1E) mutant alleles, and one of these regions was shown to be the Om(1E) gene by P element-mediated transformation experiments with D. melanogaster. The Om(1E) gene encodes a novel protein having potential transmembrane domain(s). In situ hybridization analyses demonstrates that the Om(1E) gene is expressed ubiquitously in embryonic cells, imaginal discs, and the cortex of the central nervous system of third instar larvae, and specifically in lamina precursor cells. Artificially induced ubiquitous overexpression of Om(1E) affects morphogenesis of wing imaginal disc derivatives or large bristle formation. These findings suggest that the Om(1E) gene is involved in a variety of developmental processes (Juni, 1996).

Upd3--an ancestor of the four-helix bundle cytokines

The unpaired-like protein 3 (Upd3) is one of the three cytokines of Drosophila melanogaster supposed to activate the JAK/STAT signaling pathway (Janus tyrosine kinases/signal transducer and activator of transcription). This activation occurs via the type-I cytokine receptor Domeless, an orthologue of gp130, the common signal transducer of all four-helix bundle interleukin-6 (IL-6) type cytokines. Both receptors are known to exist as preformed dimers in the plasma membrane and initiate the acute-phase response. These facts indicate an evolutionary relation between vertebrate IL-6 and the Drosophila protein Upd3. This study presents data which strengthen this notion. Upd3 was recombinantly expressed, a renaturation and purification protocol was established which allows to obtain high amounts of biological active protein. This protein is, like human IL-6, a monomeric-alpha helical cytokine, implicating that Upd3 is an 'ancestor' of the four-helix bundle cytokines (Oldenfest, 2013).

REFERENCES

Search PubMed for articles about Drosophila unpaired 1, unpaired 2 & unpaired 3

Agaisse, H., et al. (2003). Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury. Dev. Cell 5: 441-450. 12967563

Amcheslavsky, A., Jiang, J. and Ip, Y. T. (2009). Tissue damage-induced intestinal stem cell division in Drosophila. Cell Stem Cell 4: 49-61. PubMed Citation: 19128792

Arbouzova, N. I., Bach, E. A. and Zeidler, M. P. (2006). Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes. Curr. Biol. 16(1): 80-8. 16401426

Avila, F. W. and Erickson, J. W. (2007). Drosophila JAK/STAT pathway reveals distinct initiation and reinforcement steps in early transcription of Sxl. Curr. Biol. 17: 643-648. Medline abstract: 17363251

Ayala-Camargo, A., Anderson, A. M., Amoyel, M., Rodrigues, A. B., Flaherty, M. S. and Bach, E. A. (2013). JAK/STAT signaling is required for hinge growth and patterning in the Drosophila wing disc. Dev Biol. PubMed ID: 23978534

Bach, E. A., Vincent, S., Zeidler, M. P. and Perrimon, N. (2003). A sensitized genetic screen to identify novel regulators and components of the Drosophila janus kinase/signal transducer and activator of transcription pathway. Genetics 165(3): 1149-66. 14668372

Barrio, L., Dekanty, A. and Milan, M. (2014). MicroRNA-mediated regulation of Dp53 in the Drosophila fat body contributes to metabolic adaptation to nutrient deprivation. Cell Rep. PubMed ID: 25017064

Beccari, S., Teixeira, L. and Rorth, P. (2002). The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis. Mech. Dev. 111(1-2): 115-23. 11804783

Bertet, C., Rauzi, M. and Lecuit, T. (2009). Repression of Wasp by JAK/STAT signalling inhibits medial actomyosin network assembly and apical cell constriction in intercalating epithelial cells. Development 136(24): 4199-212. PubMed Citation: 19934015

Beshel, J., Dubnau, J. and Zhong, Y. (2017). A leptin analog locally produced in the brain acts via a conserved neural circuit to modulate obesity-linked behaviors in Drosophila. Cell Metab 25: 208-217. PubMed ID: 28076762

Besse, F. and Pret, A.-M. (2003). Apoptosis-mediated cell death within the ovarian polar cell lineage of Drosophila melanogaster. Development 130: 1017-1027. 12538526

Betz, A., Lampen, N., Martinek, S., Young, M. W. and Darnell, J. E., Jr. (2001). A Drosophila PIAS homologue negatively regulates stat92E. Proc. Natl. Acad. Sci. 98: 9563-9568. 11504941

Binari, R. and Perrimon, N. (1994). Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila. Genes Dev 8: 300-12. PubMed Citation: 8314084

Biteau, B., Hochmuth, C. E. and Jasper, H. (2008). JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut. Cell Stem Cell 3: 442-455. PubMed Citation: 18940735

Borensztejn, A., Boissoneau, E., Fernandez, G., Agnes, F. and Pret, A. M. (2013). JAK/STAT autocontrol of ligand-producing cell number through apoptosis. Development 140: 195-204. PubMed ID: 23222440

Buchon, N., Broderick, N. A., Chakrabarti, S. and Lemaitre, B. (2009). Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila. Genes Dev. 23(19): 2333-44. PubMed Citation: 19797770

Bunker, B.D., Nellimoottil, T.T., Boileau, R.M., Classen, A.K. and Bilder, D. (2015). The transcriptional response to tumorigenic polarity loss in Drosophila. Elife [Epub ahead of print]. PubMed ID: 25719210

Boyle, M., Wong, C., Rocha, M., and Jones, D. L. (2007). Decline in self-renewal factors contributes to aging of the stem cell niche in the Drosophila testis. Cell Stem Cell 1(4): 470-8. PubMed Citation: 18371382

Brown, S., Hu, N. and Castelli-Gair Hombria, J. (2001). Identification of the first invertebrate interleukin JAK/STAT receptor, the Drosophila gene domeless. Cur. Bio. 11: 1700-1705. 11696329

Brown, S., Hu, N. and Hombria, J. C.-G. (2003). Novel level of signalling control in the JAK/STAT pathway revealed by in situ visualisation of protein-protein interaction during Drosophila development. Development 130: 3077-3084. 12783781

Buchon, N., Broderick, N. A., Poidevin, M., Pradervand, S. and Lemaitre, B. (2009). Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation. Cell Host Microbe 5: 200-211. PubMed Citation: 19218090

Cai, X. T., Li, H., Borch Jensen, M., Maksoud, E., Borneo, J., Liang, Y., Quake, S. R., Luo, L., Haghighi, P. and Jasper, H. (2021). Gut cytokines modulate olfaction through metabolic reprogramming of glia. Nature. PubMed ID: 34290404

Carroll, S. B and Scott, M. P. (1986). Zygotically active genes that affect the spatial expression of the fushi tarazu segmentation gene during early Drosophila embryogenesis. Cell 45: 113-126. 86161687

Casper, A. L. and Van Doren, M. (2009). The establishment of sexual identity in the Drosophila germline. Development 136(22): 3821-30. PubMed Citation: 19855024

Chakrabarti, S., Dudzic, J.P., Li, X., Collas, E.J., Boquete, J.P. and Lemaitre, B. (2016). Remote control of intestinal stem cell activity by haemocytes in Drosophila. PLoS Genet 12: e1006089. PubMed ID: 27194701

Chao, J.-L., et al. (2004). Localized Notch signal acts through eyg and upd to promote global growth in Drosophila eye. Development 131: 3839-3847. 15253935

Chen, H. W., Chen, X., Oh, S. W., Marinissen, M. J., Gutkind, J. S. and Hou, S. X. (2002). mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family. Genes Dev. 16(3): 388-98. 11825879

Cline, T. W. (1993). The Drosophila sex determination signal: how do flies count to two? Trends Genet. 9(11): 385-390. PubMed Citation: 8310535

Copf, T., et al. (2011). Cytokine signaling through the JAK/STAT pathway is required for long-term memory in Drosophila. Proc. Natl. Acad. Sci. 108(19): 8059-64. PubMed Citation: 21518857

Cordero, J. B., Stefanatos, R. K., Myant, K., Vidal, M. and Sansom, O. J. (2012). Non-autonomous crosstalk between the Jak/Stat and Egfr pathways mediates Apc1-driven intestinal stem cell hyperplasia in the Drosophila adult midgut. Development 139: 4524-4535. PubMed ID: 23172913

Dominguez, M., Ferres-Marco, D., Gutierrez-Avino, F. J., Speicher, S. A. and Beneyto, M. (2004). Growth and specification of the eye are controlled independently by Eyegone and Eyeless in Drosophila melanogaster. Nat. Genet. 36: 31-39. 14702038

Eberl, D., L. Perkins, M. Engelstein, A. Hilliker, and N. Perrimon. 1992. Genetic and developmental analysis of polytene section 17 of the X chromosome of Drosophila melanogaster. Genetics 130: 569-583. PubMed Citation: 1551578

Feng, S., Thomas, S. and Wang, J. (2012). Diverse tumor pathology due to distinctive patterns of JAK/STAT pathway activation caused by different Drosophila polyhomeotic alleles. Genetics 190(1): 279-82. PubMed Citation: 22048022

Ghiglione, C., Devergne, O., Cerezo, D. and Noselli, S. (2008). Drosophila RalA is essential for the maintenance of Jak/Stat signalling in ovarian follicles. EMBO Rep. 9(7): 676-82. PubMed Citation: 18552769

Feng, S., Huang, J. and Wang, J. (2010). Loss of the Polycomb group gene polyhomeotic induces non-autonomous cell overproliferation. EMBO Rep. 12(2): 157-63. PubMed Citation: 21164514

Fregoso Lomas, M., De Vito, S., Boisclair Lachance, J.F., Houde, J. and Nilson, L.A. (2016). Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity. Curr Biol 26(19):2572-2582. PubMed ID: 27593379

Ghiglione, C., et al. (2002). The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis Development 129: 5437-5447. 12403714

Gilbert, M. M., Weaver, B. K., Gergen, J. P. and Reich, N. C. (2005). A novel functional activator of the Drosophila JAK/STAT pathway, unpaired2, is revealed by an in vivo reporter of pathway activation. Mech. Dev. 122(7-8): 939-48. Medline abstract: 15925495

Grammont, M. and Irvine, K. D. (2002). Organizer activity of the polar cells during Drosophila oogenesis. Development 129: 5131-5140. 12399305

Harrison, D., Binari, R., Nahreini, T. Gilman, M. and Perrimon, N. (1995). Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects. EMBO J. 14: 2857-2865. PubMed Citation: 7796812

Harrison, D. A., et al. (1998). Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway. Genes Dev. 12(20): 3252-63. PubMed Citation: 9784499

Hombria, J. C., Brown, S., Hader, S. and Zeidler, M. P. (2005). Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand. Dev. Biol. 288(2): 420-33. 16277982

Hou, X.S., Melnick, M.B. and Perrimon, N. (1996). marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the Mammalian STATs. Cell 84: 411-419. PubMed Citation: 8608595

Ingaramo, M. C., Sanchez, J. A., Perrimon, N. and Dekanty, A. (2020). Fat body p53 regulates systemic insulin signaling and autophagy under nutrient stress via Drosophila Upd2 repression. Cell Rep 33(4): 108321. PubMed ID: 33113367

Jiang, H., et al. (2009). Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut. Cell 137(7): 1343-55. PubMed Citation: 19563763

Jinks, T. M., et al. (2000). The JAK/STAT signaling pathway is required for the initial choice of sexual identity in Drosophila melanogaster. Molec. Cell 5: 581-587. PubMed Citation: 10882142

Johansen, K. A., Iwaki, D. D. and Lengyel, J. A. (2003). Localized JAK/STAT signaling is required for oriented cell rearrangement in a tubular epithelium. Development 130: 135-145. 12441298

Johnson, A. N., Mokalled, M. H., Haden, T. N. and Olson, E. N. (2011). JAK/Stat signaling regulates heart precursor diversification in Drosophila. Development 138(21): 4627-38. PubMed Citation: 21965617

Jung, S. H., Evans, C. J., Uemura, C. and Banerjee, U. (2005). The Drosophila lymph gland as a developmental model of hematopoiesis. Development 132(11): 2521-33. 15857916

Juni, N., Awasaki, T., Yoshida, K. and Hori, S. H. (1996). The Om(1E) mutation in Drosophila ananassae causes compound eye overgrowth due to tom retrotransposon-driven overexpression of a novel gene. Genetics 143: 1257-1270. PubMed Citation: 8807298

Kitadate, Y., Shigenobu, S., Arita, K. and Kobayashi, S. (2007). Boss/Sev signaling from germline to soma restricts germline-stem-cell-niche formation in the anterior region of Drosophila male gonads. Dev. Cell 13: 151-159. PubMed Citation: 17609117

Kolahgar, G., Suijkerbuijk, S. J., Kucinski, I., Poirier, E. Z., Mansour, S., Simons, B. D. and Piddini, E. (2015). Cell competition modifies adult stem cell and tissue population dynamics in a JAK-STAT-dependent manner. Dev Cell 34(3):297-309. PubMed ID: 26212135

Lee, J. H., Bassel-Duby, R., Olson, E. N. (2014) Heart- and muscle-derived signaling system dependent on MED13 and Wingless controls obesity in Drosophila. Proc Natl Acad Sci U S A 111: 9491-9496. PubMed ID: 24979807

Lee, J.H., Lee, C.W., Park, S.H. and Choe, K.M. (2017). Spatiotemporal regulation of cell fusion by JNK and JAK/STAT signaling during Drosophila wound healing. J Cell Sci 130(11):1917-1928. PubMed ID: 28424232

Li, Q., Li, S., Mana-Capelli, S., Roth Flach, R. J., Danai, L. V., Amcheslavsky, A., Nie, Y., Kaneko, S., Yao, X., Chen, X., Cotton, J. L., Mao, J., McCollum, D., Jiang, J., Czech, M. P., Xu, L. and Ip, Y. T. (2014). The conserved Misshapen-Warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila. Dev Cell 31: 291-304. PubMed ID: 25453828

Lin, T. H., Yeh, T. H., Wang, T. W. and Yu, J. Y. (2014). The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary. Genetics 198(3): 1087-99. PubMed ID: 25161211

Liu, S., Kim, T. H., Franklin, D. A. and Zhang, Y. (2017). Protection against high-fat-diet-induced obesity in MDM2(C305F) mice due to reduced p53 activity and enhanced energy expenditure. Cell Rep 18(4): 1005-1018. PubMed ID: 28122227

Luo, W. and Sehgal, A. (2012). Regulation of circadian behavioral output via a MicroRNA-JAK/STAT circuit. Cell 148(4): 765-79. PubMed Citation: 22305007

Manning, L.A.1, Weideman, A.M., Peercy, B.E. and Starz-Gaiano, M. (2015). Tissue landscape alters adjacent cell fates during Drosophila egg development. Nat Commun 6: 7356. PubMed ID: 26082073

McGregor, J. B., Xi, R. and Harrison, D. A. (2002). JAK signaling is somatically required for follicle cell differentiation in Drosophila. Development 129: 705-717. 11830571

Moberg, K. H., Schelble, S., Burdick, S. K. and Hariharan, I. K. (2005). Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev. Cell 9(5): 699-710. 16256744

Mukherjee, T., Schäfer, U. and Zeidler, M. P. (2006). Identification of Drosophila genes modulating janus kinase/signal transducer and activator of transcription signal transduction. Genetics 172(3): 1683-97. PubMed citation: 16387886

Oldefest, M., Nowinski, J., Hung, C. W., Neelsen, D., Trad, A., Tholey, A., Grotzinger, J. and Lorenzen, I. (2013). Upd3--an ancestor of the four-helix bundle cytokines. Biochem Biophys Res Commun 436: 66-72. PubMed ID: 23707937

Patel, P. H., Dutta, D. and Edgar, B. A. (2015). Niche appropriation by Drosophila intestinal stem cell tumours. Nat Cell Biol 17: 1182-1192. PubMed ID: 26237646

Perrimon, N. and Mahowald, A. (1986). l(1)hopscotch, A larval-pupal zygotic lethal with a specific maternal effect on segmentation in Drosophila. Dev. Biol. 118: 28-41. PubMed Citation: 3095163

Rajan, A. and Perrimon, N. (2012). Drosophila cytokine Unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion. Cell 151: 123-137. PubMed ID: 23021220

Recasens-Alvarez, C., Ferreira, A. and Milán, M. (2017). JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling. Nat Commun 8: 13815. PubMed ID: 28045022

Santabarbara-Ruiz, P., et al. (2015). ROS-induced JNK and p38 signaling is required for Unpaired cytokine activation during Drosophila regeneration. PLoS Genet 11: e1005595. PubMed ID: 26496642

Seeds, A.M., Tsui, M.M., Sunu, C., Spana, E.P. and York, J.D. (2015). Inositol phosphate kinase 2 is required for imaginal disc development in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26647185

Sefton, L., et al. (2000). An extracellular activator of the Drosophila JAK/STAT pathway is a sex-determination signal element. Nature 405: 970-973. PubMed Citation: 10879541

Seong, K. H., Ly, N. H., Katou, Y., Yokota, N., Nakato, R., Murakami, S., Hirayama, A., Fukuda, S., Kang, S., Soga, T., Shirahige, K. and Ishii, S. (2020). Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells. Commun Biol 3(1): 208. PubMed ID: 32367035

Silver, D. L. and Montell, D. J. (2001). Paracrine signaling through the JAK/STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell 107: 831-841. 11779460

Singh, S. R., Liu, W. and Hou, S. X. (2007). The adult Drosophila malpighian tubules are maintained by multipotent stem cells. Cell Stem Cell 1(2): 191-203. PubMed Citation: 18371350

Singh, S. R., Zeng, X., Zheng, Z. and Hou, S. X. (2011). The adult Drosophila gastric and stomach organs are maintained by a multipotent stem cell pool at the foregut/midgut junction in the cardia (proventriculus). Cell Cycle 10(7): 1109-20. PubMed Citation: 21403464

Sotillos, S., Díaz-Meco, M. T., Moscat, J. and Castelli-Gair Hombría, J. (2008). Polarized subcellular localization of Jak/STAT components is required for efficient signaling. Curr. Biol. 18(8): 624-9. PubMed Citation: 18424141

Staley, B. K. and Irvine, K. D. (2010). Warts and Yorkie mediate intestinal regeneration by influencing stem cell proliferation. Curr. Biol. 20(17): 1580-7. PubMed Citation: 20727758

Toledano, H., D'Alterio, C., Czech, B., Levine, E. and Jones, D. L. (2012). The let-7-Imp axis regulates ageing of the Drosophila testis stem-cell niche. Nature 485: 605-610. Pubmed: 22660319

Torres, P. L., López-Schier, H. and St Johnston, D. (2003). A Notch/Delta-dependent relay mechanism establishes anterior-posterior polarity in Drosophila. Dev. Cell 5: 547-558. 14536057

Tsai, Y.-C. and Sun, Y. H. (2004). Long-range effect by Upd, a ligand for Jak/STAT pathway, on cell cycle in Drosophila eye development. Genesis 39: 141-153. 15170700

Van de Bor, V., et al. (2011). Asymmetric localisation of cytokine mRNA is essential for JAK/STAT activation during cell invasiveness. Development 138(7): 1383-93. PubMed Citation: 21350010

Verderosa, F.J. and Muller, H.J. (1954). Another case of dissimilar characters in Drosophila apparently representing changes of the same locus. Genetics 39: 999

Vollmer, J., Fried, P., Aguilar-Hidalgo, D., Sanchez-Aragon, M., Iannini, A., Casares, F. and Iber, D. (2017). Growth control in the Drosophila eye disc by the cytokine Unpaired. Development 144(5): 837-843. PubMed ID: 28246213

Vied, C., Reilein, A., Field, N. S. and Kalderon, D. (2012). Regulation of stem cells by intersecting gradients of long-range niche signals. Dev Cell 23: 836-848. PubMed ID: 23079600

Wang, Y. H. and Huang, M. L. (2009). Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development. Mech. Dev. 126(10): 781-790. PubMed Citation: 19733656

Wawersik, M., Milutinovich, A., Casper, A. L., Matunis, E., Williams, B. and Van Doren, M. (2005). Somatic control of germline sexual development is mediated by the JAK/STAT pathway. Nature 436(7050): 563-7. 16049490

Wieschaus, E., Nüsslein-Volhard, C. and Jurgens, G. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Part III. Zygotic loci on the X-chromosome and fourth chromosome. Wilhelm Roux's Arch. Dev. Biol. 193: 296-307.

Wu, M., Pastor-Pareja, J. C. and Xu, T. (2010). Interaction between Ras^V12 and scribbled clones induces tumour growth and invasion. Nature 463(7280): 545-8. PubMed Citation: 20072127

Xi, R., McGregor, J. R. and Harrison, D. A. (2003). A gradient of JAK pathway activity patterns the anterior-posterior axis of the follicular epithelium. Dev. Cell 4: 167-177. 12586061

Xu, N., et al. (2011). EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells. Dev. Biol 354(1): 31-43. PubMed Citation: 21440535

Yang, H., Kronhamn, J., Ekström, J.O., Korkut, G.G. and Hultmark, D. (2015). JAK/STAT signaling in Drosophila muscles controls the cellular immune response against parasitoid infection. EMBO Rep [Epub ahead of print]. PubMed ID: 26412855

Yasugi, T., Umetsu, D., Murakami, S., Sato, M. and Tabata, T. (2008). Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT. Development 135: 1471-1480. PubMed Citation: 18339672

Yatsu, J., Hayashi, M., Mukai, M., Arita, K., Shigenobu, S. and Kobayashi, S. (2008). Maternal RNAs encoding transcription factors for germline-specific gene expression in Drosophila embryos. Int. J. Dev. Biol. 52: 913-923. PubMed Citation: 18956321

Zeidler, M. P., Perrimon, N. and Strutt, D. I. (1999). Polarity determination in the Drosophila eye: a novel role for Unpaired and JAK/STAT signaling. Genes Dev. 13: 1342-1353. PubMed Citation: 10346822

Zhang, Y., You, J., Ren, W. and Lin, X. (2013). Drosophila glypicans Dally and Dally-like are essential regulators for JAK/STAT signaling and Unpaired distribution in eye development. Dev Biol 375: 23-32. PubMed ID: 23313126

Zhao, X. and Karpac, J. (2017). Muscle directs diurnal energy homeostasis through a Myokine-dependent hormone module in Drosophila. Curr Biol 27(13): 1941-1955. PubMed ID: 28669758

Zhao, Y., Johansson, E., Duan, J., Han, Z., Alenius, M. (2023). Fat- and sugar-induced signals regulate sweet and fat taste perception in Drosophila. Cell Rep, 42(11):113387 PubMed ID: 37934669