Expression of HOP mRNA is ubiquitous throughout the embryo and not spatially activated (Binari, 1994).

JAK pathway and follicle cell identity

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).

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

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

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

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

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).

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).

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).

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.

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).

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).

Effects of Mutation or Deletion

The lethal phase of null alleles of hop occurs at the larval-pupal interface associated with a small imaginal disc phenotype. hop is required maternally because embryos derived from female hop mutants die with specific defects. Embryos produced from homozygous hop mutants show segment specific defects. The extent of these defects depends upon both the strength of the allele and the paternal contribution. In the most extreme case embryos exhibit defects associated with five segments T2, T3, A4, A5, and A8 [Images]. In the less extreme phenotype defects are only associated with A5. Thus, activity of hop is required both for the maintenance and continued cell division of diploid imaginal precursors and for the establishment of the full array of segments (Perrimon, 1986).

The Drosophila Tumorous-lethal (Tum-l) mutation acts as an activated oncogene, causing hematopoietic neoplasms, overproliferation, and premature differentiation. Tum-l is a dominant mutation in the hopscotch locus, which is required for cell division and for proper embryonic segmentation. The Tum-l temperature-sensitive period for melanotic tumor formation includes most of larval and pupal development (Hanratty, 1993).

A single amino acid change in HOP is associated with the Tum-l mutation. Overexpression of either wild-type hop or Tum-l in the larval lymph glands causes melanotic tumors and lymph gland hypertrophy. In addition, overexpression of Hop in other larval tissues leads to pattern defects in the adult or to lethality. Overexpression of either hop or Tum-l in Drosophila cell culture results in tyrosine phosphorylation of HOP protein (Harrison, 1995).

A true revertant of the hop/Tum-l mutation has been generated in which both the molecular lesion and the mutant hematopoietic phenotype revert back to wild type (Luo, 1995).

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

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

Gamma interferon (IFN-gamma) induces both the tyrosine and serine phosphorylation of Stat1. Stat1 serine phosphorylation is required for maximal transcriptional activity of Stat1. Stat1 tyrosine phosphorylation is not a prerequisite for Stat1 serine phosphorylation, although an active Jak2 kinase is required for both phosphorylation events. Stat1 serine phosphorylation occurs with a more delayed time course than tyrosine phosphorylation. The occurrence of serine phosphorylation without tyrosine phosphorylation suggests that serine phosphorylation takes place in the cytoplasm. Experiments performed with cells expressing either dominant-negative or constitutively active Ras protein indicate that the Ras-mitogen-activated protein kinase pathway is probably not involved in IFN-gamma-induced Stat1 serine phosphorylation. A kinase capable of correct Stat1 serine phosphorylation is detected in partially purified cytoplasmic extracts from both IFN-gamma-treated and untreated cells (Zhu, 1997).

Loss of zygotic outstretched activity causes segmentation defects in the Drosophila embryo that resemble the phenotype of hopscotch and stat92E mutant embryos. 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, zygotic os activity is essential but maternal activity is not, as evidenced by the lack of a maternal effect phenotype for os mutants 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 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 and references).

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. For example, combination of the embryonic lethal allele updYC43 with the viable allele oso results in viable adult flies with outstretched wings (Harrison, 1998 and references).

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

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, 1999a and references).

Loss of function (LOF) clones for mutations in the Drosophila JAK and STAT homologs were generated by the FLP/FRT system. Tangential sections through LOF clones of both hop and stat alleles show a regular array of ommatidia containing a wild-type complement of correctly differentiated and correctly positioned photoreceptor cells. Thus, the JAK/STAT pathway is not absolutely required for imaginal disc cell proliferation, cell fate specification, or differentiation. Mutant clones are, however, associated with stereotyped defects in ommatidial polarity (Zeidler, 1999a).

A large proportion of hop LOF clones result in polarity defects in which ommatidia straddling the polar boundary of the clone exhibit inverted DV polarity. The phenotype is strongest in larger clones and in clones in which the polar boundary runs parallel to the equator. Typically, one or two ommatidial rows are inverted, with the strongest phenotype observed showing about five inverted rows. Mutant ommatidia in the center of the clone and on the equatorial margin of the clone show a normal orientation. Both totally mutant ommatidia adjacent to the polar boundary and chimeric ommatidia comprising both wild-type and mutant cells on the clonal border can assume an inverted fate. Occasional inversions are observed in clusters immediately outside the clone in which all of the photoreceptors are wild type. LOF hop clones examined in third instar imaginal discs show the same phenotype (Zeidler, 1999a).

The downstream pathway component STAT was also tested by inducing clones of stat92E alleles. These give qualitatively identical phenotypes to hop clones, but at a lower penetrance. The frequency with which inversions are recovered is increased in a genetic background heterozygous for hop, demonstrating that removal of a single copy of hop can sensitize the pathway to loss of stat92E. The weak nature of the stat92E phenotype would appear to indicate that the stat92E gene product is only partially required to transduce the hop-mediated signal. Although unexpected, this finding is consistent with previous evidence that more than one STAT homolog exists in flies, and suggests that they act semiredundantly in ommatidial polarity determination. Thus, the juxtaposition of wild-type cells and cells unable to transduce the JAK/STAT signal can generate ectopic axes of ommatidial mirror-image symmetry that resemble the normal equator (Zeidler, 1999a).

As LOF JAK/STAT clones result in ectopic axes of ommatidial symmetry, the effects of ectopic activation of the pathway were examined by misexpression of the pathway ligand Upd/Outstretched. GOF Upd clones were generated by a combination of the FLP/FRT cassette, such that Upd is expressed in discrete groups of marked cells in the developing eye. This results in inversion of ommatidial polarity in the wild-type tissue on the equatorial side of the clone, with the greatest effect observed in clones closer to the poles of the disc. Taken together, these LOF and GOF results indicate that JAK/STAT function across the DV axis of the eye disc is necessary for the normal establishment of a single axis of ommatidial mirror-image symmetry along the DV midline, and is sufficient to define ectopic axes of mirror-image symmetry (Zeidler, 1999a).

An interesting aspect of the original P-element-mediated insertional mutation in the stat92E locus (stat92E06346) is the lacZ expression pattern produced by this enhancer detector. Eye discs from larvae carrying this insertion (subsequently referred to as stat92E-lacZ) show a gradient of lacZ activity that is highest at the poles and decreases to a low point at the DV midline. Increased expression is also seen in the ocellar spot region, and, independently, in many of the macrophage-like blood cells often associated with the eye imaginal disc complex. However, in situ hybridization experiments undertaken with probes specific for the stat92E transcript show ubiquitous expression of stat92E mRNA in third instar eye discs, suggesting that this enhancer detector might only report a subset of stat92E transcript expression (Zeidler, 1999a).

An intriguing possibility was that stat92E-lacZ expression might be related to JAK/STAT pathway activity. stat92E-lacZ staining was therefore examined in larvae carrying the constitutively active hopTuml allele of Drosophila JAK. In hopTuml eye discs with uniformly increased JAK/STAT activity, the overall level of lacZ activity is consistently lower than in discs from wild-type siblings stained in parallel. Additional experiments show that the level of stat92E-lacZ expression is inversely proportional to the level of JAK/STAT pathway activation: High activation produced by Upd expression abolishes stat92E-lacZ activity; moderate activation produced by the hopTuml allele gives reduced activity, whereas cells in which there is no JAK/STAT signaling (such as hop clones) show maximal levels of stat92E-lacZ activity. Comparing the results of these experiments with the endogenous pattern of stat92E-lacZ staining in the eye disc, it is concluded that JAK/STAT activity must be highest at the DV midline (where stat92E-lacZ activity is lowest) and low at the poles (where stat92E-lacZ activity is upregulated to levels similar to those seen in hop clones) with a gradient of JAK/STAT activity present between these extremes (Zeidler, 1999a).

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, 1999a).

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, 1999a).

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, 1999a).

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, 1999a).

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, 1999a).

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, 1999a).

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, 1999a).

The Drosophila Jak kinase Hopscotch is required for multiple developmental processes in the eye

Jak kinases are critical signaling components in hematopoiesis. While a large number of studies have been conducted on the roles of Jak kinases in the hematopoietic cells, much less is known about the requirements for these tyrosine kinases in other tissues. Loss of function mutations in the Drosophila Jak kinase Hopscotch (Hop) were used to determine the role of Hop in eye development. Hop is required for cell proliferation/survival in the eye imaginal disc, for the differentiation of photoreceptor cells, and for the establishment of the equator and of ommatidial polarity. These results indicate that hop activity is required for multiple developmental processes in the eye, both cell-autonomously and nonautonomously (Luo, 1999).

In the most extreme cases, eyes of homozygous or hemizygous hopmsv1 mutant flies are completely missing with a concomitant duplication of the antenna. A milder and also slightly more frequent abnormality is the small eye phenotype. Compared with the eye in wild-type, the small eye is reduced in size in the D/V axis, has fewer ommatidia, and exhibits irregular ommatidial arrays with duplicated bristles between ommatidia. Most frequently, the transheterozygotes show roughness along the equatorial region in which ommatidial fusion and duplicated bristles are seen. Both misrotations and changes of chirality of ommatidia are observed. For example, some of the ommatidia rotated either fewer or more than 180 degrees and display chirality defects. Finally, some of the ommatidia are fused (Luo, 1999).

The ommatidia within hop clones are generally normal, with occasional missing photoreceptor cells or polarity defects. However, many hop clones (in particular the rare larger clones) show a dramatic effect on the polarity of neighboring ommatidia. Strikingly, these clones generate an ectopic equator at the polar margin of the clone. Ommatidia at the polar margin of homozygous hop clones reverse their polarity such that the ommatidia point away from rather than toward the equator, thus creating an ectopic equator. Moreover, when a clone is observed very close to the D/V midline, the equator is deflected away and forms along the border of the clone. Most of the ommatidia with reversed polarity are composed entirely of wild-type photoreceptor cells. Thus, the requirement for hop in establishing ommatidial polarity is not cell autonomous. Since clones in both the dorsal and the ventral halves show the same phenotypes, Hop is required throughout the eye disc (Luo, 1999).

The lacZ insertion associated with a stat allele can be used as a reporter of Hop kinase activity. In wild-type eye discs, a gradient of lacZ activity is observed, with the highest and the lowest activity being observed at the poles and the equator, respectively. These studies show that Stat 92E expression is downregulated in the equatorial region of the eye disc and that Hop activity is graded, with its peak at the equator fading toward the poles. This phenomenon may suggest a negative feedback mechanism for Stat regulation (Luo, 1999).

Mutations in both wingless signaling components and hop affect equator formation nonautonomously, suggesting that a secondary diffusible signal exists downstream of them. However, their phenotypes are different in two ways: (1) mutants in wingless pathway components affect polarity on the equatorial side of the clone, whereas in hop mutant clones the polarity is reversed on the polar side, and (2) an ectopic equator forms in the center of a wingless pathway mutant clone, whereas such an ectopic equator forms outside a hop clone. The wg-signaling data suggest that (in addition to its indirect role on fng expression and thus Notch activation) Wg signaling controls a diffusible factor as a secondary signal that is either up- or down-regulated (depending on whether it is a positive or negative factor. The nonautonomy of the hop mutant clones also suggests that a secondary diffusible signal acts downstream of Hop. However, it might act in the opposite direction from the one regulated by Wg due to the opposite influence on polarity by hop- and wg-signaling components, respectively. The full understanding of the nature of the Wg- and Hop-associated phenotypes, and of their potential interactions and secondary signals activated, will only be possible when the presumptive secondary signals are identified (Luo, 1999)

Influence of fat-hippo and notch signaling on the proliferation and differentiation of Drosophila optic neuroepithelia

The Drosophila optic lobe develops from neuroepithelial cells, which function as symmetrically dividing neural progenitors. This study describes a role for the Fat-Hippo pathway in controlling the growth and differentiation of Drosophila optic neuroepithelia. Mutation of tumor suppressor genes within the pathway, or expression of activated Yorkie, promotes overgrowth of neuroepithelial cells and delays or blocks their differentiation; mutation of yorkie inhibits growth and accelerates differentiation. Neuroblasts and other neural cells, by contrast, appear unaffected by Yorkie activation. Neuroepithelial cells undergo a cell cycle arrest before converting to neuroblasts; this cell cycle arrest is regulated by Fat-Hippo signaling. Combinations of cell cycle regulators, including E2f1 and CyclinD, delay neuroepithelial differentiation, and Fat-Hippo signaling delays differentiation in part through E2f1. Roles for Jak-Stat and Notch signaling were also characterized. These studies establish that the progression of neuroepithelial cells to neuroblasts is regulated by Notch signaling, and suggest a model in which Fat-Hippo and Jak-Stat signaling influence differentiation by their acceleration of cell cycle progression and consequent impairment of Delta accumulation, thereby modulating Notch signaling. This characterization of Fat-Hippo signaling in neuroepithelial growth and differentiation also provides insights into the potential roles of Yes-associated protein in vertebrate neural development and medullablastoma (Reddy, 2010).

Both normal development and homeostasis require that cells transition from proliferating undifferentiated cells to quiescent differentiated cells. Failure to undergo this transition results in tumor formation, whereas premature differentiation results in hypotrophy. Some tissues balance proliferation and differentiation by employing stem cells that divide asymmetrically to yield both a stem cell and a progenitor cell, which will then give rise to differentiated cells. Most of the Drosophila central nervous system develops in this way: individual cells within the embryonic ectoderm become specified as neural stem cells called neuroblasts (NBs), which divide asymmetrically to yield a neuroblast and a progenitor cell called a ganglion mother cell (GMC). By contrast, much of the vertebrate central nervous system initially develops from neuroepithelia (NE), sheets of epithelial neural progenitor cells that function as symmetrically dividing neural stem cells. This provides for rapid expansion of neural tissue, and then, as development proceeds, asymmetrically dividing progenitor cells arise, although the mechanisms that govern their appearance are not well understood. The optic lobe of Drosophila is unlike the rest of the Drosophila nervous system in that, akin to the vertebrate nervous system, it develops from NE. The optic lobe may thus serve as a model in which the powerful experimental approaches available in Drosophila can be used to investigate mechanisms that control the growth and differentiation of NE (Reddy, 2010).

At the end of larval development, the optic lobes comprise the lateral half of each of the two brain hemispheres, and are organized into lamina, medulla and lobula layers. The optic lobes originate from clusters of epithelial cells that invaginate from a small region on the surface of the embryo (the optic placode). During larval development, these cells separate into an inner optic anlagen (IOA), which will give rise to the lobula and inner part of the medulla, and an outer optic anlagen (OOA), which will give rise to the outer part of the medulla and the lamina. Initially, the IOA and OOA are composed entirely of NE cells, but during the third larval instar they begin to differentiate. Along the lateral margin of the OOA, NE cells undergo cell cycle arrest in G1, and then are recruited to differentiate into lamina neurons by signals from the arriving retinal axons. Along the medial margin of the OOA, a wave of differentiation sweeps across the NE from medial to lateral, converting NE cells into medulla NBs. These NBs divide perpendicularly to the plane of the neuroepithelium, and appear to follow a NB developmental program, giving rise to additional self-renewing NBs, and to GMCs, which ultimately give rise to neurons (Reddy, 2010).

The Fat-Hippo signaling pathway encompasses distinct downstream branches that regulate planar cell polarity and gene expression. Transcriptional targets of the pathway include genes that influence cell proliferation and cell survival, and consequently Fat-Hippo signaling is an important regulator of growth from Drosophila to vertebrates. The influence of Fat-Hippo signaling on transcription is mediated by a co-activator protein, called Yorkie (Yki) in Drosophila and Yes-associated protein (YAP) in vertebrates. Warts (Wts)-mediated phosphorylation and binding to cytoplasmic proteins negatively regulate Yki by promoting its retention in the cytoplasm. Wts is regulated in at least two ways: Wts kinase activity is promoted by Hippo; and Wts protein levels are influenced by Dachs. Upstream regulators of the pathway include the large cadherin Fat, and the FERM-domain proteins Merlin (Mer) and Expanded (Ex). Fat acts as a transmembrane receptor, regulated by the cadherin Dachsous (Ds), and the cadherin-domain kinase Four-jointed (Fj). The mechanisms that regulate Ex and Mer are not completely understood, but Ex localization can be influenced by Fat, and, in mammalian cells, Mer mediates an influence of contact inhibition on Hippo signaling. Genetic studies in Drosophila have also revealed that the relative contributions of pathway components can vary among different tissues (Reddy, 2010).

Optic NE cells proliferate during larval development, but aside from a requirement for the transcription factor DVSX1 (Erclik, 2008), how this proliferation is regulated is not understood. The progression of NE cells to medulla NBs in the OOA is antagonized by Jak-Stat signaling (Yasugi, 2008), but, aside from this, the regulation of this differentiation wave is not understood. This study demonstrates that Fat-Hippo signaling regulates the proliferation and differentiation of NE cells in the optic lobe. By contrast, Fat-Hippo signaling does not detectably influence the proliferation or differentiation of NBs or their progeny. A role is identified for Notch signaling in controlling the progression of NE cells to medulla NBs, and relationships are characterized between the Fat-Hippo, Jak-Stat and Notch signaling pathways. The results indicate that a transient pause in the cell cycle is needed for cells to transition from NE cells to NBs, and suggest a model in which a cell cycle arrest modulates Notch signaling by contributing to accumulation of Delta expression. The insights these results provide into the role of Fat-Hippo signaling in NE growth and differentiation in Drosophila are likely to be relevant to recently described roles of YAP in vertebrate neural development and medulloblastoma (Reddy, 2010).

The Fat-Hippo pathway has emerged as an important regulator of growth, but has not previously been implicated in neural development in Drosophila. The observation that expression of an activated form of Yki, or mutation of tumor suppressors in the pathway (i.e. fat, ex or wts), promotes growth, whereas mutation of yki impairs growth, identify a crucial role for Fat-Hippo signaling in regulating the proliferation of optic neural progenitor cells (i.e. NE). Indeed, expression of activated Yki can result in massive overgrowths that are taken up in folded sheets of NE, which push into the central brain, forming tumors of undifferentiated NE cells. Although the influence of Fat-Hippo signaling on NE growth parallels its influence on imaginal discs, the influence of Fat-Hippo signaling on NE differentiation does not, as clones of cells mutant for tumor suppressors in the pathway can differentiate cuticle in the head, thorax and abdomen (Reddy, 2010).

In contrast to the extensive overgrowth and suppressed differentiation of NE, NBs and their more differentiated progeny appear refractory to Fat-Hippo signaling. Developing tissues that are unaffected by Fat-Hippo signaling have not been well characterized. The restriction of Fat-Hippo signaling to the NE is matched by the preferential expression of several pathway components, but even when a constitutively activated form of Yki was expressed outside of the NE, neural development in the central brain was not obviously perturbed. Given the emerging importance of Hippo signaling in cancer, determination of what makes different cell types sensitive or resistant to activated Yki is an important direction for future studies (Reddy, 2010).

The progressive nature of NE to NB differentiation in the optic lobe, with different stages displayed in a spatial pattern, make it a sensitive system for investigating differentiation. The extent of delay associated with Fat-Hippo pathway tumor suppressors varied depending on strength of the mutations, which suggests that progression of NE to NB involves a balance of positive and negative influences. The silencing of Yki expression as cells differentiate further suggests that there is negative feedback of differentiation signals onto Yki, which might normally help to ensure a sharp transition between NE and NBs. When Yki activity is further elevated, by overexpression of activated Yki, a complete block in differentiation could be achieved. The observation that a complete block in differentiation could also be achieved by combining overexpression of wild-type Yki with a mutation that influences Yki phosphorylation (wts) is intriguing in light of observations that several human cancers are associated with an increase in levels of Yki expression, rather than a simple change in its localization or phosphorylation. Thus, it is suggested that the two-hit scenario observed in the optic lobe, in which both Yki activity and Yki levels need to be affected in order to transform cells permanently, could also be relevant to human tumors (Reddy, 2010).

This analysis of optic lobe development and the influence of Fat-Hippo signaling implies that a transient pause in the cell cycle is required for cells to transition from NE to medulla NBs, and that Fat-Hippo signaling influences differentiation via an effect on the cell cycle. This model is supported by several observations: there is normally a cell cycle pause along the edge of the outer optic anlagen NE; inhibition of Fat-Hippo signaling, or activation of Yki, impairs both this cell cycle pause and differentiation; and direct manipulation of multiple cell cycle regulators can delay NE differentiation. Although multiple cell cycle regulators appear to be involved in this cell cycle pause, this analysis implicates E2f1 as a key player. PCNA-GFP is downregulated at the edge of the NE, which indicates that E2f1 activity is low there. As E2f1 activity is negatively regulated by association with Rb, and Rb is negatively regulated by phosphorylation by Cdks, expression of CycD+Cdk4 is expected to increase E2f1 activity. Thus, the significant delay in differentiation observed when CycD+Cdk4 were co-expressed with E2F1+DP could all be due to increased E2f1 activity. Importantly, E2f1 is normally regulated by Fat-Hippo signaling in the optic NE, and E2f1 is functionally important for the influence of Fat-Hippo signaling on NE differentiation, because mutation of E2f1 suppressed the wts-mediated differentiation delay. A cell cycle pause also occurs in conjunction with a wave of differentiation that sweeps across the developing eye imaginal disc; however, direct manipulation of cell cycle progression did not affect the differentiation wave in the eye disc, nor does mutation of wts, hpo or sav affect differentiation of photoreceptor cells, even though it does prevent the normal cell cycle pause in the eye disc (Reddy, 2010).

The transition from NE to NB is regulated by Notch signaling, and the results of this study suggest a model in which high level expression of Dl at the edge of the NE autonomously inhibits Notch activation, resulting in upregulation of L(1)sc, which promotes NB fate. This model is supported by the observations that activation of Notch or mutation of Dl can inhibit NE differentiation. At the same time, high-level expression of Dl should enhance Notch activation in neighboring cells, which, as Dl is upregulated by Notch activation, would contribute to the progressive spread of elevated Dl expression across the NE. This simple model allows for the input of other pathways into NE to NB progression via effects on Dl expression, and indeed this appears to be the point at which Fat-Hippo and Jak-Stat signaling intersect with Notch. As a unifying model, it is proposed that a cell cycle pause facilitates the accumulation of the high levels of Dl expression needed to autonomously block Notch signaling, and thereby to upregulate the expression of proneural genes like L(1)sc. A possible mechanism for this hypothesized effect on Delta is suggested by the recent observation in vertebrate NE that Delta1 transcripts are unstable during S-phase. The hypothesis that the influence of Fat-Hippo signaling on differentiation is due to its effect on Dl expression also provides an explanation for the specificity of this phenotype, as Dl is not generally required for the differentiation of imaginal disc cells (Reddy, 2010).

Studies of homologues of Yki, Sd, Hpo and Wts in the chick neural tube identified influences on proliferation and differentiation (Cao, 2008). These studies identified effects on Sox2-expressing neural progenitor cells, but could not distinguish between effects on NE cells versus other neural progenitor cells. A recent study has also implicated YAP in Hedgehog-associated medulloblastoma. Vertebrate NE cells give rise to progenitor cells (e.g. radial glial cells and basal progenitors) that share with neuroblasts the ability to divide asymmetrically to give rise to both another progenitor cell and a more differentiated cell. Since this analysis of the Drosophila optic lobe indicates that Fat-Hippo signaling functions specifically to regulate the proliferation and differentiation of NE, it is suggested that YAP might also function specifically within NE cells in vertebrates. Notably, the observation that depending on the level of expression, Yki can delay rather than block differentiation, provides for the possibility that YAP-dependent tumors could nonetheless contain a mixture of NE cells and more differentiated cells. In Drosophila, each of the three upstream branches of the pathway (i.e. Fat-dependent, Ex-dependent and Mer-dependent, contribute to Yki regulation in NE. Studies in vertebrates have not addressed how the pathway is normally regulated, but Fat-, Ds- and Fj-related genes are all normally expressed in vertebrate NE, consistent with the possibility that they function there (Reddy, 2010).

Artificially slowing the cell cycle can promote precocious differentiation in the cortex, although in this context increasing cell cycle length was associated with a transition from proliferative to differentiative divisions of basal progenitors, which appear functionally similar to NBs rather than to NE cells. The differentiation of optic lobe NE cells into medulla NBs also differs from the general model of increasing cell cycle length causing differentiation, because NBs proliferate even more rapidly than NE cells, and thus this step is not associated with a general lengthening of the cell cycle, but rather a transient pause. Nonetheless, it is intriguing that, in the spinal cord, overexpression of CyclinD did not block differentiation, but did appear to transiently delay it, reminiscent of the delay in NE to NB progression that this study identified in the optic lobe. Moreover, CyclinD expression is regulated by Hippo signaling in the chick neural tube, and overexpression of CyclinD inhibits differentiation there. Although further studies are required to identify the CyclinD-sensitive mechanism in the vertebrate nervous system, the reported instability of Delta1 transcripts during S phase, together with the role of Notch signaling in maintaining NE progenitors in vertebrates and the analysis of NE differentiation and Dl expression in the Drosophila optic lobe, suggest that the possibility of a general influence of cell cycle progression on Notch signaling warrants further investigation as a contributor to the link between cell cycle progression and differentiation in the nervous system across different phyla (Reddy, 2010).

Concomitant requirement for Notch and Jak/Stat signaling during neuro-epithelial differentiation in the Drosophila optic lobe

The optic lobe forms a prominent compartment of the Drosophila adult brain that processes visual input from the compound eye. Neurons of the optic lobe are produced during the larval period from two neuroepithelial layers called the outer and inner optic anlage (OOA, IOA). In the early larva, the optic anlagen grow as epithelia by symmetric cell division. Subsequently, neuroepithelial cells (NE) convert into neuroblasts (NB) in a tightly regulated spatio-temporal progression that starts at the edges of the epithelia and gradually move towards its centers. Neuroblasts divide at a much faster pace in an asymmetric mode, producing lineages of neurons that populate the different parts of the optic lobe. This paper reconstructs the complex morphogenesis of the optic lobe during the larval period, and establishes a role for the Notch and Jak/Stat signaling pathways during the NE-NB conversion. After an early phase of complete overlap in the OOA, signaling activities sort out such that Jak/Stat is active in the lateral OOA which gives rise to the lamina, and Notch remains in the medial cells that form the medulla. During the third instar, a wave front of enhanced Notch activity progressing over the OOA from medial to lateral controls the gradual NE-NB conversion. Neuroepithelial cells at the medial edge of the OOA, shortly prior to becoming neuroblasts, express high levels of Delta, which activates the Notch pathway and thereby maintains the OOA in an epithelial state. Loss of Notch signaling, as well as Jak/Stat signaling, results in a premature NE-NB conversion of the OOA, which in turn has severe effects on optic lobe patterning. These findings present the Drosophila optic lobe as a useful model to analyze the key signaling mechanisms controlling transitions of progenitor cells from symmetric (growth) to asymmetric (differentiative) divisions (Ngo, 2010).

The structure of the optic lobe primordium of the larva is highly dynamic and, towards the later stages, very complex. As a result, only a rudimentary understanding is available of how the different neuropiles and cell types of the adult optic ganglia map onto the larval optic lobe primordium. Moreover, the dynamic changes in shape that characterize the optic lobe at the different larval stages make it very difficult to interpret mutant phenotypes of genes controlling optic lobe development. This study depicts the normal development of the larval optic lobe, focusing on the outer optic anlage and its derivatives, the distal (outer) medulla and the lamina (see Topology of the developing optic lobe in the larva; Ngo, 2010).

The OOA of the early larva starts out as an expanding rectangular sheet of epithelial cells, formed dorso-ventrally oriented columns of cells. Starting at the late first instar and continuing throughout larval life, the OOA epithelium bends along the dorso-ventral-axis. As a result, cells are aligned in C-shaped curves. What this spatial transformation means when looking at optic lobes sectioned along the 'standard' frontal plane (Ngo, 2010).

During the second larval instar, the OOA becomes subdivided into two domains, visibly separated by a furrow called lamina furrow. Cells lateral of this furrow (OOAl) give rise to the lamina; the much larger medial domain (OOAm) form the distal medulla. At around the time when the lamina furrow divides the OOA into a lateral and medial domain, epithelial cells along the edges of these domains convert into asymmetrically dividing medulla neuroblasts (Mnb). This transition can be followed effectively by labelling optic lobes with anti-Crumbs (Crumbs being expressed at the apical membrane of all ectodermally derived tissues). Once cells have converted to neuroblasts, they 'bud off' progeny in the direction perpendicular to the plane defining the OOA. Because of this directed proliferation, neurons born first come to lie at ever increasing distances from the neuroblast/OOA. At the same time as the medulla neuroblasts divide, new rows of neuroblasts appear as, one by one, rows of epithelial cells along the medio-lateral-axis convert into neuroblasts. In the late larva, medulla neuroblasts start to disappear. Thus, the lineages at the medial edge of the optic lobe, which had been the first to appear, are no longer capped by a neuroblast. The fate of the medulla neuroblasts after they cease to divide has not yet been followed in detail. Similar to neuroblasts of the central brain, they are likely to undergo programmed cell death (Ngo, 2010 and references therein).

The correlation between neuron position and birth date can be visualized by BrdU pulse-chase experiments. Early pulses (24 h) result in faint labelling of medulla neurons located deep. In this experiment, BrdU is taken up by all cells of the epithelial OOA which, around 24 h, divide symmetrically. As the epithelium converts into neuroblasts, all neuroblasts 'inherit' the (by then already diluted) label. When neuroblasts start their rapid asymmetric division, only the first born neurons receive enough BrdU to maintain detectable label; these are the neurons located deeply. Pulses administered at mid-larval stages (72 h) result in strong labelling of neurons located in the medial medulla at deep and intermediate levels. In this experiment, the BrdU pulse reaches the OOA at a stage when the epithelial cells have all but ceased to divide, and the medial cells have converted into rapidly dividing neuroblasts. These are the cells that incorporate BrdU. Due to the rapid division/dilution of the label, only early born neurons, located deeply, receive enough label. Late pulses, followed by immediate fixation, result in labelling of most neuroblasts and superficially located neurons. At this stage, the most medial lineages no longer proliferate (Ngo, 2010).

The description of OOA development above indicates that two spatio-temporal gradients are in the OOAm. One gradient, directed along the medio-lateral axis of the OOAm ('ml-gradient'), describes the sequence in which rows of neuroblasts are formed; the second gradient, directed from the surface inward, perpendicular to the plane of the OOA ('z-axis'), underlies the order in which each neuroblast produces neurons ('z-gradient'). The ml-gradient correlates with the anterior-posterior axis of the retina. Thus, axons that grow towards the first-born OOAm neurons, derived from the most medial row of neuroblasts, are the R7/8 axons originating from posterior retina, as well as L- neurons from the posterior lamina. The next set of axons, arriving later, captures neurons of the next (more lateral) row of OOAm neuroblasts, etc. What this implies is that the ordered progression of NE-NB conversion may match the progression of ingrowing axons, and that this matching may be important for the formation of an ordered medulla neuropile. The significance of the z-gradient has not yet been investigated. It seems likely that, similar to what is known for lineages of the central brain and ventral nerve cord, it accounts for the sequential generation of different neuronal cell types (Ngo, 2010).

The inner optic anlage (IOA) also undergoes a NE-NB conversion, bending along the dv-axis, similar to what has been described above for the OOA. Briefly, in the late larva, the IOA consists of a C-shaped epithelial component (IOAep). Further laterally, neuroblasts derived from the IOA (IOAnb) form a mass of cells that is also bent, and therefore seen twice in a frontal section. The IOA neuroblasts produce two populations of neurons. Neurons pushed anteriorly (or outward, taking into account the curvature of the IOA) become the proximal medulla (Mp); those pushed interiorly, or centrally, become the lobula and lobula plate (Lo) (Ngo, 2010).

Neural progenitors give rise to the diversity of cell types seen in the central nervous system (CNS). Intrinsic factors expressed in progenitors, as well as extrinsic cues from neighboring cells specify cell fate. In both vertebrates and invertebrates, the specification of cell types follows a highly invariant spatio-temporal pattern. Typically, one can distinguish an early phase where the pool of progenitors (e.g., the neuroepithelium of the neural tube in vertebrates) expands by symmetric cell division. Subsequently, progenitors start leaving the pool of expanding cells and either directly differentiate into specific cell types, or undergo asymmetric divisions where one daughter cell keeps the properties of a progenitor, whereas the other differentiates (Ngo, 2010).

Neurogenesis in the Drosophila optic lobe follows a similar pattern. Segregating from the embryonic neuroectoderm as a small epithelial placode, the optic lobe anlagen undergo a phase of growth by symmetric cell division in the early larva, followed by a highly ordered transition into asymmetrically dividing neuroblasts. The medio-lateral gradient that characterizes this transition in the OOA is correlated with the posterior-anterior gradient of eye development: photoreceptor axons of the earliest developing (posterior) row of ommatidia arrive first and capture the first born neurons, formed (in case of the medulla) from the medial edge of the OOA. Later born axons occupy medulla neurons forming later, at increasingly lateral levels. It is to be assumed that this temporal match between target neuronal development and afferent axonal development plays an important role for correctly wiring the optic lobe; this hypothesis, though, requires rigorous testing (Ngo, 2010).

This study shows that the Notch pathway is critically involved in the ordered NE-NB conversion. The most significant effect resulting from decreasing Notch function in the larval brain was the reduction in size of the epithelial optic anlagen, as shown by the loss of the epithelial marker, Crb. It is therefore likely that the function of Notch in the optic anlagen is to maintain its undifferentiated, neuro-epithelial state. Clonal analysis has led to the same conclusion. This would match a similar function of Notch in the embryonic neuroectoderm, where Notch activity is also required for cells to stay epithelial. The only difference is the topology of the neuroblast (i.e., cell that moves out of the epithelium): in the embryonic neuroectoderm, neuroblasts are mostly scattered cells, surrounded on all sides by epithelial cells. In the optic anlagen, there is a continuous front where all epithelial cells convert to neuroblasts. However, this difference aside, the way in which Notch signaling acts and is controlled during the NE/NB conversion could be quite similar in the embryonic neurectoderm and the late larval optic anlagen (Ngo, 2010).

Surprisingly, Notch activity, despite of its continued expression throughout development, appears to be dispensable during the earlier phase of optic lobe development during which the epithelial optic anlagen grow by symmetric mitosis. Neither early temperature shift experiments with Nts, nor temporally restricted optic lobe expression of Su(H)DN resulted in premature neuroblast formation. Also the active lifespan of the optic lobe neuroblasts appear to be independent of Notch activity. Optic lobes of late Nts larvae raised at the restrictive temperature were mostly devoid of (Dpn-positive) neuroblasts, and lineages or overall volume of the medulla primordium were not noticeably enlarged (Ngo, 2010).

Taken together, these findings suggest the following model of Notch signaling in the larval optic lobe. Moderate levels of the Notch ligand Dl, as well as Notch activity, are present in the entire optic lobe anlage of the early larva. Starting during the mid larval stage, the proneural gene l'sc is expressed at the medial margin of the OOA. This expression sets in motion a cascade of events that result in the ordered NE/NB conversion. L'sc locally upregulates Delta and other proneural genes (ase) that promote first the conversion of OOA epithelium to neuroblasts, followed by rapid asymmetric division and neuronal differentiation. At the same time, once cells have converted to neuroblasts, L'sc and Delta are downregulated, even though N stays on in a dynamic manner in neuroblasts and neurons. L'sc remains high in a laterally moving band of cells at the medial OOA margin. A second mechanism that may act on the localized Dl upregulation, where it appears that that the cell cycle arrest in the OOAm, caused by activation of the Fat/Hippo pathway, is prerequisite for the accumulation of Dl. Throughout the third larval instar, a continued, interdependent expression of L'sc and Delta in the OOA could be the mechanism that accounts for the slow, gradual release of neuroblasts from the OOA margin. Thus, L'sc is known to upregulate Delta in other neural precursors, and this could be the case also in the OOA. The L'sc induced peak in Delta levels at the medial OOA margin would then signal to its neighbors laterally, increasing Notch activity, and thereby preventing a premature advance of L'sc towards lateral (Ngo, 2010).

How initiation and maintenance of L'sc is controlled is still unclear. Wingless (Wg), a known activator of proneural genes in other tissues, is expressed in a fairly restricted pattern in the apices of the OOA. It is possible that a long range effect of Wg could be responsible for L'sc activation along the OOA margin (Ngo, 2010).

A large number of studies in vertebrate and invertebrate systems alike suggest that the fundamental role of N in the developing nervous system is to maintain cells in an undifferentiated (neuroepithelial) state at any given moment. Cells released from N activity enter a differentiative pathway (typically accompanied by structurally visible changes, such as a switch from epithelial cell to neuroblast, and/or a switch in mitotic behavior (symmetric vs. asymmetric). The temporally controlled release/birth of neurons from the neuroepithelium is often tied to different cell fates. This has been shown very convincingly in the retina of vertebrates and Drosophila. For example, in the vertebrate retina, the first wave of differentiation results in ganglion cells, the second wave of differentiation at a later point includes photoreceptors, followed by bipolar cells, and others. If N activity is reduced at an early time point, the number of ganglion cells produced increases massively, at the expense of later born cell types. In Drosophila, the first retinal cell type to be born behind the morphogenetic furrow is the R8 photoreceptor. If at the time of R8 specification, N function is decreased, the number of R8 cells is increased, and other cell types born later are decreased. With later pulses of N depletion, one gets different phenotypes; what they all have in common is that the cell types born at the time of the pulse are increased in number, the ones born later decreased (Ngo, 2010).

In the Drosophila OOA investigated in this paper, the temporal progression of neuroblast formation that is controlled by N activity is linked to the coordinated growth between eye (and lamina) and medulla, derived from the OOA. However, it is well possible that the temporal progression is also tied into the control of different cell fates. The way in which the multitude of different medulla cell types map onto the larval optic lobe is not clear. It is most likely that (as in the lineages of the ventral nerve cord) most cell types are specified along the z-axis, which would imply that each part of the OOA (in the medio-lateral and dorso-ventral dimension) would produce the same cell types. However, it is well possible that some cell types which are actually not found in all medulla columns, such as wide field tangential neurons, are produced by different parts of the OOA. Such cell types then might be affected by premature or delayed conversion of the OOA into neuroblasts; identifying specific markers that label cell types at early stages, and using such markers in the background of Notch loss or overactivity, will help clarifying this question (Ngo, 2010).

A role of the Notch pathway has been described for later stages in neural development, that is, the specification of neurons from ganglion mother cells (GMCs). Asymmetric neuroblast proliferation in the ventral nerve cord and brain produces a series of GMCs which each divides one more time into two, often different, neurons/glial cells. It has been shown that this fate choice between sibling pairs depends on Notch activity, both during embryonic and post-embryonic stages. Such may also be the case for the neuroblasts emerging from the OOA. The relatively high level of the Notch reporter, E(spl)m8-lacZ maintained in the OOA-derived lineages would speak for a continued role of Notch in these cells; however, detailed investigations of the neurogenesis of these lineages need to be carried out in order to address the potential later Notch function (Ngo, 2010).

This study found that reduction in Stat activity causes a premature loss of the epithelial state of the OOA. This is accompanied by accelerated proliferation and gross abnormalities in the architecture of the optic lobe neuropile, as also seen in Notch mutant brains. Furthermore, continued activity of Stat in the epithelial OOA is dependent on Notch, and vice versa. The mutual interaction between both signaling pathways is most likely indirect, mediated via a number of intermediate steps. Thus, Delta levels are normal in optic lobes of Statts mutant brains up to 48 h after hatching. It is only during later stages, when the structurally visible premature change of OOA epithelium to neuroblasts occurs in the Statts mutant, that Delta expression is reduced. It remains to be seen what are the intermediate genetic events that interconnect the signaling activities of the Notch and Stat pathway (Ngo, 2010).

The larval optic lobe represents but one of many scenarios in which interdependency between Notch and Stat have been reported. The types of genetic interactions between these pathways appear to be as diverse as the developmental events or cell fates which they control. For example, in the Drosophila ovary, mutual inhibition Notch and Stat set up the boundary between the stalk (Jak/Stat dependent) and the main-body follicle cells (Notch-dependent. In the adult midgut, Notch is necessary for the differentiation of cells derived from the intestinal stem cells (ISCs) into enteroblasts and enterocytes. Thus, high Notch activity in one of the daughter cells derived from an ISC division prompts these cells to become an enteroblast (EB), which then either differentiates into enterocyte (EC) or enteroendocrine cell (EE). High Notch activity in the EB promotes the EC fate; low Notch activity allows for the formation of EEs. Jak/Stat signaling intersects with the Notch pathway at multiple steps: for example, it acts upstream in an activating manner. Thus, under stressful conditions (e.g. bacterial infection or JNK-induced stress), Stat functions to induce Notch to allow for self-renewal and proliferation. In addition, Jak/Stat is required during the differentiation of different ISC-derived cell types. The same kind of dynamic and complex relationship between the two signaling pathways can be seen in the eye, where Stat can function both upstream and downstream of Notch. It has even been suggested that Jak/Stat can function to inhibit Notch as well. From all these studies, it is clear that many of the intermediates which link Notch and Jak/Stat signaling are still unknown. One can envision scenarios where subtle differences in the spatial distribution and timing of signals may contribute to how Stat and Notch interact during development. The Drosophila optic lobe is likely to present a highly favorable system to address these complexities which impact the role of the two signaling pathways (Ngo, 2010).

An extracellular activator of the Drosophila JAK/STAT pathway is a sex-determination signal element

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

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

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

A Drosophila PIAS homologue negatively regulates stat92E

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

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

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

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

mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family

The posterior spiracle defects of the domeless/mom mutation have led to an examination of functions of the Hop/Stat92E pathway in tracheal formation. Trachea form from 10 tracheal pits, 1 per hemisegment. The trachealess gene selects the tracheal primordia in the embryonic ectoderm and drives the conversion of these planar epithelial regions into tracheal pits. The tracheal pits then sprout successively finer branches and fuse together, forming the tracheal network. The trachea is further connected to the posterior spiracle, forming a functional tracheal system. Tracheal formation was examined in mom, hop, and STAT92E mutants by using an enhancer trap line in the trachealess gene (1-eve-1) and an antibody [(mAb)2A12] that stains tracheal branches and trunks. In hop null embryos, trachealess expression is completely abolished and tracheal formation is completely blocked. In paternally rescued embryos, a defective tracheal system forms, generally with several disruptions in the main trunk and several branches. Because all of the mom and STAT92E mutants examined were enhancer trap lines, trachealess gene expression could not be examined by directly using the 1-eve-1 enhancer trap line. However, in the paternally rescued STAT92E and mom mutant embryos, similar to the hop mutant embryos, a defective tracheal system formed, generally with several disruptions in the main trunk and several branches. These data suggest that Mom and the Hop/Stat92E signal transduction pathway play an indispensable role in tracheal formation (Chen, 2002).

The JAK/STAT signal transduction pathway regulates many developmental processes in Drosophila. However, the functional mechanism of this pathway is poorly understood. The Drosophila cyclin-dependent kinase 4 (Cdk4) exhibits embryonic mutant phenotypes identical to those in the Hopscotch/JAK kinase and stat92E/STAT mutations. Specific genetic interactions between Cdk4 and hop mutations suggest that Cdk4 functions downstream of the HOP tyrosine kinase. Cyclin D-Cdk4 (as well as Cyclin E-Cdk2) binds and regulates STAT92E protein stability. STAT92E regulates gene expression for various biological processes, including the endocycle S phase. These data suggest that Cyclin D-Cdk4 and Cyclin E-Cdk2 play more versatile roles in Drosophila development (Chen, 2003).

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

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

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

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

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

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

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

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

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

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

Functional Evolution of the Vertebrate Myb Gene Family: B-Myb, but neither A-Myb nor c-Myb, complements Drosophila Myb in Hemocytes: Genetic interaction with hopscotch

The duplication of genes and genomes is believed to be a major force in the evolution of eukaryotic organisms. However, different models have been presented about how duplicated genes are preserved from elimination by purifying selection. Preservation of one of the gene copies due to rare mutational events that result in a new gene function (neo-functionalization) necessitates that the other gene copy retain its ancestral function. Alternatively, preservation of both gene copies due to rapid divergence of coding and non-coding regions such that neither retains the complete function of the ancestral gene (sub-functionalization) may result in a requirement for both gene copies for organismal survival. The duplication and divergence of the tandemly arrayed homeotic clusters have been studied in considerable detail and have provided evidence in support of the sub-functionalization model. However, the vast majority of duplicated genes are not clustered tandemly, but instead are dispersed in syntenic regions on different chromosomes, most likely as a result of genome-wide duplications and rearrangements. The Myb oncogene family provides an interesting opportunity to study a dispersed multigene family because invertebrates possess a single Myb gene, whereas all vertebrate genomes examined thus far contain three different Myb genes (A-Myb, B-Myb and c-Myb). A-Myb and c-Myb appear to have arisen by a second round of gene duplication, which was preceded by the acquisition of a transcriptional activation domain in the ancestral A-Myb/c-Myb gene generated from the initial duplication of an ancestral B-Myb-like gene. B-Myb appears to be essential in all dividing cells, whereas A-Myb and c-Myb display tissue-specific requirements during spermatogenesis and hematopoiesis, respectively. The absence of Drosophila Myb (Dm-Myb) causes a failure of larval hemocyte proliferation and lymph gland development, while Dm-Myb(-/-) hemocytes from mosaic larvae reveal a phagocytosis defect. Vertebrate B-Myb, but neither vertebrate A-Myb nor c-Myb, can complement these hemocyte proliferation defects in Drosophila. Indeed, vertebrate A-Myb and c-Myb cause lethality in the presence or absence of endogenous Dm-Myb. These results are consistent with a neomorphic origin of an ancestral A-Myb/c-Myb gene from a duplicated B-Myb-like gene. In addition, these results suggest that B-Myb and Dm-Myb share essential conserved functions that are required for cell proliferation. Finally, these experiments demonstrate the utility of genetic complementation in Drosophila to explore the functional evolution of duplicated genes in vertebrates (Davidson, 2004).

To establish whether Dm-Myb is generally required for proliferation of hemocytes, epistasis experiments were conducted using a Dm-Myb null mutation and dominant substitution mutations of the Toll receptor (Tl10b) and the Jak kinase, hopscotch (hopTuml); dominant gain-of-function mutations in these genes result in hyperactivation of their respective pathways leading to hemocyte overproliferation and abnormal lamellocyte differentiation. To determine whether Dm-Myb is required for the dysregulated overproliferation and differentiation phenotypes of Tl10b and hopTuml mutants, double mutant larvae lacking Dm-Myb in conjunction with these dominant alleles of Toll and hopscotch were generated. It was found that, in addition to an overproliferation of plasmatocytes in the primary lymph gland lobes, the secondary lymph gland lobe hemocytes aberrantly differentiate into lamellocytes in hopTuml mutants. It is thought that the normally smaller secondary lymph gland lobes serve as a reservoir of undifferentiated prohemocytes, however, in hopTuml larvae the secondary lobes enlarge with concomitant abnormal differentiation of lamellocytes. While hemocytes in the secondary lymph gland lobes of hopTuml, Dm-Myb-/- double mutants show an increased expression of the lamellocyte enhancer-trap marker, these ß-gal positive cells fail to overproliferate and do not adopt the flattened shape characteristic of differentiated lamellocytes. In summary, an activated JAK/STAT pathway cannot drive the proliferation of hemocytes in the absence of Dm-Myb. In addition, an activated Toll pathway cannot drive the proliferation of hemocytes in the absence of Dm-Myb (Davidson, 2004).

JAK/STAT pathway and the immune response

It has been proposed that germ-line-encoded pattern recognition receptors bind microbial cell wall determinants (such as lipopolysaccharides, mannans, and peptidoglycans) and initiate an immune response, either by activating associated proteases in circulation or by directly triggering intracellular signaling pathways in immune responsive cells. To date, no pattern recognition receptor has been firmly identified in Drosophila and shown to activate an immune response. A primitive complement-like system, evocative of the alternative or the lectin pathways of complement, could be involved in the activation of some of the Drosophila host defense mechanisms. This hypothesis was made attractive by the recent reports that invertebrates such as sea urchins and tunicates have a complement-like system, and produce proteins with structural similarities to vertebrate complement C3 proteins, containing an intrachain thiolester bond. Similar proteins have also been described in the horseshoe crab, a member of the class of arthropods to which Drosophila also belongs (Lagueux, 2000 and references therein).

Drosophila expresses four genes encoding proteins with significant similarities with the thiolester-containing proteins of the complement C3/alpha2-macroglobulin superfamily. The genes are transcribed at a low level during all stages of development, and their expression is markedly up-regulated after an immune challenge. For one of these genes, which is predominantly expressed in the larval fat body, a constitutive expression was observed in gain-of-function mutants of the Janus kinase (JAK) hop and a reduced inducibility in loss-of-function hop mutants. A constitutive expression was observed in gain-of-function Toll mutants. These novel complement-like proteins are likely to play roles in the Drosophila host defense (Lagueux, 2000).

In higher vertebrates, the complement system consists of about 30 serum and cell surface proteins and mediates inflammatory reactions, opsonization of microorganisms for phagocytosis, and direct killing of some pathogens. Activation can occur via the classical antibody-dependent pathway, the alternative pathway, and the lectin pathway, which all converge on the central complement C3 protein. The presence in Drosophila of several proteins with basic structural characteristics similar to complement C3 makes attractive the working hypothesis that an ancient equivalent of the alternative pathway and/or the lectin pathway exists in this species. In vertebrates, the activation of the alternative pathway is initiated by spontaneous hydrolysis of the thiolester bond of complement C3, resulting, through association with other proteins of the complement system, in an active C3 convertase that is normally inactivated by regulatory proteins present on self tissue, but absent from non-self, providing for a relative primitive mode of discriminating self from non-self. In turn, active C3 convertase activates complement C3 and, through an amplification loop, triggers the conventional effector mechanisms of complement. Activation of the lectin pathway is initiated when various sugars present on the surface of microorganisms bind to a collectin, the mannan-binding lectin (MBL), thereby inducing proteolytic cascades that activate complement C3 and the downstream events common to all three activation pathways (Lagueux, 2000).

In the mid-nineties, it became apparent that the complement system is not a unique property of the host defense armatarium of vertebrates. ESTs from cDNA libraries of sea urchin coelomocytes were found to encode a protein with structural similarities to vertebrate complement C3, including an intrachain thiolester motif, plus a homolog of vertebrate factor B, which participates in the activation of complement C3 through the alternative pathway. More recently, an ascidian species was reported to possess homologs of complement C3 and of two mannose-binding lectin-associated proteases (MASPs), plus a homolog of factor B, raising the possibility that equivalents of both the lectin and the alternative activation pathways are present in these deuterostome invertebrates. Experiments with ascidian coelomocytes further indicate that the complement C3-like molecules act as opsonic factors and are activated through a complement-like cascade (Lagueux, 2000 and references therein).

TEPs are also structurally close to alpha2-macroglobulins, which are evolutionary ancient protease inhibitors, from which complement C3 has been proposed to have arisen by gene duplication. Protease inhibitors related to alpha2-macroglobulin have been described in several invertebrates and have been particularly well studied in the horseshoe crab Limulus. Indeed, Limulus alpha2-macroglobulin has been proposed to function as a protease inhibitor, particularly of proteases released by tissue damage caused by injury or pathogens and of soluble or surface bound proteases produced by invading microorganisms. It has been suggested that the first opsonic system could have required no specific recognition or activation mechanism other than the presence of exogenous proteases causing alpha2-macroglobulin to bind directly to the protease-producing organism (Lagueux, 2000 and references therein).

Because Drosophila has four expressed genes encoding proteins with structural similarities to the superfamily of complement C3/alpha2-macroglobulin, significant functional versatilities may be expected, all the more so, because one of the Tep genes, Tep2, gives rise to five different transcripts. Interestingly, the Tep2 transcripts are identical except for a short region of 30 aa. This region is encoded by alternatively spliced exons corresponding to the hypervariable region of the TEPs; it is located in a relative position similar to the bait domain in alpha2-macroglobulins or the anaphylatoxins in complement C3. Alternative splicing has not been reported in vertebrates for members of the complement C3/alpha2-macroglobulin superfamily. By increasing the number of putative recognition motifs for microorganisms or proteases, it may contribute to the fine-tuning of recognition of noxious structural patterns in the absence of the large repertoire of receptors of the adaptive immune response in vertebrates (Lagueux, 2000 and references therein).

The Drosophila plasmatocytes are macrophage-like blood cells that readily engulf bacteria or fungal spores, as well as various cellular debris resulting from injury or apoptosis. Nothing is known about possible opsonization in this model, and a tempting working hypothesis is that the complement-like proteins described here precisely fulfill such a role in the host defense. Future efforts will be directed toward experimentally testing this hypothesis, and it is anticipated that the generation of mutants of the various Tep genes, which all map to the left arm of the second chromosome, will be invaluable in this endeavor (Lagueux, 2000).

TEP1 is produced mainly in the fat body, and its expression is up-regulated by immune challenge. It is hypothesized that, as is the case for immune-induction of antimicrobial peptides in this tissue, the up-regulation would be dependent on either the Toll or the imd pathway. This control is strongly dependent on the JAK hopscotch. Hopscotch is the only JAK identified in Drosophila, and in the gain-of-function mutant hopTum-l, Tep1 is constitutively expressed, whereas its immune-inducibility is dramatically reduced in the loss-of-function mutant hopM38. Gain-of-function mutations of hop have remarkable effects on hemopoiesis in Drosophila and result in overproliferation of blood cells, increased differentiation of lamellocytes, and aggregation of blood cells into masses, which tend to become melanized, a process referred to as melanotic tumor formation. The data thus show that these events are concomitant with an increased transcription of the Tep1 gene. Whether they are causally related remains an open question, but it will be worthwhile investigating whether the TEP1 protein can affect the aggregation of blood cells and the localized induction of melanization (Lagueux, 2000).

Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated

The mechanisms by which an organism becomes immune competent during its development are largely unknown. When infected by eggs of parasitic wasps, Drosophila larvae mount a complex cellular immune reaction in which specialized host blood cells, lamellocytes and crystal cells, are activated and recruited to build a capsule around the parasite egg to block its development. Parasitization by the wasp Leptopilina boulardi leads to a dramatic increase in the number of both lamellocytes and crystal cells in the Drosophila larval lymph gland. Furthermore, a limited burst of mitosis follows shortly after infection, suggesting that both cell division and differentiation of lymph gland hemocytes are required for encapsulation. These changes, observed in the lymph glands of third-instar, but never of second-instar hosts, are almost always accompanied by dispersal of the anterior lobes themselves. To confirm a link between host development and immune competence, mutant hosts in which development is blocked during larval or late larval stages were infected. In genetic backgrounds where ecdysone levels are low (ecdysoneless) or ecdysone signaling is blocked (nonpupariating allele of the transcription factor broad), the encapsulation response is severely compromised. In the third-instar ecdysoneless hosts, postinfection mitotic amplification in the lymph glands is absent and there is a reduction in crystal cell maturation and postinfection circulating lamellocyte concentration. These results suggest that an ecdysone-activated pathway potentiates precursors of effector cell types to respond to parasitization by proliferation and differentiation. It is proposed that, by affecting a specific pool of hematopoietic precursors, this pathway thus confers immune capacity to third-instar larvae (Sorrentino, 2002).

To confirm the correlation between ecdysone deficiency and reduction in encapsulation capacity, the effect of ecd1 on encapsulation was studied in a background in which lamellocytes are produced in lymph glands without parasitization. The temperature-sensitive semidominant lethal mutation hopTum-l causes an overproliferation of circulating hemocytes, the appearance of lamellocytes in large numbers, and the encapsulation of self tissue. The temperature-sensitive period of hopTum-l, like that of ecd1, begins in the second larval instar. The size and number of melanotic capsules was analyzed in third-instar larvae at 18, 25, and 29°C, and adults at 25°C. At all three temperatures, 100% of hopTum-l/Y larvae exhibit multiple capsules. Lowering of ecdysone levels in hopTum-l/Y; ecd1/ecd1 animals results in a mild, but significant, suppression of the hopTum-l melanotic capsule phenotype. Thus, at 18 and 25°C, over 10% of double mutants were completely clear of melanotic capsules, and at 29°C, nearly 20% were devoid of capsules. Furthermore, most double-mutant larvae that did score positively for capsules exhibited fewer and smaller capsules. Tumor penetrance in surviving double-mutant adults at 25°C (86.2%) is consistent with the corresponding larval value. Thus, ecd1 is able to partially suppress the hopTum-l-induced melanotic capsule phenotype (Sorrentino, 2002).

A model is proposed in which lymph gland development and specific steps in hematopoiesis required for the encapsulation response are both tied to a signal transduction pathway triggered by ecdysone, possibly at the L2-L3 transition. This pathway regulates the capacity of lamellocyte and crystal cell precursors to respond to infection. While the precise role of the ecdysone pathway in these steps is not clear, it is possible that ecdysone induces division of hematopoietic progenitors in order to maintain a critical basal population of immature immune effector cells. In addition, ecdysone may trigger differentiation in lamellocyte and/or crystal cell precursors. If so, third-instar ecd mutant lymph glands would have fewer lamellocyte and crystal cell precursors, most or all of which in an immature or unpotentiated state that prevents them from responding to parasitization; the aggregate effect of this would be an unresponsive lymph gland. Availability and application of molecular markers for progenitors versus differentiated lamellocytes and crystal cells will allow validation of this model and facilitate examination of whether or not lymph gland overgrowth mutants, such as hopTum-l, affect cell populations and bypass the requirement for ecdysone. Such studies can then reveal whether suppression of the hopTum-l phenotype in hopTum-l/Y;ecd1 /ecd1 double mutants (with fewer and smaller melanotic tumors than hopTum-l/Y larvae) is due to changes in these progenitor cells or unrelated effects 'downstream' in the encapsulation process (Sorrentino, 2002).

Sequential activation of signaling pathways during innate immune responses in Drosophila

Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. These transcriptional patterns can be dissected into distinct groups. In addition to signaling through the Toll/NFkappaB or Imd/Relish pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, the results may imply a close link between the control of tissue repair and antimicrobial processes (Boutros, 2002).

Lipopolysaccharides (LPS) are the principal cell wall components of gram-negative bacteria. In mammals, exposure to LPS causes septic shock through a Toll-like receptor TLR4-dependent signaling pathway. LPS treatment of Drosophila SL2 cells leads to rapid expression of antimicrobial peptides, such as Cecropins (Cec). SL2 cells resemble embryonic hemocytes and have also been used as a model system to study JNK and other signaling pathways. LPS-responsive induction of the antimicrobial peptides AttacinA (AttA), Diptericin (Dipt), and Cec relies on IKK and Relish. In order to obtain a broad overview on the transcriptional response to LPS in Drosophila, genome-wide expression profiles of SL2 cells were generated at different time points following LPS treatment. Altered expression of 238 genes was detected (Boutros, 2002).

In time-course experiments, a complex pattern of gene expression was observed that can be separated into different temporal clusters. A first group, with peak expression at 60 min after LPS, primarily consists of cytoskeletal regulators, signaling, and proapoptotic factors. This group includes cytoskeletal and cell adhesion modulators such as Matrix metalloprotease-1, WASp, Myosin, and Ninjurin, proapoptotic factors such as Reaper, and signaling proteins such as Puckered and VEGF-2. A second group, with peak expression at 120 min, includes many known defense and immunity genes, such as Cec, Mtk, and AttA, but not the gram-positive-induced peptide Drs. Interestingly, this cluster also includes PGRP-SA, which is a gram-positive pattern recognition receptor in vivo, suggesting possible crossregulation between gram-positive- and gram-negative-induced factors. A third group is transiently downregulated upon LPS stimulation. This cluster includes genes that play a role in cell cycle control, such as String and Rca1. Altogether, these results show that, in response to LPS, a defined gram-negative stimulus, cells elicit a complex transcriptional response (Boutros, 2002).

Clustering revealed a noncanonical group with small proteins that are expressed late and transiently with peak expression at 6 hr after septic injury. One of the clustered transcripts, CG11501, encodes a small Cys-rich protein that is 115 amino acids long and is strongly induced after septic injury. By RT-PCR, it was confirmed that CG11501 is upregulated after septic injury. In order to characterize how CG11501 is controlled after microbial challenge, a candidate pathway approach was undertaken. In an independent study, it was found that totM gene induction, which is part of the same cluster, is dependent on a JAK/STAT signaling pathway. Whether CG11501 induction requires JAK/STAT signaling was examined. Mutations in JAK/STAT pathways in Drosophila have been implicated in various processes during embryonic and larval development. In Anopheles, STAT is activated in response to bacterial infection. Similarly, gain-of-function STAT has been implicated in the transcriptional control of thiolester proteins. Mutant alleles of hopscotch (hop), the Drosophila homolog of JAK were examined. Quantitative PCR shows that CG11501 induction after septic injury is diminished in hop loss-of-function mutants, whereas the expression of Toll and Imd targets drs, and cec is not affected (Boutros, 2002).

This study shows that in addition to known innate immune cascades, JNK and JAK/STAT are required for the transcriptional response during microbial challenge. One transcriptional signature of small secreted peptides can be traced to JAK/STAT signaling. Additionally, JNK signaling controls cytoskeletal genes after an LPS stimulus and after septic injury in vivo. Both in cells and in vivo, JNK pathways are connected to the same upstream signaling cassette that induces NFkappaB targets. Altogether, these results suggest that innate immune signaling pathways closely link cytoskeletal remodeling, as required for tissue repair, and direct antimicrobial actions. The data also provide insights into the connection of temporal patterns and the activation of distinct signaling pathways (Boutros, 2002).

NFkappaB pathways play a central role for innate and adaptive immune response in mammals. In innate immune responses, TLRs on dendritic cells recognize microbial agents and activate NFkappaB, leading to the expression of proinflammatory cytokines and other costimulatory factors required to initiate an adaptive immune response. Additionally, other signaling pathways have been implicated at later stages during immune responses in mammals, but their physiological role in innate immunity remains rather poorly understood. For example, several cytokines, such as IL-6 and IL-11, signal through a JAK/STAT pathway to induce the expression of acute phase proteins. Similarly, JNK pathways are activated in response to TNF and IL-1, may lead to the expression of immune modulators, and are required for T cell differentiation. In Drosophila, studies have investigated two distinct NFkappaB-pathways --Toll and Imd/Rel -- that have been shown to mediate gram-positive/fungal and gram-negative responses. Both pathways induce specific antimicrobial peptides and thereby focus the response on the invading microbial agent. Genetic analysis has shown that functions of the NFkappaB-pathways are separable; flies that are mutant for only one of these pathways are susceptible to subgroups of pathogens. Could the contribution of NFkappaB-dependent and, possibly, other signaling pathways be identified by examining global expression profiles? The obtained data set demonstrates that NFkappaB-independent signaling pathways contribute to the transcriptional patterns observed after microbial infection. Both in cells and in vivo, JNK-dependent targets precede the peak expression of antimicrobial peptides that require NFkappaB. JAK/STAT targets are induced with a distinct temporal pattern that shows late, but only transient, expression characteristics. The stereotyped pathway patterns after microbial challenge suggest that the correct temporal execution of signaling events, similar to signaling during development, may play an important role in the regulation of homeostasis (Boutros, 2002).

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 (hopM38/hopmsv1), confirming the involvement of Drosophila JAK in MP1 expression (Agaisse, 2003).

Genetic analysis of contributions of dorsal group and JAK-Stat92E pathway genes to larval hemocyte concentration and the egg encapsulation response in Drosophila

Drosophila larvae defend themselves against parasitoid wasps by completely surrounding the egg with layers of specialized hemocytes called lamellocytes. Similar capsules of lamellocytes, called melanotic capsules, are also formed around 'self' tissues in larvae carrying gain-of-function mutations in Toll and hopscotch. Constitutive differentiation of lamellocytes in larvae carrying these mutations is accompanied by high concentrations of plasmatocytes, the major hemocyte class in uninfected control larvae. The relative contributions of hemocyte concentration vs. lamellocyte differentiation to wasp egg encapsulation are not known. To address this question, Leptopilina boulardi was used to infect more than a dozen strains of host larvae harboring a wide range of hemocyte densities. A significant correlation exists between hemocyte concentration and encapsulation capacity among wild-type larvae and larvae heterozygous for mutations in the Hopscotch-Stat92E and Toll-Dorsal pathways. Larvae carrying loss-of-function mutations in Hopscotch, Stat92E, or dorsal group genes exhibit significant reduction in encapsulation capacity. Larvae carrying loss-of-function mutations in dorsal group genes (including Toll and tube) have reduced hemocyte concentrations, whereas larvae deficient in Hopscotch-Stat92E signaling do not. Surprisingly, unlike hopscotch mutants, Toll and tube mutants are not compromised in their ability to generate lamellocytes. These results suggest that circulating hemocyte concentration and lamellocyte differentiation constitute two distinct physiological requirements of wasp egg encapsulation and Toll and Hopscotch proteins serve distinct roles in this process (Sorrentino, 2004).

These results suggest that the suppression of encapsulation capacity by loss of function of hop, Tl, or tub is likely to be due to distinct requirements of these genes. The suppression of lymph gland response to parasitization in the hopM4 background is consistent with the observed reduction in hopM4/Y encapsulation capacity and suggests that the Hopscotch protein is necessary for a parasite-induced signal for lamellocyte differentiation. This signal for lamellocyte differentiation is most likely mediated by the transcription factor Stat92E: Loss of function of one copy of Stat92E suppresses the penetrance of the hopTum-l-induced melanotic tumor phenotype and Stat92E is constitutively activated in Drosophila cell cultures that overexpress HopTum-l. These results are consistent with the proposed Stat92E-dependent lamellocyte signal: stat92E larvae are immune compromised and are unable to mount an efficient egg encapsulation response despite exhibiting control circulating hemocyte concentration levels. Additionally, mean circulating lamellocyte percentage in hopTum-l/Y; stat92EHJ/stat92EHJ larvae that are tumor-free is ~1%, which is indistinguishable from the control value (Sorrentino, 2004).

In contrast to Hop and Stat92E, Toll and Tube appear not to play a role in lamellocyte differentiation; rather, loss-of-function mutations in Toll or tube probably suppress encapsulation via other mechanisms. Since Toll and tube larvae have very few circulating hemocytes, reduction in encapsulation in Tl and tub mutants might be due to defects in wasp egg recognition or a reduction in hemocyte proliferation that normally follows parasitization. The effect of these mutations on crystal cells is unclear. While the possibility that these mutations reduce encapsulation capacity by reducing the crystal cell population cannot be ruled out, this is unlikely, since Black cells mutant larvae without functional crystal cells are immune competent and can still successfully encapsulate wasp eggs. The fact that lymph glands of loss-of-function Tl and tub mutant larvae can support lamellocyte differentiation suggests that the low circulating hemocyte concentration in Tl and tub larvae in itself does not hinder lamellocyte differentiation or the ability of the lymph gland to disperse after the wasp egg is introduced into the hemocoel. Given that gain-of-function Tl alleles induce lamellocyte differentiation, the lack of effect of Tl- on lamellocyte differentiation is somewhat unexpected, and it is possible that lamellocyte differentiation is in some way secondarily activated in the Tl10b background. Thus, the wasp egg encapsulation assay is a useful tool for evaluating the genetic requirements for lamellocyte differentiation (Sorrentino, 2004).

In conclusion, this study shows that while there is substantial variation in hemocyte concentration in control larvae, this variation is consistent with a log-normal distribution. Such a distribution could be a result of the inherently logarithmic process of cell division. Using this quantitative method of circulating hemocyte concentration data analysis, it was found that previously reported circulating hemocyte concentration values for mutant larvae that exhibit reduced or increased hemocyte densities are also log-normally distributed and that approximately half of each of these mutant distributions lie beyond the limits of the control distribution, allowing ranges of circulating hemocyte concentration values to be defined as being low, control, and high. In addition, encapsulation capacity in control and DV mutant larvae correlates with circulating hemocyte concentration. Evidence for biological significance of this correlation also comes from observations that D. melanogaster larvae selected for higher resistance against A. tabida have twice as many circulating hemocytes as compared to control larvae. These observations support the notion that circulating hemocytes, possibly plasmatocytes, contribute to the efficiency of the egg encapsulation response. However, high circulating hemocyte concentration alone is insufficient to trigger encapsulation; lamellocytes must be present. For example, massive 20- to 300-fold increases in circulating hemocyte concentration involving plasmatocytes and crystal cells, but not lamellocytes, are insufficient to trigger constitutive encapsulation of self tissue in the larva. The combined use of genetic and immune approaches used in this study demonstrates that different developmental signals independently contribute to the maintenance of the steady-state hemocyte concentration in circulation and the ability to differentiate lamellocytes. Together, these physiological parameters enable larval hosts to efficiently defend themselves against wasp infections (Sorrentino, 2004).

JAK/STAT pathway and oogenesis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

Interconnection between pathway(s) activated by PDGF- and VEGF-receptor related and the JAK/STAT pathway

Blood cells play a crucial role in both morphogenetic and immunological processes in Drosophila, yet the factors regulating their proliferation remain largely unknown. In order to address this question, antibodies were raised against a tumorous blood cell line and an antigenic determinant was identified that marks the surface of prohemocytes and also circulating plasmatocytes in larvae. This antigen was identified as PDGF- and VEGF-receptor related, a Drosophila homolog of the mammalian receptor for platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF). The Drosophila receptor controls cell proliferation in vitro. By overexpressing in vivo one of its putative ligands, PVF2, a dramatic increase was induced in circulating hemocytes. These results identify the PDGF/VEGF receptor homolog and one of its ligands as important players in Drosophila hematopoiesis (Munier, 2002).

The distribution of the antigenic determinant on the different Drosophila hemocyte types was examined by immunocytochemistry. Wild-type and hopTum-l mutants were used for this purpose. hopTum-l is a Janus kinase (JAK) gain-of-function mutation resulting in an overproliferation of circulating blood cells, of which a large number are lamellocytes. Plasmatocytes were stained and lamellocytes were not. In hopTum-l mutants, the most strongly reacting cells were small rounded cells that correspond to circulating progenitor blood cells (prohemocytes). In wild-type larvae, the presence of prohemocytes is mostly restricted to lymph glands. Strong staining was observed on the prohemocytes of wild-type lymph glands. In hop-Tum-l lymph glands, the small rounded prohemocytes were also strongly stained, but other cells that have been described as lamellocytes but did not react with the antibody. Stained circulating crystal cells could not be observed due to their fragility. No staining was ever observed on larval tissues other than hemocytes, namely on fat body, muscles, imaginal discs, epidermal cells, brain or trachea (Munier, 2002).

In Drosophila gain-of-function alleles in the JAK Hopscotch (e.g. hopTum-l) cause overproliferation of hemocytes. This report raises the obvious question as to what kind of interconnection exists between pathway(s) activated by PVR and the JAK/STAT pathway itself. Some cross-talk could occur, such as phosphorylation of STAT by PVF2-induced PVR activation. In mammals, for instance, PDGFR can directly activate some STATs. Conversely, evidence exists that JAK can activate the D-raf/D-MEK/MAP kinase pathway, one that is frequently activated by receptor tyrosine kinases. As is the case for PVF2, however, neither JAK nor STAT seem absolutely required for blood cell proliferation. Indeed, in loss-of-function mutants of hop or stat that permit larval development, blood cell counts are normal. This leaves open the possibility that upstream components of the JAK/STAT pathway, e.g., the receptor Domeless (DOME) and its ligand Unpaired, could act in synergy with the PVF2/PVR pathway. Both DOME and Upd are implicated in embryonic pair-rule gene expression, but their role in hematopoeisis awaits investigation (Munier, 2002).

In summary, the data indicate that PVR integrates two functions shared by mammalian receptors of the same subfamily. Like its mammalian VEGFR homologs (Flt1, KDR and Flt4), it regulates cell migration; and like c-Kit, Flt-3, c-Fms and most PDGFRs, it is implicated in the control of blood cell proliferation. In the light of the importance of hemocytes in development and in the innate immune response, it would be highly relevant to investigate further the interaction between PVFs, PVR, the JAK/STAT pathway and the downstream mitogenic factors that they induce (Munier, 2002).

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

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

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

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

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

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

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

A 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 hopC111 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 hopC111 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).

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

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

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

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

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

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

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

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

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

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

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

The Friend of GATA protein U-shaped functions as a hematopoietic tumor suppressor in Drosophila

Drosophila has emerged as an important model system to discover and analyze genes controlling hematopoiesis. One regulatory network known to control hemocyte differentiation is the Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signal-transduction pathway. A constitutive activation mutation of the Janus kinase Hopscotch (hopscotchTumorous-lethal; hopTum-l) results in a leukemia-like over-proliferation of hemocytes and copious differentiation of lamellocytes during larval stages. Friend of GATA (FOG) protein U-shaped (Ush) is expressed in circulating and lymph gland hemocytes, where it plays a critical role in controlling blood cell proliferation and differentiation. These findings demonstrate that a reduction in ush function results in hematopoietic phenotypes strikingly similar to those observed in hopTum-l animals. These include lymph gland hypertrophy, increased circulating hemocyte concentration, and abundant production of lamellocytes. Forced expression of N-terminal truncated versions of Ush likewise leads to larvae with severe hematopoietic anomalies. In contrast, expression of wild-type Ush results in a strong suppression of hopTum-l phenotypes. Taken together, these findings demonstrate that U-shaped acts to control larval hemocyte proliferation and suppress lamellocyte differentiation, likely regulating hematopoietic events downstream of Hop kinase activity. Such functions appear to be facilitated through Ush interaction with the hematopoietic GATA factor Serpent (Srp) (Sorrentino, 2007).

In wild-type lymph glands, Ush is not detectable in the second instar larva (L2) but is expressed in L3, beginning in the cortical zone and eventually spreading to the entire lymph gland. It stands to reason that during the normal dispersal of the hematopoietic organs in late L3, those lymph gland hemocytes in the cortical zone will be the first to enter circulation. In such a model, Ush-expressing lymph gland hemocytes enter the circulating hemocyte population, which (with the exception of crystal cells) already express Ush. Thus Ush can be viewed as a hemocyte maturation marker. This begs the question of why Ush is expressed in what are apparently the most mature hemocytes. The current observations strongly implicate Ush as being present in order to suppress proliferation and lamellocyte differentiation among mature plasmatocytes (Sorrentino, 2007).

The strongest evidence for a mechanism in which Ush suppresses hemocyte proliferation and lamellocyte differentiation comes from analyses of ush mutants. Reducing Ush function causes lymph gland hypertrophy, which is a direct result of an increase in the number of lymph gland hemocytes. Furthermore, in a manner similar in quality (but somewhat less in intensity) to those of hopTum-l larvae, ush mutant lymph glands disperse precociously, and cortical zone hemocytes appear to be morphologically consistent with lamellocytes. Additionally, total circulating hemocyte concentration (CHC) of ush mutants is over four-fold greater than that of wild-type larvae, and two different alleles of ush when heterozygous, induce a less severe but nonetheless significant hematopoietic phenotype. High CHC is consistent with the mechanism of a large number of hemocytes, including lamellocytes, leaving the lymph gland and entering circulation. The possibility cannot be ruled out that circulating hemocytes, which are of a different embryonic origin than lymph gland hemocytes, can also over-proliferate and/or differentiate into lamellocytes in ush mutants. Since cortical zone hemocytes (predominantly plasmatocytes) express Ush and can differentiate into lamellocytes, the fact that circulating plasmatocytes also express Ush would support the notion of circulating plasmatocytes also being able to differentiate into lamellocytes (Sorrentino, 2007).

Importantly, it was observed that wild-type L2 lymph glands do not express Ush, while L3 organs do (in the cortical zone first, then throughout the lymph gland). Clearly, a mechanism that represses a developmental decision is not necessary unless a cell has the potential to actually make the choice. Thus the existence of a Ush-regulated mechanism for suppression of hemocyte proliferation and lamellocyte differentiation in L3 cortical zone hemocytes is interpreted as supportive of the hypothesis that Ush+ cells are in a different genetic state in which they can, given the proper cues, hyperproliferate and differentiate into lamellocytes. It follows that wild-type L2 lymph gland hemocytes cannot hyperproliferate and become lamellocytes. Such a putative mechanism is consistent with previous findings; Jung (2005) observed that L3 lymph gland hemocyte proliferation takes place primarily within the cortical zone, while Sorrentino (2002) observed that, in larvae parasitized by the wasp Leptopilina boulardi, L2 lymph glands are immune-unresponsive (as indicated by mitotic index, crystal cell population size, and a lamellocyte marker) whereas L3 lymph glands do respond (Sorrentino, 2007).

Additional strong evidence for the role of Ush is provided by transgene expression data. Expression of wild-type Ush in hopTum-l/Y larvae produced an effect opposite in quality to that of ushVX22/ushr24, that being a significant 90% reduction in the hopTum-l-induced circulating lamellocyte population. The CgGAL4 driver is active in hopTum-l/Y L2 hemocytes, thus transgenic Ush has an opportunity to act on hemocytes prior to lymph gland dispersal. The significant reduction could be explained by the suppression of lamellocyte differentiation and/or the suppression of the proliferation of pro-lamellocytes. An apoptotic mechanism may also be partially involved. The reason for the observation that the hopTum-l non-lamellocyte population was not significantly affected by transgenic Ush is unknown, but one explanation would be a dosage-dependent mechanism in which experimental expression of just one copy of a UASush transgene is insufficient to suppress hopTum-l over-proliferation. It is also possible that hemocyte over-proliferation is a secondary effect of lamellocyte differentiation, and thus not under the direct control of Ush (Sorrentino, 2007).

If Ush normally suppresses crystal cell differentiation why do ush mutants not exhibit a severe overabundance of crystal cells? Using the strong amorphic ush1 background, an approximately 30% increase in mean crystal cell counts has been observed in stage-16 embryos. However, since the wild-type mean number of crystal cells in stage-16 embryos is about 24-25 per embryo, a 30% increase amounts to about 8 additional crystal cells. Since there are hundreds of plasmatocytes in an embryo, the overwhelming majority of plasmatocytes do not become crystal cells in the absence of Ush (Sorrentino, 2007).

Such findings can be explained by a model in which Ush suppresses crystal cell differentiation in a small subset of hemocytes, with the primary role of Ush in hematopoiesis being to control lamellocyte differentiation and hemocyte proliferation. In this model, the down-regulation of ush expression in embryonic crystal cells occurs because hemocytes committed to the crystal cell lineage cannot become lamellocytes, and thus require no mechanism to suppress lamellocyte differentiation (Sorrentino, 2007).

Srp is expressed in all hopTum-l/Y hemocytes, both lamellocytes and non-lamellocytes, in circulation and in the lymph gland. Thus, all Drosophila hemocyte classes studied thus far express this hematopoietic GATA factor, and the role of Srp in the differentiation of all hemocyte types is worthy of investigation. The fact that the lamellocyte population observed in ushVX22/+ larvae is strongly reduced by srpneo45/+ and completely reduced to wild-type levels by srp3/+ suggests Srp plays an active role in lamellocyte differentiation. In humans, GATA-3 determines the differentiation of Th2 cells, which like lamellocytes are the primary effectors in a cellular immune response against metazoan endoparasites. FOG-1 inhibits Th2 differentiation by inhibiting GATA-3 activity via physical interaction. However, if Srp is indeed necessary for lamellocyte differentiation, the finding that Srp is expressed in all hemocytes likely means it is not the sole determinant of lamellocyte differentiation. Thus it is considered likely that an additional transcriptional regulator, either another GATA factor (e.g., Grain, dGATAd, dGATAe) or a non-GATA factor, works in conjunction with Srp to specify the lamellocyte differentiation program (Sorrentino, 2007).

Ush232-1191, Ush302-1191, and Ush365-1191 driven by CgGAL4 are dominant inducers of hematopoietic tumor phenotypes, measurably stronger than that of the ushVX22/ushr24 loss-of-function condition. This finding is not interpreted as coincidental. In an important parallel all three of these constructs, when activated by the mesodermal twiGAL4 driver, exhibit a failure to suppress the expression of a cardiac-active Dmef2-lacZ reporter gene. Additionally, it was observed that Ush232-1191, though missing zinc finger 1, is still able to bind to Srp in vitro just as it is able to bind to Pnr. Such observations are consistent with the possibility that endogenous and transgenic Ush may compete in their binding to Srp. In such a situation, transgenic Ush232-1191, even if bound to Srp, might fail to suppress Srp-induced hemocyte proliferation and lamellocyte differentiation. Alternative explanations include: (1) Ush232-1191 bound to Srp may actually enhance normal Srp activity; (2) the Srp:Ush232-1191 complex may behave neomorphically; (3) Ush may normally dimerize while not bound to Srp, if so a Ush:Ush232-1191 complex may not be able to separate into active Ush monomers. The transgenic Ush proteins are assumed to be sufficiently stable and functional as to validate these observations, since the UASush constructs used have been shown to generate stable proteins in other cell types and also to induce measurable phenotypes (Sorrentino, 2007).

There is significantly more Ush present in the nuclei of hopTum-l/Y hemocytes than in nuclei of wild-type blood cells. Interestingly, Ush appears to be exclusively nuclear in hopTum-l/Y hemocytes, whereas there appears to be some cytoplasmic anti-Ush staining in wild-type hemocytes. It is possible that Ush function is in part determined by its cytoplasmic/nuclear ratio. Perhaps the qualitatively higher Ush concentration in hopTum-l hemocytes is the result of nuclear translocation of all cellular Ush. Based on work with human 293T and mouse erythroleukemia cell cultures, Garriga-Canut (2004) proposed a model in which TACC-3 and GATA-1 compete in binding to FOG-1, with FOG-1 bound to TACC-3 retained in the cytoplasm. Such a mechanism may also be at work in Drosophila hemocytes and the possibility of a Ush cytoplasmic sequestration phenomenon remains to be investigated (Sorrentino, 2007).

This study also found that there exist high concentrations of exclusively nuclear Ush in other tumorous backgrounds, those being in Tl10b/+ and CgGAL4>UAScol animals. Larvae carrying the dominant Tl10b allele exhibit a hematopoietic tumor phenotype similar to that of hopTum-l/Y larvae. In addition to srpDGAL4>UAScol, CgGAL4>UAScol is sufficient to induce lamellocyte differentiation (although it also induces L2 developmental arrest). All hemocytes, including lamellocytes, in both of these backgrounds also exhibit high concentrations of nuclear Ush. These observations are consistent with a model in which three different signaling pathways (Hop-Stat, Toll-Dorsal, and the early B-cell related factor Collier) all make use of a single common downstream lamellocyte induction program that involves Ush. Examination of hemocytes from additional tumorous backgrounds will reveal whether such a model is truly universal. An important question remains as to how Ush suppresses hemocyte proliferation and lamellocyte differentiation, yet is expressed so strongly in tumorous hemocytes. Taken together, the findings are supportive of Ush having an early function in repressing lamellocyte differentiation. Up-regulation of the protein in lamellocytes would be suggestive of a second, separate function for Ush within this differentiated hemocyte. Comparable multi-functional properties have been reported for FOG-1 in vertebrate hematopoiesis (Sorrentino, 2007).

Therefore, reduction of Ush function results in a classic hematopoietic tumor phenotype: lymph gland hypertrophy and early dispersal, a significant increase in total circulating hemocyte concentration, large-scale lamellocyte differentiation, and melanotic tumors. These anomalies can be partially induced by the loss-of-function of a single copy of ush. The identification of this FOG class protein as a tumor suppressor raises questions about the roles of other FOG proteins in mammalian leukemias. While mutations in murine fog1 have been associated with hematopoietic dysfunction such as the failure of megakaryopoiesis and the arrest of erythropoiesis, FOG proteins have not been implicated as a causal factor in any human leukemia. While there is no guarantee that the observations in Drosophila will directly translate to specific human hematopoietic pathologies, it may now be worthwhile to examine the state of fog gene expression and function in human leukemias (Sorrentino, 2007).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Lineage tracing of lamellocytes demonstrates Drosophila macrophage plasticity

Leukocyte-like cells called hemocytes have key functions in Drosophila innate immunity. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. In the absence of immune challenge, plasmatocytes are the predominant hemocyte type detected, while crystal cells and lamellocytes are rare. However, upon infestation by parasitic wasps, or in melanotic mutant strains, large numbers of lamellocytes differentiate and encapsulate material recognized as 'non-self'. Current models speculate that lamellocytes, plasmatocytes and crystal cells are distinct lineages that arise from a common prohemocyte progenitor. This study shows that over-expression of the CoRest-interacting transcription factor Charlatan (Chn) in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. Lamellocyte increases are accompanied by the extinction of plasmatocyte markers suggesting that plasmatocytes are transformed into lamellocytes. Consistent with this, timed induction of Chn over-expression induces rapid lamellocyte differentiation within 18 hours. Double-positive intermediates between plasmatocytes and lamellocytes were observed, and it was shown that isolated plasmatocytes can be triggered to differentiate into lamellocytes in vitro, either in response to Chn over-expression, or following activation of the JAK/STAT pathway. Finally, plasmatocytes were marked, and lineage tracing showed that these differentiate into lamellocytes in response to the Drosophila parasite model Leptopilina boulardi. Taken together, these data suggest that lamellocytes arise from plasmatocytes and that plasmatocytes may be inherently plastic, possessing the ability to differentiate further into lamellocytes upon appropriate challenge (Stofanko, 2010).

Drosophila provide a genetically tractable model system to investigate cellular innate immune function. This report examined the origins of lamellocytes, which are Drosophila hemocytes that differentiate in response to parasite infestation. Over-expression of Chn in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. The data indicate that Chn over-expression transforms plasmatocytes into lamellocytes. Consistent with this, double-positive intermediates between plasmatocytes and lamellocytes were detected, and it was shown that isolated plasmatocytes in vitro can be triggered to differentiate into lamellocytes following Chn over-expression. This property is not limited to Chn since it was observed that other stimuli, including activation of the JAK/STAT pathway and the natural response to parasitic wasp infestation, also induced lamellocyte formation from plasmatocytes (Stofanko, 2010).

The data suggest that Chn may control lamellocyte development. Previously defined regulators of lamellocyte development include the transcription factor STAT92E, the FOG-1 homologue Ush, and the NURF chromatin remodelling complex. STAT92E functions as an inducer of lamellocyte development, as gain-of-function hopTum-l mutants that activate the JAK/STAT pathway cause lamellocyte over-production. In contrast, both loss-of-function ush and Nurf mutants exhibit increased lamellocyte numbers. Like the homologous FOG-1-GATA-1 pairing, Ush modulates activity of the Drosophila GATA factor Srp to favour plasmatocyte differentiation. Recent data in mammalian systems indicates that FOG-1 mediates its effect on GATA-1 in part via recruitment of the transcriptional co-repressor NURD, suggesting that Ush functions similarly to repress expression of gene targets required for lamellocyte differentiation in plasmatocytes. Likewise, NURF also inhibits lamellocyte differentiation, in this case by preventing activation of targets of the JAK/STAT pathway (Stofanko, 2010).

The current biochemical data suggest that Chn is a transcription repressor since Chn recruits the co-repressor complex CoREST. Indeed it has been shown that Chn over-expression represses Delta expression in the eye imaginal disk, while this study has shown that Chn over-expression is accompanied by repression of some plasmatocyte markers. However, it was also shown that Chn over-expression leads to elevated expression of lamellocyte markers, and it has been demonstrated that Chn over-expression increases expression of the proneural genes Achaete and Scute. These data do not allow discrimination of whether Chn functions entirely as a transcriptional repressor or whether it may also activate transcription. However, the temporally-controlled Chn induction system (Pxn-Gal4 TARGET) that was utilized in this study will allow the primary gene targets of Chn to be determined. By analyzing transcriptional profiles of hemocytes at defined time points after Chn over-expression the primary responders to Chn over-expression will be able to be identified. It will be possible to discriminate whether these targets are preferentially activated or repressed, and also subsequently determine recruitment of transcription co-activator or co-repressor complexes such as CoREST at these targets using chromatin immunoprecipitation (Stofanko, 2010).

The data demonstrating that lamellocytes can originate from plasmatocytes sheds new light on hemocyte lineages. Current models of hemocyte lineages speculate that plasmatocytes, crystal cells and lamellocytes are distinct lineages that arise separately from a common stem cell or prohemocyte. This study proposes, however, that prohemocytes generate either crystal cells or plasmatocytes. It is suggested that plasmatocytes are a plastic population that can generate other frequently observed hemocyte types including lamellocytes. This model is strikingly reminiscent of the initial hemocyte lineages first proposed more than 50 years ago. According to that analysis prohemocytes were predicted to generate either crystal cells or plasmatocytes, with plasmatocytes differentiating further into activated cells (podocytes) and then lamellocytes. This model has support from a number of experimental studies including this study. Foremost among these are recent studies of hemocyte functions of Ush. Dominant-negative Ush variants are able to induce lamellocyte differentiation and it has been suggested that Ush regulates lamellocyte differentiation from a potential plasmatocyte. Secondly, lamellocyte differentiation in response to Salmonella infection is blocked in decapentaplegic mutants with a corresponding increase in plasmatocyte number, suggesting that lamellocytes arise from plasmatocytes or a common precursor (Stofanko, 2010).

Two recent studies also suggest that plasmatocytes are a plastic population that may be able to differentiate into lamellocytes. Marking of embryonic plasmatocytes using the gcm-GAL4 or sn-GAL4 drivers and an act5C>stop>GAL4 flip-out transgene shows that lamellocytes that arise in larvae after wasp infestation may originate from cells that had expressed gcm-GAL4 or sn-GAL4 in embryos. Similar results have also been observed using the act5C>stop>GAL4 flip-out transgene and Pxn-GAL4 and eater-GAL4. In both these cases the elicitor of the FLP/FRT activation event and the subsequent sustained marker are the same, namely GAL4 expression. However, in the current lineage tracing experiments, GAL4 expression initiates the FLP/FRT activation of a distinct marker, lacZ protein. These data, taken together with lineage tracing experiments and in vitro differentiation studies suggest that the plasmatocyte is an inherently plastic cell type that is capable of being reprogrammed to tailor immune responses to suit the infectious threats faced by the host. In humans, lymphocyte and leukocyte plasticity has a significant impact on immune responses. An important future challenge is to establish the full spectrum of Drosophila plasmatocyte heterogeneity and exploit the utility of the Drosophila genetic system to dissect the mechanisms that regulate such leukocyte plasticity (Stofanko, 2010).

Galpha73B is a downstream effector of JAK/STAT signalling and a regulator of Rho1 in Drosophila haematopoiesis

JAK/STAT signalling regulates multiple essential developmental processes including cell proliferation and haematopoiesis while its inappropriate activation is associated with the majority of myeloproliferative neoplasias and numerous cancers. Furthermore, high levels of JAK/STAT pathway signalling have also been associated with enhanced metastatic invasion by cancerous cells. Strikingly, gain-of-function mutations in the single Drosophila JAK homologue, Hopscotch, result in haemocyte neoplasia, inappropriate differentiation and the formation of melanised haemocyte-derived 'tumour' masses; phenotypes that are partly orthologous to human gain-of-function JAK2-associated pathologies. These studies show that Gα73B, a novel JAK/STAT pathway target gene, is necessary for JAK/STAT-mediated tumour formation in flies. In addition, while Gα73B does not affect haemocyte differentiation, it does regulate haemocyte morphology and motility under non-pathological conditions. This study shows that Galpha73B is required for constitutive, but not injury-induced, activation of Rho1 and for the localisation of Rho1 into filopodia upon haemocyte activation. Consistent with these results, it was also shown that Rho1 interacts genetically with JAK/STAT signalling, and that wild-type levels of Rho1 are necessary for tumour formation. These findings link JAK/STAT transcriptional outputs, Galpha73B activity and Rho1-dependent cytoskeletal rearrangements/cell motility and therefore connect a pathway associated with cancer with a marker indicative of invasiveness. As such, this study suggests a mechanism via which JAK/STAT pathway signalling may pro

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 phdel, 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, phdel , 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 phdel 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 phdel and ph505 was tested. The functions of Notch and Upd homologs in the ph505 mosaic eyes were examined with the same strategy used for phdel. 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 updΔ1-3, a deficiency line that lacks all three upd homologs in the Drosophila genome Mosaic analyses were then performed using this double mutant line. ph505 -updΔ1-3 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 phdel 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, phdel 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 phdel mosaic discs (Feng, 2012).

As controls, phdel -dome and phdel -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 phdel cells did not affect non-autonomous cell over proliferation. It did, however, mildly reduce the mutant clone size, suggesting that phdel cells might still have a weak response to Upd ligands. Adult eyes mosaic for these double mutant lines were phenotypically indistinguishable from phdel mosaic eyes, consistent with the above observations in mosaic eye discs (Feng, 2012).

Finally it was asked why phdel 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 phdel 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 phdel 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 phdel 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 phdel 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 phdel and ph505 both cause autonomous over-expression of Upd homologs in mutant cells, which represents the only driving force of cell overproliferation in phdel 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, phdel cells are largely insensitive to Upd ligands, so that Upd ligands secreted by phdel 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 phdel and ph505 cells to Upd ligands (Feng, 2012).

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

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

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

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

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

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

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

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

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

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

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

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

hopscotch: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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