outstretched
The spatial distribution of the 2.2-kb os transcript is consistent with a role in embryonic segmentation. In situ hybridization to RNA in whole mount embryos has revealed a dynamic and segmentally repetitive pattern of expression. During the syncytial blastoderm stage, the 2.2-kb transcript is not expressed at levels that are detectable above background. Shortly before cellularization, the RNA becomes abundant, but is absent from the termini, and is restricted to only the trunk of the embryo and a single incomplete head stripe. At cellularization, the trunk expression resolves into ~7 stripes, then into 14 stripes, a phenomenon seen in some pair-rule segmentation genes (Harrison, 1998).
Rearrangement of cells constrained within an epithelium is a key process that contributes to tubular morphogenesis. Activation in a gradient of the highly conserved JAK/STAT pathway is essential for orienting the cell rearrangement that drives elongation of a genetically tractable model. Using loss-of-function and gain-of-function experiments, it has been shown that the components of the pathway from ligand to the activated transcriptional regulator STAT are required for cell rearrangement in the Drosophila embryonic hindgut. The difference in effect between localized expression of ligand (Unpaired) and dominant active JAK (Hopscotch) demonstrates that the ligand plays a cell non-autonomous role in hindgut cell rearrangement. Taken together with the appearance of STAT92E in a gradient in the hindgut epithelium, these results support a model in which an anteroposterior gradient of ligand results in a gradient of activated STAT. These results provide the first example in which JAK/STAT signaling plays a required role in orienting cell rearrangement that elongates an epithelium (Johansen, 2003).
upd, encoding the ligand for the Drosophila JAK/STAT pathway, is expressed only in the small intestine and is regulated by genes controlling hindgut cell rearrangement. In drm and bowl mutants, expression of upd is missing from the small intestine, while in lin mutants, upd expression is expanded throughout much of the hindgut. These results raise the possibility that localized Upd might provide an orienting cue for rearranging hindgut cells (Johansen, 2003).
If it plays a role in hindgut cell rearrangement, upd must be expressed before and during the period of major hindgut elongation, i.e. between stages 11 and 16; genes encoding the other known components of the Drosophila JAK/STAT signaling pathway should also be expressed at the same stages, both within and adjacent to upd-expressing cells. In situ hybridization was used to characterize the expression of upd, dome, hop and Stat92E during stages just prior to and during hindgut elongation (Johansen, 2003).
Expression of upd in the hindgut is first detected at stage 9 in a narrow ring of cells that will become the small intestine. Expression in the prospective small intestine is maintained during stages 10 and 11, where it can be seen just posterior to the everting renal tubules (note that in the hindgut at these germband-extended stages, 'posterior' is toward the head). During stages 12-14, when the hindgut undergoes a major part of its elongation, upd expression is seen throughout the now distinct small intestine. Expression of upd is maintained throughout the small intestine during the remainder of embryogenesis (Johansen, 2003).
The Janus kinase hop is expressed uniformly throughout the embryo, including the hindgut as it elongates. Expression of both the receptor-encoding gene dome and Stat92E is detected weakly at the anterior of the hindgut beginning at stage 9; it becomes significantly stronger by stage 11, and is maintained through stage 14. For both the receptor- and STAT-encoding genes, expression domains in the hindgut epithelium overlap with and extend beyond the narrow domain of upd expression. Most significantly, expression of dome and Stat92E extends to a more posterior position in the hindgut epithelium than does expression of upd. Thus, the mRNA expression of the ligand, receptor and STAT components in the hindgut prior to and during its elongation is consistent with a role for JAK/STAT signaling in hindgut cell rearrangement (Johansen, 2003).
Elongation of the Drosophila hindgut by cell rearrangement requires the Upd ligand and the JAK/STAT pathway components Dome (receptor), Hop (JAK) and Stat92E. Since elongation does not occur when expression of ligand or activation of the pathway is uniform, but only when the source of ligand is localized to the hindgut anterior, the requirement for localized JAK/STAT signaling in hindgut elongation can be characterized as instructive, rather than permissive. Since patterning is normal in hindguts both lacking and uniformly expressing upd, the required role of JAK/STAT signaling in hindgut morphogenesis is likely via direct effects on cell movement (Johansen, 2003).
The rescue of the upd phenotype by anteriorly localized expression in the hindgut of upd, but not of activated JAK (Hopscotch), demonstrates that there is a requirement for upd function that is not cell autonomous. In other words, upd is required in cells (those of the large intestine that undergo the greatest rearrangement) that are different from cells that produce it (those of the small intestine). A number of examples have been described in which localized expression of a signaling molecule (including Upd) is required non-autonomously for cell rearrangement, morphogenesis or motility. In the Drosophila eye imaginal disc, expression of Upd at the midline is required to establish a dorsoventral polarity that orients ommatidial rotation. In both Drosophila tracheae and the vertebrate lung, branching morphogenesis of the epithelium depends on localized expression of FGF in adjacent mesenchyme (Johansen, 2003).
Localized activation of JAK/STAT signaling has been shown to play a role in cell motility in a number of contexts. In Drosophila, localized expression of Upd in the anterior polar cells of the egg chamber acts to coordinate the migration of the adjacent border cells. In mammals, cytokines expressed in target tissues act to attract both migrating lymphocytes and tumor. The finding that localized (only in the small intestine) expression of upd is both necessary and sufficient for rearrangement of cells in the large intestine indicates that Upd must have an organizational, action-at-a-distance function in controlling cell rearrangement during tubular morphogenesis (Johansen, 2003).
Rescue experiments establish that there is a cell non-autonomous requirement for upd in hindgut elongation. Consistent with this, there is evidence that Upd is present and required in an anteroposterior gradient in the hindgut. Prior to and during hindgut elongation, both Stat92E mRNA and Stat92E protein are detected not only in the small intestine epithelium (and the visceral mesoderm surrounding the small intestine), but also in the epithelium posterior to the small intestine; this expression of Stat92E appears to be in a gradient. In the Drosophila eye imaginal disc, a gradient of Upd is required to orient the rotation of ommatidial cell clusters; in addition, there is evidence for a gradient of Upd and Stat92E in patterning of the follicular epithelium of the Drosophila egg chamber. Since expression of Stat92E depends on upd, it is likely that Upd protein is present in the hindgut epithelium as an anteroposterior gradient, with its highest level in the upd-expressing cells of the small intestine, and lowest level in posterior, upd non-expressing cells of the large intestine. Expression of SOCS36E (suppressor of cytokine signaling at 36E), which is regulated by upd, overlaps with and extends significantly beyond the domain of upd expression, further supporting the idea that there is a gradient of Upd in the hindgut (Johansen, 2003).
In the Drosophila eye imaginal disc, anti-Upd staining and the behavior of clones of mutant cells that have lost components of the JAK/STAT pathway indicate that Upd is present in a gradient that extends at least 50 µm beyond its midline mRNA expression domain. In the Drosophila hindgut, Stat92E is a reliable reporter for the presence of Upd. Two to four hours after upd is first expressed at the anterior of the hindgut (stage 9), Stat92E can be detected at least 30-40 µm from the site of upd expression (stages 11 and 12). These time and distance parameters are similar to those observed during generation of the Upd gradient in the eye, and the Dpp and Wg gradients in wing imaginal discs, which form over distances of roughly 40-80 µm in 1-8 hours. Thus, it is reasonable to imagine that a gradient of Upd is established in the developing hindgut in a short enough time frame to affect cell rearrangement (Johansen, 2003).
The essential consequence of JAK/STAT signaling is activation of the STAT protein, which leads to altered transcriptional programs. STAT has been shown in a number of contexts to be required for cell motility, and therefore probably regulates expression of genes controlling cytoskeletal assembly and cell adhesion. In these contexts, however, activation of STAT does not appear to be required to orient cell movement, but rather to facilitate or promote it. As Stat92E is required for hindgut elongation, and its protein product appears to be present in a gradient along the anteroposterior axis, this raises the intriguing question of how a gradient of a transcription factor might orient cell rearrangement (Johansen, 2003).
Janus kinase (JAK) plays several signaling roles in Drosophila oogenesis. The gene for a JAK pathway ligand, unpaired (outstreched), is expressed specifically in the polar follicle cells, two pairs of somatic cells at the anterior and posterior poles of the developing egg chamber. Consistent with unpaired expression, reduced JAK pathway activity results in the fusion of developing egg chambers. A primary defect of these chambers is the expansion of the polar cell population and concomitant loss of interfollicular stalk cells. These phenotypes are enhanced by reduction of unpaired activity, suggesting that Unpaired is a necessary ligand for the JAK pathway in oogenesis. Mosaic analysis of both JAK pathway transducers, hopscotch and Stat92E, reveals that JAK signaling is specifically required in the somatic follicle cells. Moreover, JAK activity is also necessary for the initial commitment of epithelial follicle cells. Many of these roles are in common with, but distinct from, the known functions of Notch signaling in oogenesis. Consistent with these data is a model in which Notch signaling determines a pool of cells to be competent to adopt stalk or polar fate, while JAK signaling assigns specific identity within that competent pool (McGregor, 2002).
The somatic cells of the ovary consist of multiple subpopulations, each with its own function(s) in the developing egg. While the germline cyst is dividing and developing within the germarium, a monolayer of somatic cells surrounds the cyst as it moves posteriorly through the germarium. As the cyst becomes enveloped by the somatic cells, the egg chamber pinches off from the germarium, entering the vitellarium. At that time, approximately 5-8 somatic cells differentiate into stalk. These flattened, disc-shaped cells are stacked together to form the spacer between successive cysts. Stalk cells connect the anterior end of a more mature egg chamber to the posterior end of the next younger chamber. Also at that time, molecular markers can distinguish the stalk cells from the polar cells, which arise from the same precursors. The polar cells are arranged as two pairs of follicle cells, one pair at either end of each chamber near the stalk cells. While the stalk cells and polar cells cease proliferation at the end of the germarium, the remaining follicle cells, which are referred to as epithelial follicle cells, divide approximately five times to expand the pool of follicle cells. Those epithelial cells later differentiate into various subpopulations with specific functions in the vitellarium. Those subpopulations are pre-patterned with mirror image symmetry along the anterior-posterior axis of the egg. Imposed on that pre-pattern, signaling from the oocyte by the TGFalpha molecule Gurken stimulates the induction of posterior polarity on the somatic cells at the posterior end. The result is an egg with coordinated polarities of the somatic and germline cells. This coordination is essential for the proper localization of maternal determinants that pattern the resulting embryo (McGregor, 2002).
Strikingly, unpaired is expressed very specifically within the ovary. After egg chambers pinch off from the germarium, upd is restricted to the two pairs of polar cells found at the anterior and posterior tips of the egg. In the germarium, upd is expressed in a cluster of somatic cells at the posterior of region 3. Presumably these are the cells that give rise to the stalk and polar cells. Expression in the polar and border cells persists until egg maturation. In situ hybridization to Stat92E RNA reveals that Drosophila STAT is expressed in both the germarium and the vitellarium. Expression in the germarium occurs in all follicle cells in region 2a and 2b; it then begins to be restricted to terminal follicle cells in region 3. In the vitellarium, Stat92E is expressed weakly at the termini of the egg chamber, but in a broader domain than only the two polar cells. After stage 9, Stat92E is strongly expressed in the nurse cells, consistent with the maternal role of Stat92E in the segmentation of the early embryo. Moreover, weak ubiquitous expression of hop is detectable in the follicular epithelium. These data are consistent with a potential role for JAK signaling in oogenesis (McGregor, 2002).
What distinguishes stalk and polar cells from one another? JAK signaling induces the adoption of stalk cell fates in a subset of the stalk/polar cell precursors. Loss of JAK pathway activity expands polar cells at the expense of stalk cells, while ectopic activation of the pathway causes a reduction of polar cells. Therefore, it is proposed that JAK pathway activity determines the terminal fate of stalk and polar cells. However, JAK activity is limited in assigning stalk cell fates to only competent cells, that is, the stalk/polar cell precursor pool. Thus, another activity, perhaps N signaling, is necessary to induce competence for stalk and polar fates. Alternatively, N signaling may be primarily responsible for the assignment of polar cell fates. One could imagine a mechanism of lateral inhibition, already linked to N signaling in various tissues, in which all the cells of the precursor pool have N activity, but that the signal becomes limited to and maintained only in the polar cells. It may be the activity of the N pathway that then drives stable expression of upd and allows the induction of stalk cell fates in neighboring cells (McGregor, 2002).
When the developing cyst exits the germarium, there is a distinct change in the epithelial cell precursors. The level of Fas III, a marker for immature follicle cells, is rapidly reduced in all epithelial cell precursors. However, these cells do not begin to express markers for new cell identities until around stage 7. Therefore, it seems that the epithelial cells become committed to a fate early in the vitellarium, but do not terminally differentiate until later. This is consistent with the fact that the epithelial follicle cells continue to divide until stage 6. Furthermore, Grk/EGFR signaling does not impose posterior identity on epithelial cells until stage 6. So the loss of Fas III in epithelial cell precursors in the early vitellarium marks an intermediate step in specific epithelial identities. JAK signaling is involved in this step, because clones of JAK pathway mutations cause the persistence of Fas III in epithelial cell precursors in the early vitellarium. The normal loss of Fas III expression in epithelial precursors of the early vitellarium may indicate the establishment of a pre-pattern of epithelial identities determined by JAK signaling. It is attractive to speculate such a role because the secreted JAK pathway ligand Upd is expressed symmetrically at the termini of the chamber. It is easy to envision a scheme in which the strength of the Upd signal received by the epithelial cell precursors determines the ultimate epithelial identity. However, these epithelial cells would remain in a proliferative, undifferentiated program until stage 7. The event that allows terminal differentiation is unclear, but could also be a N signal, as suggested above for competence of stalk and polar cells. This is consistent with the report of a pulse of Delta protein, a N ligand, that occurs at stages 5-7. Additional work will determine whether JAK signaling is instructive for specific epithelial fates (McGregor, 2002).
During Drosophila oogenesis, border cells perform a stereotypic migration. Slbo, a C/EBP transcription factor, is required for this migration. Drosophila Stat92E has been identified in a screen for gain-of-function suppressors of the slbo mutant phenotype. By clonal analysis for Stat92E and hop mutants it has been found that the JAK/STAT pathway is required in border cells for their migration. The activating ligand for the pathway, Unpaired, is expressed in polar cells. Polar cells are specialized cells that can induce border cell fate in anterior follicle cells. On its own, ectopic expression of Unpaired can induce ectopic expression of border cell markers, including Slbo. However, Stat92E mutant cells still express normal levels of Slbo protein, thus Stat92E must regulate other targets critical for border cell migration (Beccari, 2002).
Production of ectopic polar cells by exposing early egg chambers to increased Hedgehog expression appears sufficient to induce ectopic migrating border cells at stage 9. A slbo-lacZ enhancer trap is induced in extra migrating clusters at stage 9. Similar ectopic border cell clusters have been observed in egg chambers with clones of follicle cells mutant for costal2, a negative regulator of the Hedgehog signal transduction pathway. Thus the presence of polar cells, and absence of posteriorizing signal from the oocyte, may be sufficient for the induction of border cells at the appropriate developmental stage. What signals from polar cells may be responsible for induction of border cell fate in adjacent follicle cells? There is good evidence that Upd is a key signal from polar cells: Upd is specifically expressed in polar cells and acts non cell autonomously; ectopic expression of Upd induces two border cell markers; and the JAK/STAT pathway is required in border cells. Previous studies of the JAK/ STAT pathway in Drosophila have indicated that Upd expression induces Stat92E activation through the JAK kinase Hop and that the effects of Upd can be explained in this manner. Ectopic expression of Upd induces ectopic expression of Slbo. Since the JAK/STAT pathway is required in border cells and thus must be active there, Upd regulated Stat92E may normally contribute to Slbo up-regulation in border cells (Beccari, 2002).
However, given that the Stat92E mutant affected border cell migration without affecting Slbo expression, the JAK/STAT pathway may not be required for Slbo expression. One reasonable explanation is that another signal from polar cells contributes to activation of Slbo in border cells. This upstream signal may by itself be required for Slbo expression or may act redundantly with Stat92E. The additional signal may be a novel effect of Upd, not mediated by the JAK/STAT pathway. However, given the inability of Upd to convert stretch cells to border cells, it is thought a different signal is more likely. Irrespective of its potential effect on Slbo, the effect of Stat92E mutant clones shows that other targets of Stat92E must be critical for border cell migration (Beccari, 2002).
Just as ectopic Slbo expression is not sufficient to convert other cells into border cells, Upd misexpression and ectopic activation of Stat92E is also not sufficient to convert stretch cells into migrating border cells. In the latter situation, the stretch cells experience both Stat92E activation and Slbo expression. The stretch cells nevertheless do not assume border cell fate. This has several possible explanations. The signal invoked above as upstream regulator of Slbo may, in addition to Slbo, have other target genes required for migration which are not being induced by Upd. Alternatively, there may be yet another signal from polar cells that is required for border cell differentiation. While one of these two explanations is favored, there are other possibilities. The stretch cells may already have been specified at the time of ectopic Upd expression, and thus be refractive to additional inductive signals. Also, when functional ectopic border cells are induced by extra polar cells, the timing and levels of signals to adjacent cells are likely to be relatively normal. This may not be the case when Upd is ectopically expressed (Beccari, 2002).
In addition to the spatial signal described above, a temporal signal must turn on expression of Slbo and other markers at the right stage. Upd and other polar cell markers are expressed in polar cells from earlier stages. Yet normal polar cells, or Hedgehog-induced ectopic polar cells, only induce border cells and border cell markers at stage 9. The temporal signal(s) may either modify polar cell signals to make them functional at the right stage, or act directly on border, stretch and centripetal cells to influence expression of target genes. Given the early expression of Upd and given that marker genes are induced in follicle cells with somewhat different temporal profiles, the latter scenario is favored. Two known candidates for supplying temporal signals are late Delta/Notch signaling and hormonal regulation (ecdysone). Analysis of a temperature-sensitive Notch allele has shown that Notch was required for Slbo expression. It has recently been shown that signaling by germ line Delta to Notch in follicle cells is required for proper differentiation of all follicle cells after stage 6. Although required for differentiation, the direct effect of Delta/Notch signaling at stage 6 is unlikely to explain the onset of Slbo expression at stage 8/9. But a cascade of events initiated at stage 6 might indirectly lead to expression of differentiation markers 16-24 h later. There is also evidence that some stage specific gene expression in egg chambers is regulated by the hormone ecdysone. In addition, the ecdysone receptor, EcR and its partner, Usp, appear to be required for border cell migration. One experiment in this study has suggested that ecdysone regulates timing of border cell migration, but apparently not timing of Slbo expression. Hormone application requires additional ectopic expression of Slbo to induce premature border cell migration. Thus the temporal regulation of anterior follicle cell differentiation may also have multiple components. Given that the stages of oogenesis are well-studied, this will be an interesting system in which to determine how temporal and spatial regulation of differentiation is coordinated (Beccari, 2002).
The Drosophila egg develops through closely coordinated activities of associated germline and somatic cells. An essential aspect of egg development is the differentiation of the somatic follicle cells into several distinct subpopulations with specific functions. The graded activity of the Janus kinase (JAK) pathway, stimulated by the Unpaired ligand, patterns the anterior-posterior axis of the follicular epithelium. Different levels of JAK activity instruct adoption of distinct anterior cell fates. Further, the coordinated activities of the JAK/STAT and epidermal growth factor receptor (EGFR) pathways are required to specify the posterior terminal cell fate. It is proposed that Upd secreted from the polar cells may act as a morphogen to stimulate A/P-derived follicular fates through JAK pathway activation (Xi, 2003).
The Drosophila egg is an intricately patterned structure with distinct specializations and polarities. These features are critical to subsequent embryonic development because the polarities of the egg are transmitted to the embryo, establishing the initial pattern in a developing zygote. The pattern of the mature egg is established by complex cellular interactions among and between both somatic follicle cells and germline cells. Each egg begins as a 16-cell germline cyst, from which one cell will become the oocyte and the remainder will become the supporting nurse cells. In the germarium, the anterior structure in which oogenesis is initiated, the germline cyst, is surrounded by a monolayer of somatic follicle cell precursors. As the encapsulated cyst exits from the germarium, approximately 10-14 of the somatic cells cease proliferation and differentiate. This group of cells forms two distinct populations: two polar cells at the anterior and posterior poles of each chamber and approximately seven stalk cells that form a bridge between the consecutive cysts. As the cyst exits the germarium, the other somatic cells covering each chamber, the epithelial follicle cells, remain undifferentiated (Xi, 2003 and references therein).
After pinching off from the germarium, each germline cyst grows, while the epithelial follicle cells proliferate. During this time, the anterior-posterior polarity that will ultimately determine all of the epithelial follicular fates is established. Elegant experiments have shown that the underlying prepattern of the follicular epithelium displays mirror image symmetry at the termini in the anterior-posterior (A/P) axis. Cells adopt one of three anterior terminal fates [border, stretched, and centripetal cells (terminal to central)], depending on proximity to the poles. In the intervening region between the terminal domains, cells will adopt a default 'main body' identity, and the posterior terminal cells form nearest the posterior pole. The symmetry of the A/P pattern is broken by EGFR signaling at the posterior. Secreted Grk from the posteriorly localized oocyte activates EGFR on the overlying follicle cells, establishing posterior terminal fate. In the absence of EGFR signaling, the anterior pattern is repeated at the posterior (Xi, 2003 and references therein).
By stage 7, the epithelial follicle cells cease proliferation and enter an endocycle. Afterward, these cells begin to show morphological and molecular signs of differentiation into the five epithelial fates: border, stretched, centripetal, posterior, and main body cells. Each of these subpopulations of follicle cells has a specific function with respect to the production of a mature egg, such that the correct number and position of each type is critical to ultimate egg morphology. These functions influence the production of structures that are essential to the egg, such as the dorsal respiratory appendages and the micropyle. These functions are also critical for proper anterior-posterior organization of the oocyte and, therefore, also for the resulting embryo (Xi, 2003 and references therein).
In early follicular differentiation, JAK activity is required for the production of stalk cells and the repression of polar cell fates. Later, JAK signaling is important for the proper recruitment and migration of border cells, a subpopulation of the follicular epithelium. Between these events, loss of JAK signaling in the follicular epithelium leads to persistent expression of Fas III, a marker for immature follicle cells (Xi, 2003 and references therein). The failure of these epithelial follicle cells to mature in hop mutant clones, as well as the persistent expression of upd in the polar cells, suggests that JAK signaling may have a role in differentiation of the entire follicular epithelium (Xi, 2003).
To address whether JAK signaling may play a role in distinguishing the terminal and main body domains, expression of mirror-lacZ (mirr-lacZ) was examined in egg chambers with aberrant JAK signaling. At ovarian stages 6-8, mirr-lacZ is strongly expressed in the main body follicle cells, with graded reduction toward the termini. In clones of hop mutant cells in the terminal regions, expression of mirr-lacZ is strongly induced, even prior to differentiation of those cells. It is concluded that JAK pathway activity distinguishes the terminal from the main body domains, at least as marked by the mirr-lacZ reporter. Specifically, JAK signaling is required to establish terminal identity and/or repress main body fate (Xi, 2003).
JAK activation is essential for specification of terminal fates, but is it also sufficient for terminal identity? To test this possibility, hop and upd were expressed in clones within the follicular epithelium, since either can activate JAK signaling. JAK pathway activation represses mirr-lacZ cell autonomously for Hop, but nonautonomously for the secreted Upd. This suggests that JAK pathway activity directly establishes the terminal domain. Furthermore, Upd expression causes graded repression of mirr-lacZ in the neighboring cells. Those cells closest to the Upd source have no expression of mirr-lacZ, but the amount of reporter in the neighboring cells increases with the distance from the Upd-expressing cells. It is concluded that JAK signaling must be activated in a graded fashion around the source of the Upd ligand, presumably because of extracellular diffusion of Upd. Moreover, the range over which the ectopic Upd can suppress mirr-lacZ is greater near the poles than in the center of the egg. Because endogenous Upd is secreted by the polar cells, it is easy to imagine that levels of JAK activity within the follicular epithelium would be greater near the poles. Consequently, the sum of the endogenous and ectopic Upd activities would be higher near the termini and could better repress mirr-lacZ. An alternative explanation is that the level of JAK signaling or another required signaling pathway is limited near the central region of the epithelium. It is concluded that JAK activation is necessary and sufficient for terminal identity in the follicular epithelium (Xi, 2003).
These results do not distinguish whether JAK signaling induces general terminal cell identity or is instructive for specific terminal fates. JAK activity could make the termini competent to respond to another signal that determines specific terminal fates or, alternatively, JAK activity could directly specify terminal fates, perhaps through varying levels of activation. To determine whether JAK signaling is essential for determining specific fates within the terminal domains, previously characterized markers for anterior subpopulations were examined. Work by others has already shown that JAK signaling can recruit the most terminal anterior fate, border cells. Here it is demonstrated that high levels of JAK activity are necessary and sufficient for border cell identity, at least within the anterior terminal domain. Consistent with the previous studies, the loss of JAK activity in clones of presumptive border cells invariably leads to failure of those cells to differentiate as border cells. However, this does not address whether JAK signaling is instructive for specific anterior fates. To address this question, the JAK pathway was activated to high levels in cells that are not at the terminus. Ectopic expression of either hop or upd stimulates additional cells to adopt border cell fate, again, in a cell-autonomous manner for hop and in a nonautonomous manner for upd. The location of the ectopic border cells suggests that they would normally have become stretched or centripetal cells. The ability of increased JAK signaling to alter fate within the anterior domain supports the hypothesis that levels of JAK signaling instruct specific fates within that domain. Increased JAK activity in the posterior terminal domain failed to induce ectopic border cells, presumably because of EGFR-mediated specification of posterior identity within this domain that would preclude expression of any anterior markers (Xi, 2003).
Involvement of JAK signaling in the specification of the more central anterior fates was examined with dpp-lacZ and MA33, which mark both stretched and centripetal cells, and BB127, which is specific for centripetal cells. JAK activity is essential for both fates. Loss of JAK signaling in either population results in a failure to express the population-specific markers. In addition to loss of the stretched cell marker, hop mutant cells also fail to migrate, spread out, and adopt the squamous morphology distinctive for stretched cells. On the basis of the expression of mirr-lacZ in JAK mutant clones above, it is presumed that these cells adopt a default main body fate. Moreover, effects of a weak general reduction of JAK signaling in the egg can be examined in ovaries of females transheterozygous for one severe and one weak allele of upd. In such eggs, the number of cells expressing a border cell marker is reduced. But, in addition, a marker for the stretched/centripetal fates is prominently expressed in the remaining border cells. Comparable expression of stretched cell markers in border cells is never observed in wild-type. Furthermore, the defective 'border cells' of upd mutant chambers also show aberrant migration. It is concluded that high levels of JAK activity are required for border cell fate, while lower levels direct stretched cell fate. Consistent with this, the border cells of upd mutant chambers did not express the centripetal cell-specific marker. Moreover, aberrant border cell specification in the Upd mutants indicates that Upd must normally activate JAK signaling during this process (Xi, 2003).
As with loss-of-function, clones of JAK-activated cells fail to express stretched and centripetal markers appropriate for their positions within the egg. On the basis of the morphology of the misexpressing cells and the complementary evidence with border cell markers, the presumptive stretched or centripetal cells with increased JAK activation are converted to the terminal-most border cell fate. Furthermore, JAK activation in the presumptive main body can induce the adoption of centripetal cell fate. However, it is somewhat surprising that JAK activation is unable to induce the most terminal (border cell) fate. One possible reason is that the endogenous JAK activity is likely highest near the poles and lowest in the central region, making it easier to convert cells closer to the terminus to border cell fate. This model assumes that the levels of ectopic activity must be lower than the highest levels of endogenous JAK activity, though evidence below suggests that this may not be true. A second possibility suggests that downstream components or cofactors required for high-level activation of JAK or for another required pathway may be in limited supply in the central region. Despite limited response in the central region, all the epithelial follicle cells are responsive to changes in levels of JAK activation. This indicates that the JAK pathway plays an active role, not a permissive role, in assigning specific terminal fates within the follicular epithelium (Xi, 2003).
The transformation of hop mutant cells in the posterior terminal domain into main body cells, as marked by mirr-lacZ, suggests that JAK signaling is essential for posterior terminal identity, as well as anterior fates. To address this hypothesis, two enhancer trap markers for posterior cells were analyzed in hop mutant clones. The pnt-lacZ marker is normally expressed strongly at the posterior, with a graded reduction in posterior cells farther from the pole. Cells mutant for hop show complete loss of pnt-lacZ expression in a cell-autonomous fashion. Moreover, because the cells at the posterior do not undergo the dramatic migrations seen at the anterior, it is possible to analyze the mutant and wild-type cells in their relative positions to one another. Significantly, it can be seen that wild-type cells express normal levels of pnt-lacZ, even when mutant cells intervene between them and the polar cells. Similar results were seen for a second posterior marker, blot01658. This suggests that the wild-type cells are receiving a signal directly from the polar cells and not via a local signal relay or 'bucket brigade' mechanism from neighboring cells (Xi, 2003).
To confirm that JAK activity influences posterior terminal fate, the function of those terminal cells was examined. At approximately stage 7, the posterior terminal cells send a signal to the underlying oocyte that stimulates microtubule reorganization in the oocyte. This microtubule reorganization is important for the migration of the oocyte nucleus to the dorsal anterior and for the proper sequestration of A/P determinants that direct development of the resulting embryo. After reorganization, one of these determinants, Staufen, is tightly associated with the posterior end of the oocyte. However, in egg chambers that lack JAK activity at the posterior terminus, Staufen fails to localize and is dispersed in the cytoplasm. Furthermore, in eggs with JAK mutant clones that cover only a portion of the presumptive posterior terminal cells, Staufen becomes localized directly underneath the wild-type cells at the posterior. This suggests that high JAK activity in the posterior follicle cells very precisely stimulates aggregation of a Staufen-bound complex in the underlying membrane. Despite the consistent mislocalization of Staufen in eggs with hop mutant clones at the posterior, the oocyte nucleus rarely fails to migrate to the dorsal anterior. This may indicate either that global microtubule reorganization is separable from Staufen localization or that Staufen is more sensitive to perturbations in microtubule reorganization (Xi, 2003).
Conversely, cell clones that express either hop or upd are able to activate pnt-lacZ, but only near the posterior of the chamber. Once again, the activation is autonomous for hop and nonautonomous for upd, supporting a direct role for JAK signaling in determining cell fates. Moreover, as with the mirr-lacZ reporter, the nonautonomous activity of Upd results in a graded response in the marker, such that the level of pnt-lacZ decreases as the distance from the ectopic Upd source increases. Again, this points to a gradient of JAK activity being established around the upd-expressing cells. Interestingly, activation of the posterior marker in cells neighboring upd-expressing clones was stronger on the posterior side of the clone. Again, this may be due to additive influences of the endogenous Upd signal coming from the polar cells and the ectopically expressing cells (Xi, 2003).
The initial A/P pattern in the follicular epithelium has a mirror image symmetry, such that cells at either end that are equidistant from the polar cells have equivalent identities. Subsequently, Grk from the oocyte, which always lies at the posterior of the egg, breaks the symmetry by stimulating EGFR in the follicle cells, inducing posterior terminal fate. Loss of EGFR activation in the posterior cells causes adoption of the underlying anterior fates. The requirement for both EGFR and JAK activation explains the failure of ectopic JAK activation to induce posterior identity at the anterior. But clones of cells that express activated EGFR can induce pnt-lacZ at the anterior. However, as in the posterior, induction of the marker at the anterior is graded, with highest levels closest to the pole. Furthermore, in the main body, activated EGFR is unable to induce posterior fate. This suggests that another factor essential for posterior identity is normally present, but limiting, in the anterior region. These domains that are competent to respond to activated EGFR coincide with the JAK activation. So, is EGFR limited in specifying posterior fate by the underlying activity of the JAK pathway? Consistent with this supposition, the coexpression of activated EGFR and JAK is capable of inducing posterior fate in all follicular epithelial cells. Thus, the coordinated activities of the two pathways are necessary and sufficient for induction of posterior identity (Xi, 2003).
Graded response of the mirr-lacZ marker and the ability of altered levels of JAK activity to change anterior fates are consistent with a model in which graded levels of JAK activity specify different follicular fates along the A/P axis. This model predicts that an overall increase or decrease of JAK activity would alter the number of cells adopting fates for each of the anterior subpopulations. Specifically, an overall reduction of JAK activity should reduce the number of border cells while shifting and/or reducing the number of cells adopting the more central stretched and centripetal fates and expanding the main body domain. To test this hypothesis, egg chambers from reduced function mutants of upd and hop were examined for the number and distribution of cells within each of the anterior subpopulations. Reduction of JAK activity dramatically reduces the number of border cells. Combination of one weak and one strong mutant allele of upd reduces the number of border cells by nearly half. Furthermore, combination of two weak hop alleles completely eliminates all border cells, despite producing morphologically normal eggs. Moreover, stretched cells are somewhat reduced in the hop mutant, while centripetal cells are only slightly affected. Similar results were seen at the posterior, where reduced hop activity results in marker expression that is only detectable to about four cell diameters from the posterior, rather than the normal eight cell diameters. However, graded marker expression is maintained, just shifted toward the posterior. A more substantial reduction of the most terminal fates strongly supports existence of graded JAK activity that is highest at the termini (Xi, 2003).
A model is presented for anterior-posterior follicular patterning. Patterning of the follicular epithelium requires the coordination of several signaling pathways. In the A/P axis, prominent roles for the EGFR and Notch pathways have been established. By incorporating the functions of the JAK pathway, an integrated model of A/P patterning in the follicular epithelium is proposed. The activation of the JAK pathway in the follicular epithelium is graded, with highest levels at the anterior and posterior poles. This is consistent with the production of the secreted ligand, Upd, from the polar cells, which is then received by cells of the follicular epithelium. The expression of Upd in the polar cells begins even within the germarium, so it is established as a potential graded signal from the earliest stages of follicular epithelial development. The polar cells have an organizer function in the establishment of A/P pattern. This organizer activity is consistent with the functions and behaviors described for JAK signaling in the surrounding epithelium. It is proposed that the gradient of JAK activity from both termini determines the presumptive border, stretched, and centripetal cells, on the basis of thresholds of JAK activity that define each fate, establishing a symmetrical prepattern. However, JAK signaling may not be the only patterning element in this process. Ectopic JAK activation in the main body domain is insufficient to induce the most terminal fate, the border cells. Though this could arise because of an inherent limitation to JAK signaling in the main body, the induction of Stat92E and Dome to high levels in similar activation clones argues against this. Alternatively, the main body may have low levels of some downstream coactivator for JAK signaling or of an independent patterning element, perhaps another signaling pathway. With the exception of this reduced response in the main body, adoption of each epithelial follicular fate can be simply ascribed to varying thresholds of JAK pathway activity (Xi, 2003).
The symmetrical prepattern established by JAK signaling is broken by EGFR activation in the posterior follicle cells stimulated by the secreted Grk ligand from the oocyte. The combined activation of JAK and EGFR signaling at the posterior defines posterior terminal follicle cell identity, overriding the default anterior fates specified by JAK activity alone. By the end of stage 6, when proliferation ceases, the cell fates of the follicular epithelium must already be determined. At that time, Notch pathway activation in all epithelial follicle cells triggers the transition from active division to an endocycle. By stage 9, the epithelial cells express markers for the various fates, begin migrations toward the posterior, and undergo morphological changes appropriate for ultimate function of that fate. Thus, the combined and sequential functions of the JAK, EGFR, and Notch pathways establish a series of anterior and posterior fates in the follicular epithelium (Xi, 2003).
The essential nature of morphogens, signals that have the ability to induce cell fates on the basis of levels of activity, is a central theme in animal development. Yet, despite this centrality, very few proteins have been demonstrated to have morphogenic function. Interestingly, most of the known morphogens have retained that activity throughout animal evolution. In both vertebrates and invertebrates, well-known signaling proteins of the Wnt, Hedgehog, and TGF-ß families act as morphogens. Though not all of the criteria have been explored, it is suggested that the properties of Upd and its stimulation of the JAK pathway in follicular epithelial cells are consistent with function as a morphogen. While the JAK intracellular cascade is highly conserved from flies to man, no proteins with significant homology to the Upd ligand have been found in other organisms. Therefore, Upd may be an unusual example of a morphogen that has rapidly diverged evolutionarily (Xi, 2003).
Morphogens are generally regarded to have four defining characteristics. (1) They are released from a localized source. In the ovary, Upd is secreted by the polar cells. (2) Morphogens form a concentration gradient from nearby to distant cells that respond directly to the signal, not through a relay mechanism. Although a gradient of Upd has not been directly visualized, the underlying gradient of JAK activation is apparent. Moreover, the response of cells to Upd activity requires downstream components of the JAK pathway in a cell-autonomous manner, demonstrating that the response to Upd is direct and not relayed. (3) Cells within the region of the gradient must show at least two different responses in addition to the default. In the follicular epithelium, the region that corresponds to the presumed JAK gradient gives rise to the border cells, stretched cells, and centripetal cells, in addition to the default main body cells. (4) Over- and underexpression should change cell fates in opposite directions. Clonal analysis clearly demonstrates that the anterior terminal and main body cell fates can be influenced by gain or loss of JAK pathway activity in an opposite and predictable manner. Thus, despite no direct visualization of a Upd gradient, the characteristics of the JAK pathway are consistent with a system that transduces a morphogenic signal (Xi, 2003).
The anterior-posterior axis of Drosophila becomes polarized early in oogenesis, when the oocyte moves to the posterior of the germline cyst because it preferentially adheres to posterior follicle cells. The source of this asymmetry is unclear, however, since anterior and posterior follicle cells are equivalent until midoogenesis, when Gurken signaling from the oocyte induces posterior fate. Asymmetry is shown to arise because each cyst polarizes the next cyst through a series of posterior to anterior inductions. Delta signaling from the older cyst induces the anterior polar follicle cells, the anterior polar cells signal through the JAK/STAT pathway to induce the formation of the stalk between adjacent cysts, and the stalk polarizes the younger anterior cyst by inducing the shape change and preferential adhesion that positions the oocyte at the posterior. The anterior-posterior axis is therefore established by a relay mechanism, which propagates polarity from one cyst to the next (Torres, 2003).
The follicle stem cells reside in region 2b of the germarium and give rise to two distinct lineages: the epithelial follicle cell precursors, which proliferate until stage 6 to generate most of the cells that surround each cyst, and the polar/stalk precursors. The latter exit mitosis at stage 1 to 2 of oogenesis and give rise to the symmetric pairs of polar cells at the anterior and posterior poles of the cyst and to the stalk that separates each cyst from the adjacent one. Delta mutant germline clones and Notch follicle cell clones fail to form polar cells, indicating that Delta signals from the germline to activate the Notch receptor in the polar/stalk precursors to induce them to adopt the polar cell fate. This induction requires fringe, which is upregulated in the polar/stalk precursors and renders these precursors competent to respond to the Delta signal. Once the polar cells are specified, they express Unpaired, the ligand for the JAK/STAT pathway, and the resultant activation of JAK/STAT signaling plays two key roles in patterning the rest of the follicle cells. (1) The polar cells induce uncommitted polar/stalk cell precursors to become stalk cells. Overexpression of Unpaired causes all polar/stalk cell precursors to differentiate as stalk, whereas loss-of-function mutations in hopscotch (JAK) or STAT92E cause a loss of the stalk. (2) Unpaired signaling from the polar cells induces the adjacent epithelial follicle cells at each pole of the egg chamber to adopt a terminal fate. This induction is essential for axis formation because only the terminal cells are competent to respond to Gurken by becoming posterior. Unpaired also acts as a morphogen to specify three distinct terminal cell types at the anterior: the border cells, the stretched follicle cells, and the centripetal cells. In the absence of Gurken signaling, all three cell types also form at the posterior of the egg chamber, indicating that the graded activity of JAK/STAT pathway creates a symmetric prepattern at both poles (Torres, 2003 and references therein).
Polar cells are pairs of specific follicular cells present at each pole of Drosophila egg chambers. They are required at different stages of oogenesis for egg chamber formation and establishment of both the anteroposterior and planar polarities of the follicular epithelium. Definition of polar cell pairs is a progressive process since early stage egg chambers contain a cluster of several polar cell marker-expressing cells at each pole, while as of stage 5, they contain invariantly two pairs of such cells. Using cell lineage analysis, it has been demonstrated that these pre-polar cell clusters have a polyclonal origin and derive specifically from the polar cell lineage, rather than from that giving rise to follicular cells. In addition, selection of two polar cells from groups of pre-polar cells occurs via an apoptosis-dependent mechanism and is required for correct patterning of the anterior follicular epithelium of vitellogenic egg chambers. Prevention of pre-polar cell death and subsequent generation of supernumerary polar cells may lead to production of an excess of signaling molecules, such as Unpaired, and alteration of endogenous morphogen gradients which could explain why both squamous cells and border cells exhibit aberrant behavior when pre-polar cell death is blocked (Besse, 2003).
Thus, each pair of mature polar cells derives from a pool of precursor pre-polar cells within which supernumerary cells are eliminated via an apoptosis-dependent mechanism. This mechanism probably requires both caspase activity and the 'death' gene reaper, since death is inhibited by ectopic expression of the bacculoviral p35 protein and is associated with specific induction of reaper expression. However, whereas the self-death machinery appears to be evolutionary conserved, a wide range of distinct signaling mechanisms can be used to elicit apoptosis. Cellular interactions within or without the pre-polar cell cluster may also be crucial for regulation of the selective pre-polar cell loss. In the present study, no correlation could be made between pre-polar cell position and cell removal, at least for apoptosis events occurring after egg chamber budding. It would be interesting nonetheless to examine Notch signaling as a survival factor in this system. Indeed, induction of Notch loss-of-function clones in prefollicular cells is associated with absence of polar cells. Conversely, egg chambers with terminal clones expressing an activated form of Notch contain up to 6 polar cell marker-positive cells. Such phenotypes, interpreted as reflecting a role for Notch signaling in polar cell specification, could also correspond to a Notch-dependent control of apoptosis within the pre-polar cell lineage (Besse, 2003).
Considering both the embryonic and oogenesis systems, the question can be asked as to the biological significance of creating supernumerary cells to remove them afterwards. One proposed explanation for what is observed in the embryonic CNS is that a differential number of glial cells may be required depending on the developmental stage. By analogy, at least 6 pre-polar cells may be required in the ovary to assume early germarial functions, whereas only 2 polar cells may be needed during later egg chamber maturation. Another possibility is that the process of polar cell production corresponds to an evolutionarily conserved mechanism to which removal of deleterious cells would have been added later. Further studies are now needed to determine whether the pre-polar cell clusters have a functional role in early oogenesis (Besse, 2003).
Polar cells are involved in several important signaling processes during oogenesis, including posterior positioning of the oocyte, induction of stalk cells in the germarium, organization of follicular cell epithelial planar polarity during mid-oogenesis and anteroposterior patterning of follicular epithelial cells at stage 9 (Besse, 2003 and references therein).
Strikingly, polarization of basal actin filament bundles in the follicular epithelium arises progressively, starting at stage 5 and proceeding from the poles, suggesting the existence of a diffusible signal produced by polar cells. Consistent with this, ectopic polar cells generated upon hedgehog overexpression have the capacity to reorganize actin bundles locally. However, orientation of planar actin filaments probably does not require a precise level of polarizing signal since it is shown here that an increase in polar cell number does not affect the establishment or maintenance of planar polarity (Besse, 2003).
In contrast, it has been found that the restriction in the number of polar cells seems to be required for correct anterior patterning of follicular epithelial cells. Indeed, preventing the elimination of supernumerary pre-polar cells results in morphogenetic defects affecting both stretching of anterior squamous cells and migration of border cell clusters. The latter is also accompanied by an increase in the number of recruited border cells. Production of ectopic polar cells has been described to induce ectopic and poorly migrating border cells at stage 9. Yet, this phenomenon was observed upon ectopic activation of hedgehog signal transduction (patched and Costal 2 mutant contexts). Therefore it is not clear in these cases whether migration defects are a direct consequence of extra border cell number or whether they reflect an additional effect of the Hedgehog signaling pathway on border cell migration and/or specification (Besse, 2003).
The fused gene encodes a serine/threonine kinase identified as a positive effector of the Hedgehog signal transduction pathway. In the ovary, Hedgehog signal transduction controls somatic stem cell (SSC) proliferation. Indeed, SSC self-renewing properties are not maintained in the absence of Hh signaling, whereas excessive Hh signaling produces supernumerary stem cells and leads to the accumulation of poorly differentiated somatic cells between egg chambers. Analysis of fu mutations had indicated that fu function is not involved in this process. Rather, fu-dependent Hedgehog signal transduction is necessary for somatic prefollicular cell differentiation and morphogenesis. In particular, fu function seems to be required for correct timing of the polar cell differentiation program. Indeed, fu mutant females exhibit a global shift in the dynamics of A101 staining, as visualized after anti-ß-galactosidase staining of fuJB3/fuJB3; A101/+ females. (1) The appearance of A101 staining is delayed, since 28% of stage 2 fu egg chambers do not exhibit any marked anterior cells compared to 19% in heterozygous sisters. (2) Restriction of A101 staining to 2 polar cells is also delayed, since 60% of stage 3 and 19% of stage 4 fu egg chambers contained 3 or more stained anterior cells compared to 33% and 4%, respectively, for fu+ egg chambers. Strikingly, 100% of stage 5 fu mutant egg chambers exhibit only 2 A101+ cells, indicating that restriction in the number of polar cells does eventually occur as in wild-type ovarioles. Altogether, these results suggest that fu mutations lead to a delay in the polar cell differentiation program (Besse, 2003).
Close examination of fu ovarioles has revealed that a higher proportion of groups containing 4 A101+ cells, 5 A101+ cells (8/156), and even 6 A101+ cells (1/156) can be found in stage 2 as well as stage 3 fu mutant egg chambers compared to the wild-type situation. It was reasoned that the presence of such groups of cells could result either from abnormally slow apoptosis-dependent elimination of pre-polar cells, or from an overproduction of pre-polar cells or their precursors. The first hypothesis could not be tested directly because the relatively low number of TUNEL-positive cells found in both wild-type and fused females (possibly due to rapid elimination of apoptotic cells) made it impossible to compare quantitatively the dynamics of polar cell apoptotic cell death between these two contexts. Therefore the second hypothesis was tested; defects were sought in polar and pre-polar cell proliferation, or in the number of polar cell precursor cells. First, polar cell proliferative properties do not seem to be altered in the vitellarium of fu ovarioles since (1) no increase in the size of A101+ terminal clusters is observed with increasing age of fu egg chambers from stage 2 to 5, and (2) no prolongation beyond stage 6 of somatic cell mitotic activity is observed in fu ovarioles. Second, using a dominantly marked clone approach, it has been shown that early clusters of 4-6 A101+ cells found in fu females never contain more than 2 GFP+ cells, and therefore that they do not result from extra divisions of precursor cells within the germarium. Third, it was reasoned that preventing apoptosis in the polar cell lineage in a fu mutant context should give an indication about the number of polar cell precursors present in these flies. If the number of such precursors is greater in fu females than in wild-type females, then blocking apoptosis should result in a greater number of 'rescued' cells in polar cell clusters than in a wild-type context (that is more than 6 cells). Therefore, the flp-out/Gal4 system was used to generate large somatic clones of p35 overexpressing cells in a fu mutant context. Although an increase in the average size of the terminal Fas III+ cell cluster was observed after p35 overexpression, only groups containing 2 to 6 cells were recovered. This indicates that fused females contain the same number of polar cell precursors as wild-type females (Besse, 2003).
Therefore, the supernumerary polar cells in both wild-type and fused mutant contexts is interpreted to represent pre-polar cells, and it is proposed that slower apoptosis-mediated reduction in the number of these cells in a fused context allows easier visualization of these cells. Thus, fused mutations, by delaying the somatic cell differentiation program, confirm the existence of pre-polar cell clusters and allow detection of up to 6 pre-polar cells. However, restriction in the final number of polar cells is achieved by stage 5 and is probably also mediated by apoptosis since TUNEL-positive A101+ cells are found in fused females (Besse, 2003).
In this study, defective border cell migration was detected after having prevented cell death specifically within neuralized-expressing pre-polar and polar cells. This suggests, first, that rescued pre-polar cells are not inert and, second, that final polar cell number per se is critical for both border cell recruitment and migration. Interestingly, polar cells show specific expression of Unpaired (Upd), an extracellular ligand that activates the conserved JAK/Stat signaling pathway. In the absence of positive effectors of this pathway, such as Unpaired, Hopscotch or STAT92E, defects in both recruitment and migration of border cells and sometimes also in stretching of squamous cells are observed. In addition, ectopic expression of Upd in a subset of anterior somatic cells is sufficient to induce expression of border cell markers in adjacent squamous cells. Interestingly, high levels of Upd result in the formation of egg chambers in which both normal and supernumerary border cells frequently fail to migrate. Altogether, this suggests that Upd could act as a morphogen produced by polar cells and necessary for establishing anteroposterior patterning of the follicular epithelium. In the present study, prevention of pre-polar cell death and subsequent generation of supernumerary polar cells may lead to production of an excess of signaling molecules, such as Upd, and alteration of endogenous morphogen gradients, which could explain why both squamous cells and border cells exhibit aberrant behavior. These results therefore provide further evidence for a non cell-autonomous role for anterior polar cells in patterning of the follicular epithelium (Besse, 2003).
Germ cells must develop along distinct male or female paths to produce the sperm or eggs required for sexual reproduction. In both mouse and Drosophila, sexual identity of germ cells is influenced by the sex of the surrounding somatic tissue, but little is known about how the soma controls germline sex determination. This study shows that the JAK/STAT pathway provides a sex-specific signal from the soma to the germline in the Drosophila embryonic gonad. The somatic gonad expresses a JAK/STAT ligand, unpaired (upd), in a male-specific manner, and activates the JAK/STAT pathway in male germ cells at the time of gonad formation. Furthermore, the JAK/STAT pathway is necessary for male-specific germ cell behavior during early gonad development, and is sufficient to activate aspects of male germ cell behavior in female germ cells. This work provides direct evidence that the JAK/STAT pathway mediates a key signal from the somatic gonad that regulates male germline sexual development (Wawersik, 2005).
While investigating communication between the somatic gonad and germline, the JAK/STAT pathway was found to be specifically activated in male, but not female, germ cells. In Drosophila, JAK/STAT signaling is initiated when an UPD or UPD-like ligand binds a transmembrane receptor (Domeless), activating the JAK Hopscotch (HOP), which phosphorylates the STAT92E transcription factor. STAT activation has been shown to regulate stat gene expression and can induce upregulation of the STAT92E protein, which can be used as an assay for JAK/STAT pathway activation. STAT92E is upregulated specifically in male, but not female germ cells at the time of gonad formation. This reflects male-specific activation of the JAK/STAT pathway since (1) the activated form of STAT92E (phospho-STAT92E) is also detected in only male germ cells, and (2) JAK activity is necessary and sufficient for STAT92E expression. Expression of a JAK inhibitor, Socs36E, results in loss of STAT92E expression in male germ cells and expression of constitutively active JAK (hopTumL) induces STAT92E in female germ cells. The male-specific activation of STAT92E at this time is distinct from STAT92E activation in germ cells in the early embryo, which is not sex-specific and is regulated by the MAP kinase pathway (Wawersik, 2005).
It was also found that STAT92E expression in male germ cells is dependent on their association with the somatic gonad. STAT92E is not detected in germ cells that are migrating to the gonad, but is detected in male germ cells after they contact the somatic gonad. STAT92E expression is greatly reduced or absent in eya mutants, where somatic gonad identity is initiated, but not maintained. Furthermore, STAT92E is not detected in germ cells found outside the gonad in wild type embryos or in mis-localized germ cells in wunen and HMG-CoA reductase mutants which lack guidance cues that target germ cells to the somatic gonad. However, in these same mutants, STAT92E is detected in the few germ cells that contact the somatic gonad in male embryos (Wawersik, 2005).
STAT92E expression in the germline is dependent on the sex of the surrounding soma. When XX (normally female) germ cells were present in a soma that was masculinized by expression of the male form of the somatic sex determination gene doublesex (dsx), germ cells now expressed STAT92E. dsx does not play an autonomous role in germ cells themselves, indicating that STAT92E induction in these embryos is caused by masculinization of the soma. Conversely, when the somatic gonad of an XY (normally male) embryo is feminized by expression of the sex determination gene transformer (tra) in the mesoderm, but not germ cells, STAT92E expression is no longer observed in XY germ cells. Taken together, these data indicate that the male somatic gonad is necessary and sufficient to activate the JAK/STAT pathway in either XX or XY germ cells (Wawersik, 2005).
Consistent with this, it was found that the JAK/STAT ligand, upd, is expressed specifically in the male, but not female, somatic gonad. Expression of STAT92E in male germ cells was no longer detected in embryos in which upd and two homologs, upd2 and upd3, are deleted [Df(os1a]. Since male germ cells from embryos mutant for upd alone still express STAT92E, JAK/STAT activation in the germline may be regulated redundantly by upd and one or more of its homologs. In addition, expression of upd in either the mesoderm or germ cells is sufficient to induce STAT92E expression in XX germ cells. Expression of upd2 or upd3 is also capable of inducing STAT92E in germ cells (Wawersik, 2005).
upd is also important for embryonic patterning and somatic sex determination. Interestingly, upd promotes female identity in the soma, but promotes male development in the germline. To verify that the effects of upd on the germline are not indirectly caused by other effects of upd, indicators of embryonic segmentation (Engrailed), somatic sex determination (Sex lethal), somatic gonad identity (Eyes absent), and somatic gonad sexual identity (Sox100B) were examined. Df(os1a) hemizygous male embryos exhibit segmentation defects as expected, but form gonads that express normal somatic and sex-specific markers. Embryos ectopically expressing upd are normal in all respects examined (Wawersik, 2005).
Whether activation of the JAK/STAT pathway by the male somatic gonad regulates male-specific development of germ cells was examined. In adult testes, the JAK/STAT pathway is required for maintenance of germline stem cells, making it difficult to assess the role of this pathway on male germ cell identity at this stage. Instead, germ cells were examined during embryogenesis and early larval stages, when germ cell development first becomes sexually dimorphic. In the mouse, the earliest manifestation of sex determination in the germline is differential regulation of the germline cell cycle by the soma. In Drosophila, germ cells undergo 1-2 divisions after their formation, but are arrested in the cell cycle during germ cell migration and only resume division shortly after the gonad has formed. Since larval testes contain more germ cells than larval ovaries, whether proliferation is regulated differently in male and female germ cells was examined. Indeed, sex-specific analysis of a mitotic marker (phosphohistone-H3) in the germline indicates that germ cell proliferation is entirely male-specific during early stages of gonad development. Furthermore, male-specific germ cell division is dependent on the male somatic gonad. Male germ cells do not proliferate in eya mutants that lack the somatic gonad, or in lost germ cells within wunen mutant embryos. XX germ cells in a masculinized soma (dsxD/ dsx1) proliferate, while XY germ cells in a feminized soma (UAS-traF; twist-Gal4) do not. Thus, the pattern of germ cell proliferation correlates exactly with activity of the JAK/STAT pathway in germ cells (Wawersik, 2005).
To assess whether JAK/STAT signaling regulates male-specific germ cell division, embryos lacking zygotic Stat92E activity were examined and a dramatic decrease was observed in male germ cell proliferation. Similar reductions in germ cell proliferation are observed in the upd/upd-like mutant (Df(os1a)) and in embryos where the JAK inhibitor Socs36E is expressed in germ cells. Thus, JAK/STAT activity is required within germ cells for proper male-specific germ cell division in the gonad. Expression of upd in the germline is sufficient to induce proliferation in female germ cells. Thus, the JAK/STAT pathway can induce XX germ cells to exhibit this male-specific germ cell behavior (Wawersik, 2005).
Whether the JAK/STAT pathway regulates other aspects of male germ cell development was examined. male germline marker-1 (mgm-1) is a lacZ enhancer trap line that is expressed in male germ cells, but not female germ cells, and therefore is a marker for male germ cell identity. Inhibiting the JAK/STAT pathway by removing zygotic Stat92E activity does not affect mgm-1 expression in the embryo, which is as expected since initial mgm-1 expression is dependent on germ cell autonomous cues. However, removal of zygotic Stat92E activity completely abolished mgm-1 expression in first instar larvae. In wild-type first instar male larvae, mgm-1 expression is observed in most germ cells, which are likely to be developing male germline stem cells and spermatogonia. No mgm-1 expression is observed in Stat92E-mutant larvae, and β-galactosidase expression is only observed in the soma, not the germline, in the pattern expected from the Stat92E P element allele. In an experiment where 25% of larvae were expected to be both male and contain the mgm-1 enhancer trap, 23.2% (n=262) of wild type larvae exhibited mgm-1 expression in the germ cells, while no Stat92E mutant larvae exhibited germ cell mgm-1 expression; this is significantly different from wild type siblings. Thus, Stat92E mutants exhibit a strong effect on male germline development, and some male germline cell types are either missing, or have an altered identity (Wawersik, 2005).
Finally, the extent to which activation of the JAK/STAT pathway can masculinize female germ cells was assessed. Female germ cells expressing upd are not expected to be fully masculinized because, although a male-specific signal is being activated, these germ cells are otherwise still in a female somatic environment and retain female germ cell autonomous cues. Indeed, such embryos give rise to fertile adult females, indicating that at least some germ cells retain, or revert back to, a female identity. This may be due, in part, to the failure of the upd construct to be expressed in the adult female germline. However, upd is sufficient to induce male-specific gene expression in embryonic XX germ cells. While mgm-1 is normally expressed only in germ cells in males, mgm-1 was expressed in all embryos when upd was ectopically expressed. In addition, two new male germline markers, disc proliferation abnormal (dpa) and minichromosome maintenance 5 (mcm5), were identified, that can also be induced by upd. Whereas these genes are normally expressed in germ cells only in males, female embryos exhibit germ cell expression of these genes when upd is ectopically expressed. In an experiment where only 50% of embryos are expected to express ectopic upd in the germline, 32.5% of female embryos expressed dpa and 21.3% expressed mcm5. Therefore, upd expression is sufficient to activate male-specific gene expression in female germ cells (Wawersik, 2005).
These data indicate that the JAK/STAT pathway mediates a critical signal from the male somatic gonad that is required for male germ cell development. This signal likely acts together with male germ cell autonomous cues to promote male germline identity and spermatogenesis. This signal is also sufficient to activate the male pattern of proliferation and gene expression in female germ cells, even when these germ cells retain female germ cell autonomous cues and are present in an otherwise female soma. It will be very interesting in the future to identify additional (e.g. female) somatic signals, along with germ cell autonomous cues, and to assess the relative contribution of these factors to proper germline sexual development. Since one of the earliest aspects of sex-specific germ cell behavior in both Drosophila and mouse is the regulation of the germline cell cycle by the somatic gonad, it will be of further interest to determine if the somatic signals operating in Drosophila play a similar role in germline sex determination in mammals (Wawersik, 2005).
X-linked signal elements (XSEs) communicate the dose of X chromosomes to the regulatory-switch gene Sex-lethal (Sxl) during Drosophila sex determination. Unequal XSE expression in precellular XX and XY nuclei ensures that only XX embryos will activate the establishment promoter, SxlPe, to produce a pulse of the RNA-binding protein, SXL. Once XSE protein concentrations have been assessed, SxlPe is inactivated and the maintenance promoter, SxlPm, is turned on in both sexes; however, only in females is SXL present to direct the SxlPm-derived transcripts to be spliced into functional mRNA. Thereafter, Sxl is maintained in the on state by positive autoregulatory RNA splicing. Once set in the stable on (female) or off (male) state, Sxl controls somatic sexual development through control of downstream effectors of sexual differentiation and dosage compensation. Most XSEs encode transcription factors that bind SxlPe, but the XSE unpaired (upd) encodes a secreted ligand for the JAK/STAT pathway. Although STAT directly regulates SxlPe, it is dispensable for promoter activation. Instead, JAK/STAT is needed to maintain high-level SxlPe expression in order to ensure Sxl autoregulation in XX embryos. Thus, upd is a unique XSE that augments, rather than defines, the initial sex-determination signal (Avila, 2007).
The question of how embryos differentiate between precise 2-fold differences in X-linked signal element (XSE) doses is central to understanding how genetic constitution defines sexual fate. Current X-chromosome-counting models posit that the female fate is set when XSE proteins exceed a threshold concentration and activate SxlPe. The XSE threshold is set by interactions between the XSEs and other proteins in the embryo. Some XSEs interact with maternally supplied proteins to form dose-sensitive transcription factors, such as Scute/Daughterless, that bind SxlPe, but XSE doses are also assessed with reference to maternally and zygotically expressed repressors. Three XSE proteins, SisA, Scute, and Runt, are viewed as acting similarly by binding directly to and activating SxlPe. The fourth XSE, unpaired (upd, also called outstretched or sisC), encodes a secreted ligand that signals through the JAK kinase (hopscotch) to activate the Stat92E transcription factor. Although upd meets the criteria of an XSE, its effects on sex determination are weaker than those of sisA, scute, and runt, and changes in its gene dose have only moderate effects on Sxl. To understand how this comparatively dose-insensitive XSE regulates sex, when and where upd, JAK, and STAT act on the Sxl switch was examined (Avila, 2007).
Using in situ hybridization, the early embryonic expression pattern of upd was defined. No evidence was found for maternally supplied transcripts and it was observed that upd mRNA was first detectable in nuclear cycle 13. The fact that the first upd transcripts are present throughout the embryo, including at the poles, is consistent with the distribution of phosphorylated Stat92E. As cellularization progresses past early cycle 14, the upd pattern resolves into indistinct stripes that developed into a 14 stripe pattern during gastrulation. These results show that upd expression begins later than that of the other XSEs (sisA in cycle 8; scute in cycle 9) and also, paradoxically, that it begins after the onset of transcription of its target, Sxl, in cycle 12 (Avila, 2007).
To understand how upd functions in Sxl activation and how it differs from other XSEs, upd mutations were examined for their effects on SxlPe by using in situ hybridization and on Sxl protein levels by using immunostaining with SXL antibody. Significantly, the RNA probes detected nascent Sxl transcripts, allowing monitoring of both the spatial and temporal responses of SxlPe on a cell-cycle by cell-cycle basis (Avila, 2007).
updsisC1, a loss-of-function mutation that appears to specifically affect sex determination was examined, because it has no observable effect on later upd functions. Consistent with the fact that upd has a modest effect on SxlPe, it was found that two-thirds of homozygous updsisC embryos expressed SxlPe in a manner indistinguishable from that of the wild-type. A small proportion of embryos, 15%, had within their middle sections several clusters of 5–15 nuclei that did not express SxlPe, whereas the remaining 18% had severe defects, with SxlPe expression being absent from most of the central regions of the embryos. Despite early aberrations in SxlPe activity, immunostaining revealed no lasting defect in the expression of SXL, because updsisC1 embryos that reached germband extension stained in a 1:1 male:female ratio. To determine the effects of a complete loss of zygotic upd activity, updYC43, a probable null mutation, and the deficiency Df(1)ue69, which deletes upd and the upd-like gene, upd3, were examined. With respect to SxlPe, it was found that upd-null-mutant females were more severely affected than were updsisC1 embryos. At cellularization, the defects ranged from embryos containing large clusters of nuclei that did not express SxlPe in the central part of embryo to those in which the entire central region failed to express the promoter. The poles, however, expressed SxlPe normally. Immunostainings of updYC43 and Df(1)ue69 embryo collections revealed that these alleles had strong but incompletely penetrant effects on the later distribution of SXL. The fact that an estimated 40% of mutant female embryos stage 6 and older failed to express SXL in their central regions is consistent with the observed defects in SxlPe activity. The remainder eventually expressed normal levels of SXL in all their tissues, indicating that most upd mutant females were able to compensate for reduced SxlPe activity and ultimately engaged autoregulatory Sxl mRNA splicing. Two upd-like genes, upd2 and upd3, map adjacent to upd. Loss of zygotic upd2 had no effect on SxlPe, and the effects of Df(1)ue69 (upd3−,upd−) appeared identical to those of updYC43 when analyzed in a common genetic background. This shows that XSE activity in this region of the X is due to upd alone (Avila, 2007).
Except for the ligands, each component of the JAK/STAT pathway is maternally deposited into the embryo. To eliminate JAK/STAT activity completely, the dominant female-sterile technique was used to generate females lacking maternal hopscotch (hop) or Stat92E, which encode the only JAK kinase and STAT in Drosophila. It was expected that by removing maternal hop, STAT would remain unphosphoryated, allowing a determination of the effects of the loss of the entire pathway on SxlPe (Avila, 2007).
When Sxl expression was examined in cycle 14 embryos derived from hopC111 germline clones, it was found that SxlPe was active in the anterior and posterior regions of the embryos but almost completely inactive in the central region of the embryos. In contrast to the results with upd mutants and deficiencies, all of which exhibited considerable embryo-to-embryo variation, loss of maternal hop had nearly identical effects on SxlPe in every embryo. This more potent effect of maternal hopC111 as compared to upd mutants suggests that zygotic Upd might not be the only activator of JAK in the precellular embryo (Avila, 2007).
The findings with hopC111 were confirmed by using the Stat92E06346 mutation. Cycle 14 embryos derived from Stat92E06346 germline clones also lacked nearly all SxlPe expression in their central regions, but they were even more strongly affected than hopC111 females because SxlPe activity was also reduced in the termini. These findings are contrary to predictions of a linear JAK/STAT pathway going from zygotic Upd through receptor and kinase to activated STAT. Instead, the progressive weakening of SxlPe by removal of upd and Stat92E suggests that there is hop-independent control of Stat92E function in sex determination. The possibility of cross-talk between signaling systems is supported by the finding that the torso receptor-tyrosine-kinase pathway activates STAT92E in the embryo termini (Avila, 2007).
Although the hopC111 and Stat92E06346 mutations had large effects on SxlPe during cycle 14, the period of maximum SxlPe expression, it was found that these mutations had little effect on SxlPe at earlier stages. In wild-type females, SxlPe is first activated in cycle 12. Expression increases throughout cycle 13 and reaches a peak in the first minutes of cycle 14. In embryos from hopC111 mothers, SxlPe was expressed as in the wild-type during cycles 12 and 13. However, upon entry into cycle 14, SxlPe activity ceased in the middle sections of the embryos. A similar phenomenon was observed in embryos carrying strong upd mutants and in those derived from Stat92E06346 germline clones. These results show that JAK/STAT, and thus upd XSE function, is not needed for the initial activation of SxlPe. Instead, upd must function as a different kind of XSE: one dispensable for the initial assessment of X-chromosome dose, but needed to maintain SxlPe activity in the final stage of the X-counting process (Avila, 2007).
When the progeny of hopC111 mutant mothers were examined for Sxl protein, it was found that defects in SxlPe expression led to a permanent failure to express SXL in the central regions in 35% of female embryos. This suggests that the loss of SxlPe activity in cycle 14 can reduce the level of early Sxl to below the threshold normally required to activate autoregulatory mRNA splicing. Although 35% of female embryos were defective for later Sxl expression, most females that completed gastrulation expressed Sxl uniformly. This striking discordance between the effects of hop (and upd and Stat92E) mutants on SxlPe activity and ultimate Sxl levels suggests that stable Sxl autoregulation can be established even when SxlPe function has been seriously compromised. Although some rescuing Sxl mRNA or protein may have diffused from the poles, an alternative explanation is that expression of SxlPe during cycles 12 and 13 might often have provided sufficient Sxl to trigger autoregulation once the maintenance promoter, SxlPm, had been activated (Avila, 2007).
SxlPe is thought to have two main functional elements: a proximal 390 bp X-counting region responsible for sex-specific activation, and a more distal (to −1.4 kb) element that elevates Sxl transcription. Three predicted STAT-binding sites are located in these elements at positions −253, −393, and −428 bp. To test their roles, consensus TTC sequences were changed to TTT because such changes block binding by STAT92E and the mammalian homologs STATs 5 and 5a. In situ hybridizations revealed that the mutation in the proximal STAT site, S1, greatly reduced the number of nuclei expressing SxlPe-lacZ, creating a patchy staining pattern and lower overall mRNA levels. Mutations in S1 and S2, or in all three sites together, caused a strong but variable loss of SxlPe-lacZ expression in most nuclei, resulting in dramatically reduced accumulation of lacZ mRNA. Although the S1, S2, S3 mutant appeared to have a slightly stronger effect than the double mutant, both transgenes exhibited phenotypes reminiscent of those seen in embryos derived from Stat92E06346 germline clones. These results show that STAT92E acts through the consensus binding sites at SxlPe (Avila, 2007).
SxlPe is remarkable for both its rapid response and exquisite sensitivity to X-chromosome dose. In male embryos, it is always off. In female embryos, SxlPe is strongly expressed, but only during a 35–40 min period from mid cycle 12 until about 10–15 min into cycle 14. Given these time constraints, many have assumed that all XSEs would function to establish the initial on or off state of SxlPe. However, it was found that upd behaved very differently than sisA and scute, both of which are required for SxlPe activation and expression. Loss of upd or the JAK/STAT pathway had little or no effect on SxlPe during cycles 12 or 13. Instead, JAK/STAT mutations blocked SxlPe expression late in the process, during cycle 14. This observation is interpreted as revealing that SxlPe is regulated in two mechanistically distinct phases: the first controlling the initial response to X-chromosome dose, and the second acting to maintain or reinforce the initial decision (Avila, 2007).
The relatively late actions of upd and hop offer explanations for several puzzling aspects of upd's function in sex determination. First, upd is considered a weak XSE. This is both because Sxl is comparatively insensitive to upd dose and because loss of upd or JAK/STAT function doesn't eliminate Sxl expression. Both effects are consistent with expectations of a two-step, initiation and maintenance, model for SxlPe function. JAK/STAT mutations would not be expected to eliminate all Sxl function in a two-step model because the STAT-independent initiation step would produce Sxl mRNA and protein. The exact gene dose of upd would not be particularly important for sex because excess active STAT could not induce SxlPe without the prior actions of the initiating XSEs and because even a single dose of upd+ could provide enough active STAT to augment an earlier decision to become female. Thus, the proposed STAT maintenance function explains both the failure of the constitutively active hoptum-l allele to induce ectopic SxlPe expression in males and the ability of hoptum-l to further stimulate SxlPe activity in females. Likewise, the requirement for STAT site S2, located just distal to the 390 bp X-counting region of SxlPe, and the finding that upd is first expressed after Sxl can be explained if STAT's role is to bolster transcription from SxlPe in embryos that already have counted two Xs. Although neither essential for SxlPe expression nor highly dose sensitive, upd, hop, and Stat92E nonetheless play important roles at SxlPe. In their absence, the period of SxlPe activity is cut short, reducing the concentration of Sxl and preventing a large fraction of embryos from engaging the maintenance mode of Sxl expression (Avila, 2007).
How might STAT92E function in a two-step model? One possibility is that STAT might antagonize the late-acting repressor Dpn. Alternatively, the STAT transcription factor might augment, stabilize, or replace earlier-acting XSE activator complexes as their concentrations diminish in cycle 14. BAP60, a core component of the Brahma chromatin-remodeling complex, has been shown to interact with two components of the sex-determination signal. If STAT92E also interacts with the Brahma complex, it might maintain SxlPe chromatin in an active state, facilitating the restoration of transcription after the 13th mitosis (Avila, 2007).
Understanding the commonalities and unique mechanisms STATs employ in their multitude of roles is a fundamental goal of research on this ubiquitous signaling pathway. It is also essential for understanding why the pathway has so often been co-opted into new roles during evolution. STATS seem primarily permissive rather than instructive. They are rarely the primary signals defining cell fate. In these respects, comparison of the even-skipped (eve) stripe 3 enhancer and SxlPe reveals interesting parallels. Both SxlPe and eve stripe 3 are regulated by the balance between several activators and repressors. The responses of both elements to JAK/STAT signaling are extremely rapid, occurring within the dynamic environment of the precellular embryo. Stat92E is important for each, but its roles augment the actions of other factors, rather than being responsible for defining the initiating signals (Avila, 2007).
With respect to the evolution of the sex signal, it has been proposed that a diffusible JAK/STAT signal might have been recruited to allow non linear signal amplification or, alternatively, that a diffusible ligand might render SxlPe less sensitive to random fluctuations in cell-autonomous XSE protein concentrations. Although the weak dose dependence of upd argues against signal amplification, a buffering function is consistent with existing data. These findings suggest another possibility. STAT proteins respond rapidly to a range of regulatory signals; it may be this ability to act within a matter of minutes that brought JAK/STAT into the temporally dynamic X-chromosome-counting process (Avila, 2007).
Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from their precursors are defined. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis (Jung, 2005).
In the late embryo, the lymph gland consists of a single pair of lobes containing ~20 cells each. These express the transcription factors Srp and Odd skipped (Odd), and each cluster of hemocyte precursors is followed by a string of Odd-expressing pericardial cells that are proposed to have nephrocyte function. These lymph gland lobes are arranged bilaterally such that they flank the dorsal vessel, the simple aorta/heart tube of the open circulatory system, at the midline. By the second larval instar, lymph gland morphology is distinctly different in that two or three new pairs of posterior lobes have formed and the primary lobes have increased in size approximately tenfold (to ~200 cells. By the late third instar, the lymph gland has grown significantly in size (approximately another tenfold) but the arrangement of the lobes and pericardial cells has remained the same. The cells of the third instar lymph gland continue to express Srp (Jung, 2005).
The third instar lymph gland also exhibits a strong, branching network of extracellular matrix (ECM) throughout the primary lobe. This network was visualized using several GFP-trap lines in which GFP is fused to endogenous proteins. For example, line G454 represents an insertion into the viking locus, which encodes a Collagen IV component of the extracellular matrix. The hemocytes in the primary lobes of G454 (expressing Viking-GFP) appear to be clustered into small populations within pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as the uncharacterized GFP-trap line ZCL2867, also highlight this branching pattern. What role this intricate ECM network plays in hematopoiesis, as well as why multiple cells cluster within these ECM chambers, remains to be determined (Jung, 2005).
Careful examination of dissected, late third-instar lymph glands by differential interference contrast (DIC) microscopy revealed the presence of two structurally distinct regions within the primary lymph gland lobes that have not been previously described. The periphery of the primary lobe generally exhibits a granular appearance, whereas the medial region looks smooth and compact. These characteristics were examined further with confocal microscopy using a GFP-trap line G147, in which GFP is fused to a microtubule-associated protein. The G147 line is expressed throughout the lymph gland but, in contrast to nuclear markers such as Srp and Odd, distinguishes morphological differences among cells because the GFP-fusion protein is expressed in the cytoplasm in association with the microtubule network. Cells in the periphery of the lymph gland make relatively few cell-cell contacts, thereby giving rise to gaps and voids among the cells within this region. This cellular individualization is consistent with the granularity of the peripheral region observed by DIC microscopy. By contrast, cells in the medial region were relatively compact with minimal intercellular space, which is also consistent with the smoother appearance of this region by DIC microscopy. Thus, in the late third instar, the lymph gland primary lobes consist of two physically distinct regions: a medial region consisting of compactly arranged cells, which was termed the medullary zone; and a peripheral region of loosely arranged cells, termed the cortical zone (Jung, 2005).
Mature hemocytes have been shown to express several markers, including collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter Collagen-gal4 (Cg-gal4), which is expressed by both plasmatocytes and crystal cells, is restricted to the periphery of the primary lymph gland lobe. Comparison of Cg-gal4 expression in G147 lymph glands, in which the medullary zone and cortical zone can be distinguished, reveals that maturing hemocytes are restricted to the cortical zone. In fact, the expression of each of the maturation markers mentioned above is found to be restricted to the cortical zone. The reporter hml-gal4 and Pxn, which are expressed by the plasmatocyte and crystal cell lineages, are extensively expressed in this region. Likewise, the expression of the crystal cell lineage marker Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the cortical zone was verified by several means, including the distribution of melanized lymph gland crystal cells in the Black cells background and analysis of the terminal marker ProPOA1. The cortical zone is also the site of P1 antigen expression, a marker of the plasmatocyte lineage. The uncharacterized GFP fusion line ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that the homeobox transcription factor Cut is preferentially expressed in the cortical zone of the primary lobe. Although the role of Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut are known to be regulators of the myeloid hematopoietic lineage in both mice and humans. Cells of the rare third cell type, lamellocytes, are also restricted to the cortical zone, based upon cell morphology and the expression of a msn-lacZ reporter (msn06946). In summary, based on the expression patterns of several genetic markers that identify the three major blood cell lineages, it is proposed that the cortical zone is a specific site for hemocyte maturation (Jung, 2005).
The medullary zone was initially defined by structural characteristics and subsequently by the lack of expression of mature hemocyte markers. However, several markers have been identified that are exclusively expressed in the medullary zone at high levels but not the cortical zone. Consistent with the compact arrangement of cells in the medullary zone, it was found that Drosophila E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant expression of DE-cadherin was observed among maturing cells in the cortical zone. E-cadherin, in both vertebrates and Drosophila, is a Ca2+-dependent, homotypic adhesion molecule often expressed by epithelial cells and is a crucial component of adherens junctions. Attempts to study DE-cadherin mutant clones in the medullary zone where the protein is expressed were unsuccessful since no clones were recoverable. The reporter lines domeless-gal4 and unpaired3-gal4 are preferentially expressed in the medullary zone. The gene domeless (dome) encodes a receptor molecule known to mediate the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The unpaired3 (upd3) gene encodes a protein with homology to Unpaired and has been associated with innate immune function. These gal4 lines are in this study only as markers that correlate with the medullary zone and, at the present time, there is no evidence that their associated proteins have a role in lymph gland hematopoiesis. Other markers of interest with preferential expression in the medullary zone include the molecularly uncharacterized GFP-trap line ZCL2897 and actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary zone. It is therefore reasonable to propose that this zone is largely populated by prohemocytes that will later mature in the cortical zone. Prohemocytes are characterized by their lack of maturation markers, as well as their expression of several markers described as expressed in the medullary zone (Jung, 2005).
The posterior signaling center (PSC), a small cluster of cells at the posterior tip of each of the primary (anterior-most) lymph gland lobes, is defined by its expression of the Notch ligand Serrate and the transcription factor Collier. During this analysis, several additional markers were identified that exhibit specific or preferential expression in the PSC region. For example, it was found that the reporter Dorothy-gal4 is strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which belongs to a class of enzymes that function in the detoxification of metabolites. The upd3-gal4 reporter, which has preferential expression in the medullary zone, is also strongly expressed among cells of the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has made it clear that the PSC is a distinct zone of cells that can be defined by the expression of multiple gene products (Jung, 2005).
The PSC can be defined just as definitively by the characteristic absence of several markers. For example, the RTK receptor Pvr, which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise, dome-gal4 is not expressed in the PSC, further suggesting that this population of cells is biased toward the production of ligands rather than receptor proteins. Maturation markers such as Cg-gal4, which are expressed throughout the cortical zone, are not expressed by PSC cells. Additionally, the expression levels of the hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are dramatically reduced in the PSC when compared with other hemocytes of the lymph gland. Taken together, both the expression and lack of expression of a number of genetic markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).
In contrast to primary lobes of the third instar, maturing hemocytes are generally not seen in the secondary lobes. Correspondingly, secondary lobes often have a smooth and compact appearance, much like the medullary zone of the primary lobe. Consistent with this appearance, secondary lymph gland lobes also express high levels of DE-cadherin. The size of the secondary lobe, however, varies from animal to animal and this correlates with the presence or absence of maturation markers. Smaller secondary lobes contain a few or no cells expressing maturation markers, whereas larger secondary lobes usually exhibit groups of differentiating cells. Direct comparison of DE-cadherin expression in secondary lobes with that of Cg-gal4, hml-gal4 or Lz revealed that the expression of these maturation markers occurs only in areas in which DE-cadherin is downregulated. Therefore, although there is no apparent distinction between cortical and medullary zones in differentiating secondary lobes, there is a significant correlation between the expression of maturation markers and the downregulation of DE-cadherin, as is observed in primary lobes (Jung, 2005).
The relatively late 'snapshot' of lymph gland development in the third larval instar establishes the existence of spatial zones within the lymph gland that are characterized by differences in structure as well as gene expression. In order to understand how these zones form over time, lymph glands of second instar larvae, the earliest time at which it was possible to dissect and stain, were examined for the expression of hematopoietic markers. As expected, Srp and Odd are expressed throughout the lymph gland during the second instar since they are in the late embryo and third instar lymph gland. Likewise, the hemocyte-specific marker Hemese is expressed throughout the lymph gland at this stage, although it is not present in the embryonic lymph gland (Jung, 2005).
To determine whether the cortical zone is already formed or forming in second instar lymph glands, the expression of various maturation markers were examined in a pair-wise manner to establish their temporal order. Of the markers examined, hml-gal4 and Pxn are the earliest to be expressed. The majority of maturing cells were found to be double-positive for hml-gal4 and Pxn expression, although a few cells were found to express either hml-gal4 or Pxn alone. This indicates that the expression of these markers is initiated at approximately the same time, although probably independently, during lymph gland development. The marker Cg-gal4 is next to be expressed since it was found among a subpopulation of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in the early third instar. Interestingly, the early expression of each of these maturation markers is restricted to the periphery of the primary lymph gland lobe, indicating that the cortical zone begins to form in this position in the second instar. Whenever possible, each genetic marker was directly compared with other pertinent markers in double-labeling experiments, except in cases such as the comparison of two different gal4 reporter lines or when available antibodies were generated in the same animal. In such cases, the relationship between the two markers, for example dome-gal4 and hml-gal4, was inferred from independent comparison with a third marker such as Pxn (Jung, 2005).
By studying the temporal sequence of expression of hemocyte-specific markers, one can describe stages in the maturation of a hemocyte. It should be noted, however, that not all hemocytes of a particular lineage are identical. For example, in the late third instar lymph gland, the large majority of mature plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the remainder express only Pxn (~15%) or hml-gal4 (~5%) alone. Thus, while plasmatocytes as a group can be characterized by the expression of representative markers, populations expressing subsets of these markers indeed exist. It remains unclear at this time whether this heterogeneity in the hemocyte population is reflective of specific functional differences (Jung, 2005).
In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar reveals an interesting developmental progression. A group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4, as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal. These cells resemble earlier precursors in the embryo, except they express the marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively (Jung, 2005).
The cells of the PSC are already distinguishable in the late embryo by their expression of collier. It was found that the canonical PSC marker Ser-lacZ is not expressed in the embryonic lymph gland and is only expressed in a small number of cells in the second instar. This relatively late onset of expression is consistent with collier acting genetically upstream of Ser. Another finding was that the earliest expression of upd3-gal4 parallels the expression of Ser-lacZ and is restricted to the PSC region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar, similar to what is seen in the third instar (Jung, 2005).
To determine whether maturing cortical zone cells are indeed derived from medullary zone prohemocytes, a lineage-tracing experiment was performed in which dome-gal4 was used to initiate the permanent marking of all daughter cell lineages. In this system, the dome-gal4 reporter expresses both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ under the control of the actin5C promoter. At any developmental time point, GFP is expressed in cells where dome-gal4 is active, while lacZ is expressed in all subsequent daughter cells regardless of whether they continue to express dome-gal4. In this experiment, cortical zone cells are permanently marked with ß-galactosidase despite not expressing dome-gal4 (as assessed by GFP), indicating that these cells are derived from a dome-gal4-positive precursor. This result is consistent with and further supports independent marker analysis that shows that dome-gal4-positive prohemocytes downregulate dome-gal4 expression as they initiate expression of maturation markers representative of cortical zone cells. As controls to the above experiment, the expression patterns of two other gal4 lines, twist-gal4 and Serrate-gal4 were determined. The reporter twist-gal4 is expressed throughout the embryonic mesoderm from which the lymph gland is derived. Accordingly, the entire lymph gland is permanently marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the third instar lymph gland. Analysis of Ser-gal4 reveals that PSC cells remain a distinct population of signaling cells that do not contribute to the cortical zone (Jung, 2005).
Genetic manipulation of Pvr function provides valuable insight into its involvement in the regulation of temporal events of lymph gland development. To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were generated in the lymph gland early in the first instar and then examined during the third instar for the expression of maturation markers. It was found that loss of Pvr function abolishes P1 antigen and Pxn expression, but not Hemese expression. The crystal cell markers Lz and ProPOA1 are also expressed normally in Pvr-mutant clones, consistent with the observation that mature crystal cells lack or downregulate Pvr. The fact that Pvr-mutant cells express Hemese and can differentiate into crystal cells suggests that Pvr specifically controls plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL positive but do express the hemocyte marker Hemese and can differentiate into crystal cells, all suggesting that the observed block in plasmatocyte differentiation within the mutant clone is not due to cell death. Additionally, Pvr-mutant clones were large and not significantly different in size from their wild-type twin spots. Thus, the primary role of Pvr is not in the control of cell proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same phenotypic features, confirming that Pvr controls the transition of Hemese-positive cells to plasmatocyte fate (Jung, 2005).
Entry into S phase was monitored using BrdU incorporation and distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In the second instar, proliferating cells are evenly distributed throughout the lymph gland. By the third instar, however, the distribution of proliferating cells is no longer uniform; S-phase cells are largely restricted to the cortical zone. This is particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary zone cells, which can be identified by the expression of dome-gal4, rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second instar lymph gland quiesce as they populate the medullary zone of the third instar. As prohemocytes transition into hemocyte fates in the cortical zone, they once again begin to expand in number. This is supported by the observation that the medullary zone in white pre-pupae does not appear diminished in size, suggesting that the primary mechanism for the expansion of the cortical zone prior to this stage is through cell division within the zone. Proliferating cells in the secondary lobes continue to be distributed uniformly in the third instar, suggesting that secondary-lobe prohemocytes do not reach a state of quiescence as do the cells of the medullary zone. These results indicate that cells of the lymph gland go through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).
This analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, termed the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Jung, 2005).
Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. It is proposed that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types (Jung, 2005).
Based on this analysis a model is proposed by which hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland are first distinguishable as Srp+, Odd+ (S+O+) cells. These will eventually give rise to a primary lymph gland lobe where the steps of hemocyte maturation are most apparent. During the first or early second instar, these S+O+ cells begin to express the hemocyte-specific marker Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called pre-prohemocytes and, in the second instar, cells expressing only these markers occupy a narrow region near the dorsal vessel. Subsequently, a subset of these Srp+, Odd+, He+, Pvr+ (S+O+H+Pv+) pre-prohemocytes initiate the expression of dome-gal4 (dg4), thereby maturing into prohemocytes. The prohemocyte population (S+O+H+Pv+dg4+) can be subdivided into two developmental stages. Stage 1 prohemocytes, which are abundantly seen in the second instar, are proliferative, whereas stage 2 prohemocytes, exemplified by the cells of the medullary zone, are quiescent. As development continues, prohemocytes begin to downregulate dome-gal4 and express maturation markers (M; becoming S+O+H+Pv+dg4lowM+). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S+O+H+Pv+dg4-M+), found generally in the cortical zone. Thus, the maturing hemocytes of the cortical zone are derived from prohemocytes previously belonging to the medullary zone. This is supported by lineage-tracing experiments that show cells expressing medullary zone markers can indeed give rise to cells of the cortical zone. In turn, the medullary zone is derived from the earlier, pre-prohemocytes. Early cortical zone cells continue to express successive maturation markers (M) as they proceed towards terminal differentiation. Depending on the hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1, msn-lacZ, etc. These studies have shown that differentiation of the plasmatocyte lineage requires Pvr, while previous work has shown that the Notch pathway is crucial for the crystal cell fate. Both the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).
Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis (Jung, 2005).
Loss of zygotic os activity causes segmentation defects in the Drosophila embryo that resemble the
phenotype of hopscotch and stat92E mutant embryos (Wieschaus, 1984). These defects
always include loss of the fifth abdominal denticle band and the posterior mid-ventral portion of the
fourth band. Defects in other segments are variable, but often include reduction of the second thoracic
and eighth abdominal denticle bands and fusion of the sixth and seventh bands. In contrast to hop or
stat92E (Perrimon and Mahowald 1986; Hou et al. 1996), zygotic os activity is essential
but maternal activity is not, as evidenced by the lack of a maternal effect phenotype for os mutants
(Eberl et al. 1992). The similarity between embryos that lack zygotic os and those that lack
maternal hop or stat92E suggests that os is a component of the JAK signaling pathway. This
hypothesis is further supported by genetic interactions between these genes. It has been observed
previously (Perrimon, 1986) that certain allelic combinations of hop are viable, but have adult defects. The partial loss of hop activity in such animals causes reduced viability,
held-down wings, reduced production of mature eggs, and/or defects in eggs produced. Each of the
heteroallelic combinations results in a consistent and predictable degree of severity with respect to
these phenotypes. To test whether the hop and os genes interact genetically, one copy of os was
removed from animals carrying allelic combinations of hop. Altering the dose of os activity
exacerbates the defects observed for these hop mutant combinations. Such
enhancement is likely to occur if the two gene products are active in the same pathway (Harrison, 1998).
Strong alleles of unpaired are embryonic lethal, but weaker alleles show an
outstretched (os) phenotype, resulting in adult flies with wings held out away from the body. Allelism
of upd and os is based on the failure of zygotic lethal upd alleles to complement the wing phenotype of
os alleles (Harrison, 1998 and Eberl, 1992). For example, combination of the embryonic lethal
allele updYC43 with the viable allele oso results in viable adult flies with outstretched wings (Harrison, 1998).
The JAK/STAT pathway is central to the
establishment of planar polarity during Drosophila eye development. A localized source of the pathway ligand, Unpaired/Outstretched, present at the midline of the developing eye, is capable of activating the JAK/STAT pathway over long distances. A gradient of
JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized
Unpaired influences the position of the equator via repression of mirror (Zeidler, 1999).
Given the known function of Upd as a JAK/STAT pathway
ligand during Drosophila embryonic development, and the
results showing that expressing Upd during eye development can mimic
activation of the JAK/STAT pathway, it was decided to
investigate whether Upd was likely to be acting as the endogenous
JAK/STAT pathway ligand during eye patterning. Therefore, the time course of Upd expression during eye
development was examined, using a polyclonal anti-Upd antibody. In first instar discs, only very weak expression is observed,
in a faint horseshoe-shaped pattern around the poles and posterior of
the disc. By second instar, expression is
seen localized to the posterior margin of the disc, lying on the DV
midline adjacent to the optic stalk. This pattern of
expression persists into the third instar stage, however,
by late third instar, an additional patch of staining is seen at the
anteroventral margin of the eye disc adjacent to the junction with the
antennal disc. Higher magnification views of an early third
instar disc reveal that the Upd protein is highly expressed in only a
small group of cells on the DV midline, where it can be seen
cytoplasmically localized, but consistent with it being a
secreted ligand, the protein is seen around the periphery of cells away
from the site of expression in a concentration gradient toward the
poles of the disc (Zeidler, 1999).
Considering the expression domain of endogenous Upd in the eye and the
negative regulation of stat92E-lacZ by ectopically expressed
Upd, it seems likely that the wild-type pattern of stat92E-lacZ is at least partly a consequence of endogenous
Upd expression. The expression pattern of stat92E-lacZ in the third instar disc does not seem to be the
exact inverse of the Upd expression pattern. However, it must be borne
in mind that it is difficult to predict the exact stat92E-lacZ pattern that would be expected, because
stat92E-lacZ is probably a lagging indicator of Upd repression (due to the perdurance of the beta-galactosidase gene
product) and the cells in the posterior of the disc (behind the furrow)
undergo fewer cell divisions, which would tend to distort the pattern seen. On the basis of this evidence, it has been proposed that the Upd expression at the optic stalk is sufficient to set up a gradient of JAK/STAT activation across the DV axis of the developing eye disc (Zeidler, 1999).
Further experiments were carried out to test the hypothesis that
localized expression of Upd at the DV midline and the consequent gradient of JAK/STAT expression is important for normal
eye patterning. The normal pattern of Upd expression is altered by
ectopically expressing Upd in the developing eye imaginal disc using
the GAL4/UAS system and the 30A-GAL4 line, which drives
expression at the dorsal and ventral poles of th