hopscotch

DEVELOPMENTAL BIOLOGY

Embryonic

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).

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 upd^YC43 with the viable allele os^o 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 45ƒ 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 hop^Tuml allele of Drosophila JAK. In hop^Tuml eye discs with uniformly increased JAK/STAT activity, the overall level of lacZ activity is consistently lower than in discs from wild-type siblings stained in parallel. Additional experiments show that the level of stat92E-lacZ expression is inversely proportional to the level of JAK/STAT pathway activation: High activation produced by Upd expression abolishes stat92E-lacZ activity; moderate activation produced by the hop^Tuml allele gives reduced activity, whereas cells in which there is no JAK/STAT signaling (such as hop clones) show maximal levels of stat92E-lacZ activity. Comparing the results of these experiments with the endogenous pattern of stat92E-lacZ staining in the eye disc, it is concluded that JAK/STAT activity must be highest at the DV midline (where stat92E-lacZ activity is lowest) and low at the poles (where stat92E-lacZ activity is upregulated to levels similar to those seen in hop clones) with a gradient of JAK/STAT activity present between these extremes (Zeidler, 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 hop^msv1 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)

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, Sxl_Pe. A new XSE called sisterlessC (sisC) is described whose mode of action differs from that of previously characterized XSEs, all of which encode transcription factors that activate Sxl _Pe directly. In contrast, sisC encodes a secreted ligand for the Drosophila Janus kinase (JAK) and 'signal transducer and activator of transcription' (STAT) signal transduction pathway and is allelic to outstretched (os, also called unpaired). sisC works indirectly on Sxl through this signaling pathway because mutations in sisC or in the genes encoding Drosophila JAK or STAT reduce expression of Sxl_Pe similarly. The involvement of os in sex determination confirms that secreted ligands can function in cell-autonomous processes. Unlike sex signals for other organisms, sisC has acquired its sex-specific function while maintaining non-sex-specific roles in development, a characteristic that it shares with all other Drosophila XSEs (Sefton, 2000).

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

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

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. hop^Tum-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 hop^Tum-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 hop^Tum-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 hop^Tum-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 hop^Tum-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 hop^msv1 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, hop^Tum-l, was used for this experiment. When grown above 29°C, flies heterozygous for hop^Tum-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 hop^Tum-l and Cdk4 with females heterozygous only for hop^Tum-l. An improved survival rate was obtained by removing a single copy of Cdk4 in hop^Tum-l heterozygous females. However, the formation of melanotic tumors is affected less by removing a single copy of Cdk4 in hop^Tum-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 (hop^C111) 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 hop^C111 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 hop^C111 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 hop^Tum-l and CycE with females heterozygous only for hop^Tum-l. An improved survival rate was observed by removing a single copy of CycE in hop^Tum-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 hop^Tum-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 (Tl^10b) and the Jak kinase, hopscotch (hop^Tuml); 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 Tl^10b and hop^Tuml 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 hop^Tuml mutants. It is thought that the normally smaller secondary lymph gland lobes serve as a reservoir of undifferentiated prohemocytes, however, in hop^Tuml larvae the secondary lobes enlarge with concomitant abnormal differentiation of lamellocytes. While hemocytes in the secondary lymph gland lobes of hop^Tuml, 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 hop^Tum-l, Tep1 is constitutively expressed, whereas its immune-inducibility is dramatically reduced in the loss-of-function mutant hop^M38. 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 hop^Tum-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 hop^Tum-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 hop^Tum-l/Y larvae exhibit multiple capsules. Lowering of ecdysone levels in hop^Tum-l/Y; ecd¹/ecd¹ animals results in a mild, but significant, suppression of the hop^Tum-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 25ƒC (86.2%) is consistent with the corresponding larval value. Thus, ecd¹ is able to partially suppress the hop^Tum-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 hop_Tum-l, affect cell populations and bypass the requirement for ecdysone. Such studies can then reveal whether suppression of the hop_Tum-l phenotype in hop_Tum-l/Y;ecd₁ /ecd₁ double mutants (with fewer and smaller melanotic tumors than hop_Tum-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 (hop^M38/hop^msv1), 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 hop^M4 background is consistent with the observed reduction in hop^M4/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 hop^Tum-l-induced melanotic tumor phenotype and Stat92E is constitutively activated in Drosophila cell cultures that overexpress Hop^Tum-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 hop^Tum-l/Y; stat92E^HJ/stat92E^HJ 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 Tl^10b 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 hop^Tum, many of which invaded the nurse cell cluster. These extra cells did not result from excess proliferation because follicle cells cease dividing at stage 6, at least 12 hr prior to border cell differentiation. Furthermore, no difference in phospho-histone H3 antibody labeling was observed in cells overexpressing upd or in cells lacking stat, ehrn compared to wild-type. Moreover, it was possible to obtain large clones lacking upd, hop, or stat activity, indicating that homozygous mutant cells retain the ability to divide numerous times. Thus, activation of the JAK/STAT pathway leads to border cell specification and migration, without effects on proliferation. In addition, while extra follicle cells could become migratory as a secondary consequence of ectopic polar cell formation, activation of the JAK/STAT pathway results in the appearance of additional migratory cells in the absence of extra polar cells (Silver, 2001).

The question of whether signaling through this pathway might be sufficient to cause epithelial cells to become invasive was addressed ectopically expressing Upd, Hopscotch (Hop), or the constitutively active form of Hop, Hop^Tum1, using the GAL4/UAS expression system. In this method, the yeast transcriptional activator GAL4 is expressed under the control of a cell type-specific enhancer, in this case slbo-GAL4 and c306-GAL4. In stage 9 egg chambers, slbo-GAL4 induces expression of genes that are under the control of the yeast upstream activating sequence (UAS) in approximately 20 anterior follicle cells, a subset of which normally become the border cells. This is nearly identical to the ß-gal expression from an enhancer trap insertion into the slow border cells (slbo) locus, even though Slbo protein expression is normally restricted to the border cells at stage 9. C306-GAL4 drives expression in a larger number of anterior, as well as posterior, follicle cells, compared to slbo-GAL4. C306-GAL4 also begins expressing earlier in oogenesis than slbo-GAL4 (Silver, 2001).

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

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).

JAK signaling is somatically required for follicle cell differentiation in Drosophila

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

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

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

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

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

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

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

Several signaling pathways have been implicated in the patterning of the follicular epithelium. The best characterized are the Notch, EGFR and Hedgehog pathways. In the earliest of these activities, strong expression of hh in the terminal filament and cap cells at the anterior tip of the germarium stimulates the proliferat