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).
Three protein complexes control polarization of epithelial cells: the apicolateral Crumbs and Par-3 complexes and the basolateral Lethal giant larvae complex. Polarization results in the specific localization of proteins and lipids to different membrane domains. The receptors of the Notch, Hedgehog, and WNT pathways are among the proteins that are polarized, with subcellular receptor localization representing an important aspect of signaling regulation. For example, in the WNT pathway, differential DFz2 receptor localization results in activation of either the canonical or the planar polarity pathway. Despite the large body of research on the vertebrate JAK/STAT pathway, there are no reports indicating polarized signaling. By using the conserved Drosophila JAK/STAT pathway as a system, it was found that the receptor and its associated kinase are located in the apical membrane of epithelial cells. Unexpectedly, the transcription factor STAT is enriched in the apicolateral membrane domain of ectoderm epithelial cells in a Par-3-dependent manner. These results indicate that preassembly of STAT and the receptor/JAK complex to specific membrane domains is a key aspect for signaling efficiency. These results also suggest that receptor polarization in the ectoderm cell membrane restricts the cell's response to ligands provided by neighboring cells (Sotillos, 2008).
Besides setting up epithelial polarity, apicobasal complexes also modulate the subcellular compartmentalization or localized activation of various signaling molecules. The JAK/STAT signaling pathway is involved in processes ranging from immune response to organogenesis. In the vertebrate-signaling model, inactive STAT is shuttling from the cytoplasm to the nucleus. Ligand binding to the dimerized receptor results in the activation of JAK bound to the receptor. JAK phosphorylates itself and the receptor, creating docking sites for STAT. Inactive cytoplasmic STAT now binds to the phosphoreceptor/JAK complex, where it is phosphorylated by the kinase. Phosphorylated STAT is imported to the nucleus, where it activates the transcription of target genes. In contrast to vertebrates, in which the JAK/STAT core-signaling elements are highly redundant, the Drosophila pathway is composed of only three ligands, Unpaired (Upd), Unpaired2, and Unpaired3; one receptor, Domeless (Dome); one JAK, Hopscotch (Hop); and one transcription factor, STAT92E. Therefore, Drosophila was used as a model to investigate the polarization of the pathway (Sotillos, 2008).
dome, hop, and stat92E mRNAs are maternally provided and ubiquitously transcribed in the embryo. To analyze their protein subcellular localization, specific antibodies were used or functional tagged proteins were expressed by using UAS-dome, UAS-hop-Myc, and UAS-STAT92E-GFP. These constructs were expressed by using either mesodermal or ectodermal Gal4 drivers, and the subcellular localization of the proteins was analyzed, paying special attention to three organs where the endogenous ligand is expressed and the pathway is active: the posterior spiracles (ectodermal origin), the pharyngeal musculature (mesodermal), and the hindgut (an ectodermal tube surrounded by mesoderm) (Sotillos, 2008).
In the pharynx, as expected for a receptor, Dome localizes to the membrane, and does so in a dotted pattern that could correspond to endocytic vesicles. Hop-myc localizes to the cytoplasm, obscuring any membrane localization. This is due to the high levels of Hop-myc expressed, saturating the receptor binding sites and accumulating in the cytoplasm, as simultaneous coexpression of Hop-myc with the receptor relocates Hop to the membrane. This depends on the cytoplasmic domain of Dome, as it also occurs with a construct missing the extracellular domain but not with constructs missing the intracellular domain. STAT is detected in the cytoplasm and is more concentrated in the nuclei, as expected from the activation of the pathway in the pharynx. All of these observations agree with current knowledge of JAK/STAT activation based on vertebrate studies (Sotillos, 2008).
In contrast to the mesoderm, analysis of ectoderm cells shows a different picture. Both in the hindgut and the posterior spiracles, the Dome receptor localizes on the apical membrane. Hop is again cytoplasmic, but after coexpression with Dome both proteins localize to the apical membrane. Surprisingly, by using a specific antibody it was observed that STAT concentrates on the apical membrane of all embryonic ectodermal cells irrespective of the level of activation of the pathway. And, in cells in which the pathway is active, STAT also localizes to the nucleus. The signal detected by the antibody is specific; the same result by using a STAT-GFP fusion protein. STAT membrane localization is more prominent in cells in which the pathway is inactive; for instance, in the trunk epidermis or the spiracle after stage 15. This suggests that STAT translocates from the subapical membrane to the nucleus after pathway activation, returning to the membrane after inactivation (Sotillos, 2008).
To determine if STAT-GFP membrane localization is due to any other of the pathway's components, STAT-GFP localization was analyzed in upd, dome, or hop null mutants. STAT does not disappear from the membrane in a deficiency that removes all three Upd ligands. STAT membrane localization is not affected in null mutants for either dome or hop, demonstrating that apical STAT localization is independent of the pathway (Sotillos, 2008).
STAT localizes to the membrane domain in which the apical complexes are located. This, and the fact that STAT does not localize to the membrane in the mesoderm where Crb and Par-3 complexes are not formed, suggests the apical complexes could be recruiting STAT. To test this, different apical complex proteins were expressed in the mesoderm, and their capacity to modify STAT subcellular localization was studied. Neither the expression of Crb nor aPKC (another member of Par-3 complex) is able to translocate STAT to the membrane. In contrast, expression of Par-3 results in efficient membrane translocation of STAT and STAT-GFP. Moreover, STAT-GFP and Par-3 coimmunoprecipate from embryo extracts overexpressing STAT-GFP and par-3, pointing to Par-3 as the molecule responsible of STAT apical localization. In accordance, STAT-GFP is lost from the membrane in par-3 zygotic mutants, whereas in crb null mutants, where the polarity is highly compromised and Par-3 localization is severely affected, STAT remains in the membrane of cells only where Par-3 is still present. Similarly, in null aPKC embryos, STAT-GFP exclusively remains apical in cells in which Par-3 still localizes at the membrane. Thus, STAT recruitment is independent of Crb or aPKC and may directly depend on Par-3 (Sotillos, 2008).
To analyze if JAK/STAT polarization is functionally relevant, genetic interactions with polarity mutants were tested. Heterozygous polarity mutants or stat92E embryos are viable and normal. In contrast, embryos simultaneously heterozygous mutant for stat92E and either par-3, aPKC, or crb present phenotypes associated to JAK/STAT loss of function, including malformation of the posterior spiracles and abnormal segmentation. A specific readout of the pathway's activity was studied, analyzing the expression of a crb-spiracle enhancer that is directly activated by JAK/STAT. The expression of this enhancer is severely reduced in zygotic par-3 mutants simultaneously heterozygous for stat92E, compared to its expression in heterozygous stat92E embryos or zygotic par-3 mutants. In contrast, the expression of the JAK/STAT independent ems-spiracle enhancer is not affected in the same genetic backgrounds. The capability of Par-3 to induce STAT membrane localization and the strong genetic interaction between stat92E and cell-polarity mutations indicate that the apical polarization of JAK/STAT components is required for full-signaling efficiency in the ectoderm (Sotillos, 2008).
Next, whether the apical localization of all JAK/STAT transducer components in the ectoderm results in signaling occurring exclusively through this membrane domain was tested. For this purpose the posterior hindgut, where JAK/STAT is required in the ectoderm and in the mesoderm surrounding it, was analyzed. Upd expressed from the most anterior ectodermal cells of the hindgut activates in the ectoderm ventral veinless (vvl) and upregulates in the mesoderm dome through the dome-MESO enhancer. Thus, vvl and the dome-MESO autoregulatory enhancer can be used as readouts for JAK/STAT activation in the different hindgut tissues (Sotillos, 2008).
If signaling in the ectoderm were transduced exclusively through the apical membrane, it would be expected that vvl activation on the hindgut would not be possible if Upd is presented from the basal side. To test this Upd was expressed either in the ectoderm or in the mesoderm, and its effect on vvl activation in the ectoderm was analyzed. As a positive control the expression of dome-MESO was analyzed. When expressed throughout the ectoderm, Upd induces ectopic expression of dome-MESO in the mesoderm and of vvl in the ectoderm, behaving as the endogenous Upd. In contrast, when Upd is expressed throughout the mesoderm, dome-MESO is ectopically activated, whereas vvl is not. The unresponsiveness of the ectoderm cells to Upd from the mesoderm is consistent with the endogenous receptor being apically localized in the hindgut ectoderm and, thus, unable to receive any mesoderm signal (Sotillos, 2008).
Many proteins involved in the establishment and maintenance of cell polarity also modulate signaling pathways by modifying or restricting the localization of their signaling components. Precise subcellular distribution may help the activation of the pathway or restrict its activity by sequestering key elements. This study has shown that in the epithelial cells the localization of JAK/STAT components is highly polarized. The apical restriction of the receptor can influence transduction, since only ligand presented to the apical side of the epithelium would be detected. This may be of relevance after septic injury, when circulating haemocytes secrete the Upd3 cytokine into the haemolymph. In this case, the secreted ligand would activate its targets in the fat body without stimulating the ectoderm epithelial cells, since the cell junctions efficiently block Upd diffusion to the apical side (Sotillos, 2008).
Par-3-dependent STAT apical localization is intriguing. The localization of STAT to the subapical membrane seems important for signal transduction, since mutations reducing the amount of cell polarity proteins enhance stat loss of function phenotypes and reduce the activation of direct pathway targets. It is proposed that in ectodermal cells, where the receptor and the kinase locate apically, the existence of a subapical pool of STAT facilitates its rapid translocation to the activated receptor, increasing signaling efficiency. Future research should resolve whether this is achieved simply by the increased local concentration of apical STAT facilitating receptor binding or if there exists some dedicated machinery to translocate STAT from the subapical region to the active receptor similar to the one involved in nuclear import. It is interesting to note that crb expression is upregulated by JAK/STAT signaling in the follicle cells and in the posterior spiracles. Since Crb helps maintaining Par-3 in the apical membrane, upregulation of crb by STAT might increase apical Par-3, reinforcing signal transduction by increasing the apical concentration of STAT (Sotillos, 2008).
There are few reports of polarized vertebrate JAK/STAT signaling. However, analysis of the subcellular localization of two IL-6 receptors in MDCK epithelial cells has shown that gp130 localizes basolaterally and CNTF-R apically. Also, in the mammary glands, the IL-4Ra receptor is localized apically in luminal cells during gestation and lactation. Recently, activated STAT3 has been transiently detected at the membrane in the nascent cell-cell contacts of squamous cell carcinoma of the head and neck. In vertebrates the Par-3 complex functions as a regulator of junction biogenesis. It will be interesting to investigate whether Par-3 also mediates the localization of STAT3 in the membrane. The results suggest that JAK/STAT polarization in epithelia may be a general feature (Sotillos, 2008).
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 the developing eye disc. In wild-type eyes, the array of facets
appears externally regular, however, misexpression of Upd at
the poles of the eye gives rise to adults in which the normal regular
array of ommatidial facets is externally disrupted at both the dorsal and ventral poles of the eye. Sections through either the dorsal or ventral regions of such eyes
show that although equatorially ommatidial polarity is normal, at
the poles ommatidia have an inverted
orientation. The inversions generated by
misexpressing Upd in this manner do not form a regular and straight
equator but rather appear to define a field of inversion with no clear
boundary between dorsal and ventral ommatidial fates. This confused
region may well represent the area in which ommatidia are responding to
competing polarity signals, produced either by endogenous Upd
expression at the optic stalk and Upd at the poles or possibly by other
independent signaling mechanisms (Zeidler, 1999).
Next, the effect of completely removing Upd
expression from the developing eye was investigated. Clones homozygous mutant for two
independent Upd alleles were generated. These were recovered at high
frequency and none of the clones analyzed (those which lay within the eye
field) displayed any visible phenotype, either in imaginal discs or adult
eyes. However, clones that overlapped the region of Upd
expression adjacent to the optic stalk produced almost complete
dorsalization of the eye field as assayed by ommatidial polarity. In an effort to better understand the ommatidial dorsalization observed
following removal of Upd, the expression of mirr was examined.
Wild-type mirr expression is restricted to the dorsal hemisphere of the eye, and via repression of fng results in activation of the Notch pathway
at the DV midline, thereby defining the position of the endogenous equator. To investigate mirr regulation,
P-element insertions in mirr were used that function as enhancer
detectors and express both the lacZ gene and the P-element
mini-white+ marker gene specifically in the dorsal half of
the adult eye, henceforth referred to as mirr-lacZ. When mirr-lacZ is present in a background in which clones
removing upd are generated, a low frequency of flies are
recovered in which all or almost all of the eye field expresses the
dorsal fate-specific white+ mirr reporter. When the eyes of flies showing this external dorsalization phenotype are sectioned, the dorsal fate of the ommatidia within the region ectopically expressing mirr is confirmed, although intriguingly, such eyes show occasional ommatidia still exhibiting ventral fate in the mirr-expressing region. Therefore, it is concluded that Upd expression at the optic stalk during normal eye patterning is required for restriction of mirr expression to the dorsal hemisphere of the eye. This restriction then determines the position of the equator along the DV midline of the eye disc via activation of Notch (Zeidler, 1999).
Given the role of Upd in restricting mirr expression, one
possible mechanism by which JAK/STAT LOF clones might
induce ectopic axes of mirror-image symmetry would be through the
generation of ectopic boundaries of mirr expression. The expression of mirr-lacZ was examined in
hop clones. Many clones lying both dorsally and ventrally were
examined in eye discs, and in no case was an alteration in
mirr-lacZ expression observed. Additionally, hundreds of adults carrying mirr-lacZ
were examined, in which hop clones had been induced, and,
again, in no case was a change in mirr-regulated
white+ expression observed (Zeidler, 1999).
Thus, ommatidial polarity inversions generated by hop clones
are mirr independent. It is therefore concluded that the process of midline equator definition by dorsally restricted mirr expression and the regulation of ommatidial polarity by the JAK/STAT pathway are separable processes. It is also noted that these results suggest that Upd might act independently of Hop to
regulate mirr expression (Zeidler, 1999).
These results show that LOF JAK/STAT clones give
nonautonomous inversions of ommatidial polarity on the polar clonal
boundary, and that JAK/STAT activity is highest at the DV
midline as a result of localized Upd expression. This situation is
reciprocal to that seen for LOF Wg pathway clones that cause
nonautonomous inversions of ommatidial polarity on the equatorial
clonal boundary. An important question is
whether the Wg and JAK/STAT pathways are acting
independently to regulate ommatidial polarity decisions, or whether one
acts through the other. Experiments were carried out to test directly whether Upd and
Wg might regulate each other's expression. Ectopic Upd
expression has no effect on Wg expression in the developing eye disc and also ectopic expression of Wg
adjacent to the optic stalk does not alter Upd expression.
Thus, Upd cannot be producing its phenotype via negative regulation of
Wg and, similarly, Wg does not act via regulation of Upd.
These results indicate that Wg and Upd do not regulate each other's
expression, and, thus, that one of these pathways is not likely to be
downstream of the other. Instead, it is surmised that Wg and Upd act in
parallel to one another in the regulation of ommatidial polarity (Zeidler, 1999).
The ommatidial polarity phenotype produced by removal of JAK
activity in mosaic clones has a number of important features: (1)
the phenotype observed is an inversion of ommatidial polarity in which
either the dorsal rotational form is seen in the ventral hemisphere of
the eye or vice versa; (2) the phenotype is only observed on the
polar boundary of the mosaic tissue; (3) the strength of the
phenotype (in terms of the number of inverted ommatidia seen) is
dependent on the size and shape of the clone; (4) the phenotype
is cell nonautonomous as either fully mutant, fully wild-type, or as
mosaic clusters that can manifest the phenotype (Zeidler, 1999).
From these characteristics, the following can be deduced: the
nonautonomy of the phenotype produced by removal of the autonomously acting pathway component JAK, and its dependence on clone size and shape, suggests that JAK/STAT affects ommatidial polarity via a secreted downstream signal (which subsequently will be referred to as a second signal, most likely detected by Frizzled). The direction of the nonautonomy (only in a polar direction) and the strict DV nature of the polarity inversions
indicates that this second signal must be graded in its activity along
the DV axis, with a change in direction of the gradient at the equator.
The direction of this gradient would then be the instructive cue to
which ommatidia respond when rotating to establish their mature polarity (Zeidler, 1999).
Since Upd expression does not regulate Wg and
vice versa, the possibility that these two pathways act
sequentially can be excluded, and so it is proposed that they must act in parallel. An
attractive possibility is that the Upd and Wg pathways might act in
parallel to regulate the concentration of a single second signal. In
this case, Upd expression at the DV midline would activate the signal
and Wg expression at the poles would repress the signal. Adding
together the effect of two such opposing signals produces a predicted
second signal concentration that has a fairly even slope from the DV
midline to the poles. In contrast, a single signal that is
high at the DV midline and decays to zero at the poles has a very
shallow gradient in the polar regions. Since reading the
slope of a steep gradient is presumably easier than reading the slope
of a shallow gradient, the use of two opposing gradients to set up second signal concentration is thus highly advantageous. Therefore, this presents a possible biological explanation for the proposed redundant use of both an Upd and a Wg concentration gradient in determining ommatidial polarity (Zeidler, 1999).
The most likely candidate for a receptor of the second signal is the
seven pass transmembrane protein encoded by the frizzled (fz) locus. fz function is
required in the presumptive R3/4 cells of the
pre-ommatidium, and clonal analysis suggests that it interprets a
gradient of positional information that is high at the equator and low
at the poles. Recent results suggest that
differential activation of Fz signaling in R3/4 results
in asymmetric Notch activation in this photoreceptor pair, which
ultimately leads to a binary cell-fate decision such that the cell
closest to the equator takes on the R3 fate and the ommatidial unit as
a whole adopts the correct rotational fate (Zeidler, 1999 and references).
The simplest model would be that there is a single second signal secreted from the equator, which is downstream of mirr/fng/Notch, and that Wg and
Upd/JAK/STAT feed into this pathway upstream of Notch. This is consistent with the roles of Wg and Upd as regulators of mirr expression and, thus, in positioning
the endogenous equator. However, it is not consistent with the observed ommatidial polarity inversions produced in the eye field both dorsally and ventrally by
Wg-pathway and JAK/STAT-pathway LOF and GOF clones. These phenotypes indicate that second-signal concentration is dependent on Wg pathway and
JAK/STAT pathway activity across the whole of the eye field, and thus the second signal cannot be only secreted from the DV midline as a
consequence of localized Notch activation. It is conceivable that Notch is activated on the polar boundary of JAK/STAT LOF clones, but in this context the only
known mechanism of Notch activation is via mirr/fng interactions, and this possibility has been ruled out (Zeidler, 1999).
Instead, the data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localize
activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, becausefng LOF clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity (Zeidler, 1999).
Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a
gradient(s) of one or more second signals that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of
ommatidia away from the equator and the localized Notch-dependent polarizing signal. A number of observations provide a great deal of support for such a model. (1) It is consistent with the known timing of the events involved. The requirement for fng function has been
shown to lie between late first instar and mid second instar, which coincides with the first appearance of high levels of Upd expression at
the optic stalk. However, the ommatidia are not formed (and thus do not respond to the polarity signal) until the start of the third instar, a stage when localized Upd
expression still persists. Furthermore, extracellular Upd protein can be seen in a concentration gradient many cell diameters from the optic stalk at the early third instar stage, consistent with Upd being at least partly responsible for setting up the long-range gradient of JAK/STAT activity across the DV axis of the eye disc that is revealed by the stat92E-lacZ reporter. (2) This model does not require that a single source of second signal secreted by a narrow band of cells at the equator should be capable of determining ommatidial polarity across the whole of the DV axis of the disc during the third instar stage of development. Instead, the band of activated Notch at the equator
would serve to draw a straight line between the fields of dorsally and ventrally polarized ommatidia, and need only secrete a localized source of second signal to polarize ommatidia in this region. Further from the equator, the opposing gradients of Upd and Wg signaling would provide a robust mechanism for maintenance of
correct ommatidial polarity across the DV axis. Conversely, without the mirr/fng/Notch mechanism to draw a straight line, it would be impossible to imagine how Upd at the posterior margin and Wg at the poles alone could provide the perfectly straight equator that is ultimately formed. (3) The phenotypes that are observed are consistent with multiple competing mechanisms responsible for determining ommatidial polarity. When
inversions of ommatidial polarity are induced by generating hop clones or ectopically expressing Upd, straight equators are not produced, such that two cleanly abutting fields of dorsal and ventral ommatidia are produced. Instead, there is usually some confusion of ommatidial identities as if they might be
receiving conflicting signals. Additionally, when upd activity is removed from the optic stalk, an equator still forms (albeit at the ventral edge of the disc), but some ommatidia dorsal to the equator still adopt a ventral fate as if the determination of ommatidial polarity is less robust in the absence of Upd (Zeidler, 1999).
It is concluded that Upd is required to position the future equator via dorsal repression of mirr and localized activation of Notch (as a consequence of the
restricted expression of the fringe (fng) gene product in the ventral half of the disc and Mirror in the dorsal half of the
disc) at the DV midline. Upd also appears to regulate
ommatidial polarity via activation of a gradient of JAK/STAT pathway activity and secretion of an unidentified second signal. Intriguingly, in both of these contexts,
Wg secreted from the poles of the disc appears to cooperate with Upd. It will be interesting to see whether this represents a general mechanism for cooperation of
these signaling pathways in pattern formation (Zeidler, 1999).
Metazoans use diverse and rapidly evolving mechanisms to determine sex.
In Drosophila an X-chromosome-counting mechanism
determines the sex of an individual by regulating the master switch gene,
Sex-lethal (Sxl). The X-chromosome dose is communicated
to Sxl by a set of X-linked signal elements (XSEs), which activate
transcription of Sxl through its 'establishment' promoter,
SxlPe. A new XSE called sisterlessC
(sisC) is described whose mode of action differs from that of previously characterized
XSEs, all of which encode transcription factors that activate Sxl
Pe directly. In contrast, sisC encodes a secreted ligand
for the Drosophila Janus kinase (JAK) and 'signal transducer
and activator of transcription' (STAT) signal transduction pathway and
is allelic to outstretched (os, also called unpaired). sisC works indirectly on Sxl through this signaling
pathway because mutations in sisC or in the genes encoding Drosophila
JAK or STAT reduce expression of SxlPe similarly.
The involvement of os in sex determination confirms that secreted ligands
can function in cell-autonomous processes. Unlike sex signals for other organisms,
sisC has acquired its sex-specific function while maintaining non-sex-specific
roles in development, a characteristic that it shares with all other Drosophila
XSEs (Sefton, 2000).
The two copies of XSEs present in XX individuals in Drosophila specify
female development by transiently activating SxlPe
in the young embryo. A positive autoregulatory feedback loop acting on RNA
splicing keeps Sxl active in females thereafter. Male development ensues
in XY individuals because their single set of XSEs is insufficient to activate
SxlPe. Because Sxl controls the vital process
of X-chromosome dosage compensation as well as sex determination, sexually
inappropriate expression of Sxl is lethal. For example, simultaneous
duplication of sisA and sisB kills males, as a female dose of
these two XSEs in males causes Sxl to be expressed in its female mode,
thereby reducing X-linked gene expression (Sefton, 2000).
Because XSEs act additively, males that would be killed by an excess dose
of one group of XSEs can be rescued by compensating mutations that reduce
the dose of other XSEs. A genetic screen based on this principle
of additivity has generated five new mutations that define a
new XSE, sisC. The first four sisC alleles recovered, including
an apparent null, sisC1, have no phenotype by themselves,
even in trans to deficiencies of the region. In contrast, sisC
5, which is also null for sex-determination, exhibits phenotypes
unrelated to sex: variably reduced viability (females more than males), female
sterility and tergite defects. All mutations were mapped by recombination
and deficiency analysis close to os based on their interactions with
mutations in other XSEs (Sefton, 2000).
sisC and os are now shown to be the same gene, but this
possibility seemed to have been excluded by the initial characterization of
the putative sisC region. Df(1)os1a seemsto be wild-type for sex-determination
function. Moreover, construction of an osssisC1
chromosome placed sisC centromere-proximal to os and the phenotype
of the double mutant adults gives no hint that both lesions affect the same
gene. Using DNA centromere-proximal to os, a DNA breakpoint has been identified for the atypical allele sisC5 precisely
where the genetics had predicted sisC to be -- outside the region
deleted by Df(1)os1a; however, complications were suspected
when no candidate sisC RNAs or other sisC lesions near
this breakpoint could be found (Sefton, 2000).
The subsequent discovery that embryonic lethal os
alleles (osupd by convention) fail to complement
sisC5 female sterility and tergite defects indicates that
the non-sex-determination defects of this atypical sisC allele must
be due to a different lesion that disrupts os slightly. The finding that embryonic lethal os alleles are deficient for XSE function
indicates that this second lesion in sisC5 might also
be responsible for the sex-determination defect. This finding coincides with
the recovery of a candidate os complementary DNA (lambdaKZ-GR) (Sefton, 2000).
With this cDNA as a probe, the anticipated second change was found in
sisC5, 100 kilobases (kb) centromere-distal to the
first and within Df(1)os1a, just upstream of the 5'
end of lambdaKZ-GR. DNA sequencing has showen
sisC5 to be a simple inversion, and allows the design of
polymerase chain reaction (PCR) primers that will amplify only os
upd DNA from sisC5/osupd
females. The discovery that osupd-3 and os
upd-4 are a 2-base-pair (bp) insertion at codon 143 and a C-to-T
change causing a stop at codon 60, respectively, shows that lambdaKZ-GR
corresponds to os (Sefton, 2000).
Characterization of os DNA for the other sisC mutants shows that sisC and os are the same
gene and that the first sisC5 breakpoint and the
Df(1)os1a XSE test results were misleading. sisC
1 is a 1.6-kb DNA insertion and a ~100-bp duplication just
upstream of lambdaKZ-GR. All weaker sisC lesions also fall within
os: sisC3 deletes 7 bp upstream of the translation
start; sisC4 inserts tyrosine after alanine 128, and
sisC2 substitutes CGG for GTT 5 bp downstream of
a 5' splice site (decreasing RNA splicing efficiency as determined by
reverse transcription [RT]-PCR). The contradiction
that Df(1)os1a is sisC+ was resolved by showing
that the Df chromosome carries a closely linked, maternal-effect suppressor. Mutations that only disrupt sex determination
are now designated as ossisC, but the locus is referred to as sisC when
discussing os as an XSE (Sefton, 2000).
The 5' end of os transcripts was defined to guide construction
of transgenes essential for showing the role of os in sex determination
and for understanding sex-specific mutations in a non-sex-specific gene. 5'
rapid amplification of cDNA ends (RACE) indicates two potential transcription
start sites 1,316 and 1,439 bp upstream of the 5' end of lambdaKZ-GR,
with the product for the +1,316 species terminating in a non-coding G that
could correspond to the 5' methyl cap of a bona fide messenger RNA end.
RNase protection shows that the two RACE products
correspond to the major and minor os mRNA species present during
the first half of embryonic development. Sequencing the RACE products, other
RT-PCR products and genomic DNA reveal the true first exon and redefine
the start of the exon designated II. The
5' end of lambdaKZ-GR may be artifactual, as it was not found in mRNA
from embryos (Sefton, 2000).
Identification of these transcription start sites shows that sisC
fits a pattern established for all other XSEs that control Sxl throughout
the embryo: sisC has three copies of the sequence CAGGTAG less than
0.5 kb upstream of its first transcription start site (-242, -424 and
-450 bp). Several conserved copies of
this sequence or its complement have been found previously upstream of the transcription
start sites for sisA, sisB and Sxl, leading to speculation that
this sequence might be involved in the unusually early onset of XSE transcription (Sefton, 2000).
Three genomic transgenes were constructed with different amounts of 5'
sequence but the same 3' end. Lines with 6.5 kb or 0.6 kb of genomic
sequence upstream of the transcription start sites (P{sisC}
10 and P{sisC}5.8, respectively) provide high XSE
function but only partially complemente osupd lethals.
In contrast, transgenes (P{sisC}4.8) lacking the transcription
start sites and most of exon 1, but still containing 43 bp more uninterrupted
5' sequence than In(1)ossisC-5, have no XSE and
os activity. Hence, the significant level of os+
activity provided by In(1)ossisC-5 despite its disrupted
transcription start sites is unlikely to be due to an undiscovered endogenous
os promoter, but rather to introduction of a foreign promoter providing
activity sufficient only for non-sex-specific functions (Sefton, 2000).
The data show that os is an XSE.
To merit XSE status, lowering the zygotic dose of the gene must decrease the
probability of Sxl+ activation in females, but increasing
the dose of the same gene must also increase that probability in males. The two larger sisC transgenes
can compensate for mutations that lower perceived X-chromosome dose in females.
Females simultaneously homozygous for a sisC null and heterozygous
for a mutation in sisA would die but are rescued by the two larger
transgenes. Line-to-line variation in rescue probably reflects the effects
of different insertion-site positions. os+ transgenes can kill males by increasing
perceived X-chromosome dose. Males were sensitized to increased sisC
+ by an increase in sisA+ dose. A double
dose of sisC+ is more lethal to males than a single
dose, reflecting the additive behaviour expected of XSEs. That this expected lethality is Sxl dependent is shown by the fact that Sxl
- males are fully viable regardless of XSE dose. The effect of
sisC+ transgenes is comparable to that of a chromosomal
duplication of sisC+, Dp(1;3)JC153, but is smaller than seen for extra doses of sisA+
or sisB+ (Sefton, 2000).
As is true for other Drosophila XSEs, eliminating sisC activity
reduces expression of SxlPe, but less than eliminating
sisA or sisB. Like sisA and sisB mutations, but unlike
runt mutations, sisC mutations affect SxlPe throughout
the embryo. Mutations in the Drosophila JAK/STAT signaling pathway reduce
expression of the Sxl 'establishment' promoter Sxl
Pe throughout the embryo. In non-mutant situations, most SxlPe:lacZ females
stain darkly and comprise
the expected 50% of the progeny. The other 50% are males whose light staining matches that of embryos lacking P
{SxlPe:lacZ}. Most Df(1)os/os-
females stain lighter than os
+ female controls but darker than males, showing that os
- generally reduces but does not eliminate SxlPe
expression. The reduction is not always uniform across the embryo. Any region could be affected, but anterior
expression seemed to be reduced the least. The range of effects is considerable:
fewer than 50% of the mutant embryos stained above background; some mutant
females may not have expressed SxlPe at all, but Sxl
Pe expression in a few others matched that of their os
+ sisters. In contrast to the Df(1)os/osupd
females, Df(1)os/ossisC-1
females are fully viable, but they show a comparable reduction
in SxlPe expression. This observation confirms
that ossisC-1 is near null with respect to sex determination,
but still supports normal development (Sefton, 2000).
If os acts on SxlPe indirectly through effects
on Drosophila JAK (encoded by hopscotch [hop]) and on
Drosophila STAT (encoded by Stat92E), then the effect on Sxl
Pe of eliminating either hop or Stat92E should
be the same as eliminating os. This prediction was confirmed. Because
only maternal rather than zygotic hop and Stat92E are likely
to be relevant at the very early embryonic stage when SxlPe
is activated, the maternal contribution
of these two genes was eliminated by inducing homozygous mutant germline clones in mothers
heterozygous for null alleles. Expression of SxlPe:lacZ
in these experimentals was compared with that for control embryos derived
from hop-/+ and Stat92E-/+ germ
cells. Loss of maternal hop+ does not eliminate Sxl
Pe expression, but expression is substantially reduced: although
49% of the experimental embryos expressed SxlPe:lacZ
, essentially identical to the 50% figure for the controls, 32% of the experimental embryos were in the intermediate
staining class compared with only 6% for the controls. The reduction was generally
more uniform across the embryos than in the os experiment. Similar results were seen for Stat92E. Sixteen per cent of controls stained in the intermediate range, compared with
45% for the experimentals; thus, SxlPe expression was clearly
reduced. Curiously, the fraction of experimental embryos staining above background
is greater than 50%, suggesting that although loss of maternal Stat92E
decreases SxlPe expression in females, it might also
increase SxlPe expression in males. Alternatively, this
increase might be due to effects on the lacZ enhancer trap present
in Stat92E6346. The
observation that Drosophila STAT is a regulator of SxlPe
is consistent with the finding of STAT binding sites (TTCNNNGAA)
253, 393 and 428 bp upstream of the SxlPe transcription
start site. The tandem arrangement of these sites in Sxl would facilitate
the kind of cooperative binding of STAT dimers shown to be important in some
systems (Sefton, 2000).
With the discovery of sisC, the collection of fly XSEs may be nearly
complete. The impression given by this collection is that
Drosophila relies on biochemically diverse proteins to assess X-chromosome
dose, but they all act on Sxl at the level of transcription. In contrast,
the XSEs of Caenorhabditis elegans include both transcriptional and
post-transcriptional regulators of their target, xol-1. Characterization of sisC reveals that both
C. elegans and Drosophila XSEs seem to include proteins that work
extracellularly (Sefton, 2000).
The JAK/STAT signaling pathway, renowned for its effects on cell proliferation and survival, is constitutively active in various human cancers, including ovarian. JAK and STAT are required to convert the border cells in the Drosophila ovary from stationary, epithelial cells to migratory, invasive cells. The ligand for this pathway, Unpaired (Upd), is expressed by two central cells within the migratory cell cluster. Mutations in upd or jak cause defects in migration and a reduction in the number of cells recruited to the cluster. Ectopic expression of either Upd or JAK is sufficient to induce extra epithelial cells to migrate. Thus, a localized signal activates the JAK/STAT pathway in neighboring epithelial cells, causing them to become invasive (Silver, 2001).
Polar cells emit a short-range signal that causes adjacent follicle cells to surround them and acquire the ability to migrate through the nurse cells. The results reported here suggest that Upd is the major signal secreted by the polar cells that both recruits adjacent follicle cells into the cluster and causes them to become migratory. Both of these functions are carried out by activation of JAK and STAT in the neighboring follicle cells. Signaling through this pathway is necessary, both for recruitment of border cells to the cluster and for motility once the cells are recruited. This is based on the observations that in the majority of mutant egg chambers, border cell clusters contain fewer than the normal number of cells, and that even clusters with normal numbers of cells fail to migrate normally (Silver, 2001).
It is worth noting that while some migration is observed in JAK and STAT border cell mutants, the loss of Upd in the polar cells completely prevents migration. This may reflect greater perdurance of JAK and STAT proteins in the mosaic clones, compared to Upd, if Upd is normally present at lower levels and/or is more labile. Alternatively, these differences may imply that in addition to its activation of JAK and STAT, Upd can activate other signaling pathways (Silver, 2001).
Activation of the JAK/STAT pathway is not only necessary but is also sufficient to convert epithelial follicle cells to become migratory. Numerous extra border cells were observed following overexpression of upd, hop, or hopTum, many of which invaded the nurse cell cluster. These extra cells did not result from excess proliferation because follicle cells cease dividing at stage 6, at least 12 hr prior to border cell differentiation. Furthermore, no difference in phospho-histone H3 antibody labeling was observed in cells overexpressing upd or in cells lacking stat, ehrn compared to wild-type. Moreover, it was possible to obtain large clones lacking upd, hop, or stat activity, indicating that homozygous mutant cells retain the ability to divide numerous times. Thus, activation of the JAK/STAT pathway leads to border cell specification and migration, without effects on proliferation. In addition, while extra follicle cells could become migratory as a secondary consequence of ectopic polar cell formation, activation of the JAK/STAT pathway results in the appearance of additional migratory cells in the absence of extra polar cells (Silver, 2001).
The question of whether signaling through this pathway might be sufficient to cause epithelial cells to become invasive was addressed ectopically expressing Upd, Hopscotch (Hop), or the constitutively active form of Hop, HopTum1, using the GAL4/UAS expression system. In this method, the yeast transcriptional activator GAL4 is expressed under the control of a cell type-specific enhancer, in this case slbo-GAL4 and c306-GAL4. In stage 9 egg chambers, slbo-GAL4 induces expression of genes that are under the control of the yeast upstream activating sequence (UAS) in approximately 20 anterior follicle cells, a subset of which normally become the border cells. This is nearly identical to the ß-gal expression from an enhancer trap insertion into the slow border cells (slbo) locus, even though Slbo protein expression is normally restricted to the border cells at stage 9. C306-GAL4 drives expression in a larger number of anterior, as well as posterior, follicle cells, compared to slbo-GAL4. C306-GAL4 also begins expressing earlier in oogenesis than slbo-GAL4 (Silver, 2001).
Egg chambers from c306-GAL4; UAS-hop females exhibit a dramatic increase in the number of border cells compared to wild-type. Up to 90 slbo expressing cells are produced, about 60 of which invade the nurse cell cluster and 20 of which have completed migration by early stage 10. Similar, though less dramatic, phenotypes are observed when the constitutively activated kinase is expressed with either slbo-GAL4 or c306-GAL4. Likewise, slbo-GAL4;UAS-upd and c306-GAL4;UAS-upd females contain numerous extra slbo-expressing cells compared to wild-type, in the absence of extra polar cells. This is in marked contrast to the effect of excessive Hedgehog pathway signaling, which causes ectopic border cells to form as a secondary consequence of ectopic polar cell specification. Overexpression of upd does not appear to cause excess cell proliferation, sinces no difference was detected in phospho-histone H3 antibody labeling, which marks mitotic cells, as compared to wild-type (Silver, 2001).
Egg chambers from females heterozygous for any of the stat alleles have a semi-dominant border cell migration phenotype. Advantage was taken of this slight haploinsufficiency to test for dominant genetic interactions with other genes required for border cell migration. Dominant genetic interactions were observed with slbo, hop, and upd alleles. A mutation in the gene coding for DE-cadherin, shotgun, also exhibited a dominant interaction with stat. These interactions appeared to be specific, since stat does not interact with other known border cell migration genes, such as tai, jing, or PZ6356 (Silver, 2001).
Recently, a candidate transmembrane receptor for Upd has been identified. Mutation of this gene, which is named domeless, causes embryonic phenotypes that are indistinguishable from those of upd, hop, and stat mutants. In addition, the gene encodes a protein with sequence homology to mammalian cytokine receptors that mediate JAK/STAT signaling. A dominant negative form of Domeless has been generated, which mimics the loss-of-function phenotype (Brown, 2001). Upon expression of the dominant negative receptor specifically in the outer border cells, using slbo-GAL4, dramatic recruitment and migration defects are observed. The average number of outer border cells in these egg chambers was 0.5 and the migration index was 2.6. These results provide further support for the proposal that Upd from the polar cells activates signaling in the surrounding epithelial cells for their recruitment to the cluster and migration (Silver, 2001).
Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as
well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and
are specified by the activation of Notch. Their role in follicle patterning has been investigated by creating clones of
cells mutant for the Notch modulator fringe. In the absence of fng or Notch function, polar cells do not form, and the requirement for these genes in polar cell fate is strictly cell autonomous. This genetic ablation of polar cells results in cell fate defects within
surrounding follicle cells. At the anterior, the border cells, the immediately adjacent follicle cell fate, are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. These observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis. Only one gene, upd, is known that encodes for a signaling molecule that is expressed by polar cells. Although loss of upd, or other components of the JAK-STAT pathway, reduces the number of border cells, this contrasts markedly with the complete elimination of border cells observed in the absence of polar cells. Moreover, loss of upd does not have obvious effects on any of the other terminal cell fates that are polar-cell dependent. Thus, the existence of additional signaling molecules must be invoked to account for the organizing activity of the polar cells (Grammont, 2002).
domeless was identified using a screen for suppressors of
an eye phenotype caused by overexpression of unpaired.
Overexpression of upd using a
UAS-upd and GMR-Gal4 driver causes
compound eye dramatic overgrowth in the adult eye because of an
increase in the number of ommatidia. The average number of
ommatidia in the compound eye of UAS-upd/GMR-Gal4 female
flies is 978 ± 10 compared with 745 ± 7 in wild-type flies. Histological sections through the overgrown eyes reveal that most
ommatidia have normal photoreceptor cells and regular cell size, indicating that Upd activity mainly regulates cell proliferation in the compound eye. However, the ommatidia look more
crowded and have irregular space and arrangement, and several big
vacuoles are integrated into the ommatidia lattice. The
severity of eye morphology appears proportional to the strength of the
Hop/Stat92E-mediated signaling, because removing one copy of
hop partially suppresses the big eye phenotype; the
average number of ommatidia is 854 ± 9). The advantage of this
sensitized system lies in the possibility of conducting a screen for mutations that reduce
(suppressors) or increase (enhancers) the degree of eye size. It was
reasoned that a twofold reduction in the dose of a gene (by mutating
one of its two copies) that functions downstream of Upd should
dominantly alter signaling strength, which, in turn, should visibly
modify the eye size. Based on this assumption,
available X-chromosome P-element insertion mutations were screened and one complementation group of suppressors with four alleles was identified at the cytological location 18E. Based on its presumed role in the
Hop/Stat92E signal transduction pathway, this novel gene was named master of marelle (mom). The relative strength of four mom alleles in suppressing the UAS-Upd/GMR-Gal4 fly big eye phenotype is
mom1 > mom2 >
mom3 = mom4, and
mom1 is the strongest allele. mom is indeed the same gene as domeless (Chen, 2002).
To investigate whether dome has the genetic characteristics expected of the JAK/STAT receptor, dome interactions with upd, the known JAK/STAT ligand, were tested. To do this, advantage was taken of the fact that when the h-GAL4 line is used for ectopic expression of upd in the embryo, the result is abnormal head formation in 81% of the embryos. When upd is expressed ectopically in dome zygotic mutant embryos, this proportion is reduced to 16%. This result is consistent with dome being necessary to transduce the upd signal (Brown, 2001).
unpaired (upd) encodes a ligand for the Jak/STAT signaling pathway in Drosophila. In the second instar and early third larval eye disc, upd is expressed in the center of the posterior margin. upd loss-of-function mutations causes eye size reduction and upd overexpression causes eye enlargement. Upd regulates eye size through the Dome/Jak(Hop)/STAT92 signaling pathway to promote cell proliferation. Interestingly, the effect of Upd is only on cells located anterior to the morphogenetic furrow (MF), but has no effect on the second mitotic wave, which is posterior to MF. Overexpression of upd behind the MF can nonautonomously induce cell proliferation up to 20 rows of cells anterior to MF. The G1 cyclin, cycD transcript level is also enhanced anterior to MF. Consistent with the long-range effect, it was found that the extracellular Upd protein can be detected over a comparable long range, suggesting that Upd acts directly over a long distance as a signaling molecule (Tsai, 2004).
To characterize the features of JAK/STAT signaling in Drosophila immune response, totA was identified as a gene that is regulated by the JAK/STAT pathway in response to septic injury. Septic injury triggers the hemocyte-specific expression of upd3, a gene encoding a novel Upd-like cytokine that is necessary for the JAK/STAT-dependent activation of totA in the Drosophila counterpart of the mammalian liver, the fat body. In addition, totA activation is shown to require the NF-KB-like Relish pathway, indicating that fat body cells integrate the activity of NF-KB and JAK/STAT signaling pathways upon immune response. This study reveals that, in addition to the pattern recognition receptor-mediated NF-kappaB-dependent immune response, Drosophila undergoes a complex systemic response that is mediated by the production of cytokines in blood cells, a process that is similar to the acute phase response in mammals (Agaisse, 2003).
In order to identify genes that are regulated by the JAK/STAT pathway in response to septic injury in adult flies, a screen was performed for candidates that display an inducible expression upon immune challenge and that are constitutively expressed in flies carrying a gain-of-function mutation in the JAK/STAT pathway. To this end, custom-made cDNA microarrays were used to compare gene expression profiles of nonchallenged wild-type flies to gene expression profiles of challenged wild-type flies and to gene expression profiles of nonchallenged TumL flies displaying a gain-of-function mutation in the Drosophila JAK kinase Hopscotch. MP1 was identified as a gene that fulfilled both criteria for induction upon challenge and constitutive expression in a JAK/STAT gain-of-function mutation. MP1 expression was not induced in challenged flies displaying loss-of-function mutation in hop (hopM38/hopmsv1), confirming the involvement of Drosophila JAK in MP1 expression (Agaisse, 2003).
Sequence analysis of MP1 cDNA reveals that MP1 codes for Turandot A (TotA), a polypeptide that is produced by the larval fat body and accumulates in hemolymph in response to various stress conditions in flies. totA expression is mainly fat body specific in adult flies. totA was weakly expressed in the fat body of unchallenged flies and strongly induced after septic injury (Agaisse, 2003).
Activation of the JAK/STAT pathway culminates in translocation of phosphorylated STAT dimers from the cytoplasm to the nucleus. In mosquitoes, it has been shown that AgSTAT, the homolog of Drosophila STAT, translocates into the nucleus of fat body cells in response to bacterial infection. To further confirm the activation of the JAK/STAT pathway in Drosophila fat body in response to septic injury, the subcellular location of STAT protein was examined in fat body cells by immunostaining. In unchallenged flies, STAT protein is located both in the cytoplasm and nucleus. In challenged flies, STAT substantially clears the cytoplasm and accumulates in the nucleus. In contrast, there was no STAT nuclear translocation in Drosophila JAK mutant adult flies. Conversely, a very strong staining was detected both in the cytoplasm and nucleus of flies carrying a Drosophila JAK gain-of-function mutation. These results demonstrate that totA activation correlates with the JAK-dependent activation of STAT in Drosophila fat body cells in response to septic injury (Agaisse, 2003).
A Drosophila homolog of the vertebrate cytokine class I receptor, Dome (a.k.a. Mom), has been identified. Mutations in dome result in embryonic defects similar to the embryonic phenotype associated with mutation in the JAK/STAT pathway components. A truncated version of Dome, DomeΔCYT, has been generated by deletion of the intracellular region that is involved in signal transduction. This mutated receptor still contains the extracellular cytokine binding module and acts as a signaling antagonist, probably by titrating the ligand. Accordingly, DomeΔCYT overexpression during embryogenesis mimics the loss-of-function phenotype of dome mutants. To test whether Dome plays a role in totA expression, the GAL4/UAS system was used to express the dominant-negative form of Dome, DomeΔCYT, in adult fat body. Northern blot analysis has revealed that totA expression upon immune challenge is totally abolished in the corresponding animals. It is concluded that totA activation in response to bacterial infection is the result of a signaling event that is transduced by the Drosophila homolog of the vertebrate cytokine receptor, Dome, in the fat body (Agaisse, 2003).
The involvement of dome in totA expression strongly suggests the existence of a cytokine-like molecule involved in the control of totA expression. upd has been characterized as a gene encoding the cytokine that activates the JAK/STAT pathway during Drosophila embryogenesis. Strong alleles of upd are embryonic lethal, but weaker alleles, such as outstrechted (os), give rise to adult flies that hold their wings at right angles and have small eyes. totA activation was found to be strongly decreased in os flies, suggesting that upd might be involved in totA expression. However, totA activation is nearly wild-type in transheterozygous flies displaying the os mutation over a null mutation in upd (updYM55), suggesting that a defect in upd expression is not responsible for the lack of totA activation in the os genetic background. Interestingly, totA activation is abolished in transheterozygous flies displaying the os mutation over a large deficiency (os1A) of the upd locus. This suggests that the os mutation affects the expression of a gene involved in totA activation that maps to the os/upd locus, but that is not upd. Blast search analysis reveals the presence of two other upd-like cytokine-encoding genes at the upd/os locus. upd2 corresponds to CG5988 and maps 50 kb downstream from upd. upd3 corresponds to CG15062 (for the first and second exon) and CG5963 (for the third exon) and maps 25 kb downstream from upd. These observations prompted a hypothesis that upd2 and/or upd3 might be involved in totA expression (Agaisse, 2003).
To further investigate the potential role of upd2 and/or upd3 in totA expression, whether the expression of the upd-like genes was inducible upon septic injury was examined. No upd2 expression was detected in adult flies by using RT-PCR analysis. In contrast, the level of upd3 expression, which was very low in control animals, was found to be significantly increased after septic injury. The pattern of GFP expression was examined in flies harboring a upd3 promoter region-GAL4 fusion and a UAS-GFP reporter. No GFP production was detected in fat body cells of control or challenged animals. However, GFP production was strongly increased in blood cells after challenge, indicating that hemocytes might be the main site of upd3 expression. To investigate the functional importance of upd3 hemocyte-specific expression in totA expression, an in vivo RNAi strategy was designed to silence upd3 expression in a tissue-specific manner. The hemolectin-GAL4 and the yolk-GAL4 constructs were used to drive the expression of the UAS-iupd3 hairpin construct in hemocytes and fat body, respectively. While the production of upd3 dsRNA in fat body did not interfere with totA expression, upd3 dsRNA expression in hemocytes led to a strong decrease in totA activation upon septic injury. Altogether, these experiments suggest that upd3 activation in hemocytes subsequently leads to totA activation in the fat body (Agaisse, 2003).
To further analyze the regulation of totA expression in fat body cells, totA expression was monitored in response to clean injury, septic injury with gram-negative bacteria (E. coli), or septic injury with gram-positive bacteria (M. luteus). Clean injury and septic injury with M. luteus resulted in a modest but significant induction of totA expression: 4-fold induction 6 hr after challenge and 7-fold induction 18 hr after challenge. In sharp contrast, septic injury with E. coli resulted in a robust induction of totA expression: 25-fold induction at 6 hr and 35-fold induction at 18 hr. Gram-negative bacteria therefore constitute the best inducer for totA expression. It is well established in flies that immune response to gram-negative bacteria is mediated by the Imd pathway through activation of TAK1 and the NF-KB-like transcription factor Relish. Therefore totA expression was analyzed in TAK1 and in relish mutant flies. totA activation after challenge was totally abolished in these mutants, indicating that, in addition to being JAK/STAT dependent, totA expression also requires the activity of the Relish pathway. Whether the activity of the Relish pathway is specifically required in the fat body was analyzed. To this end, relish dsRNA was overexpressed in fat body using the UAS-irel construct and the yolk-GAL4 driver. dsRNA-mediated silencing of relish expression leads to a failure in totA activation, indicating that Relish activity is specifically required in the fat body. Finally, whether Relish activation in the fat body is sufficient to activate totA expression was analyzed. Overexpression of Imd in fat body cells has been shown to lead to activation of Relish and therefore constitutive expression of the antimicrobial peptide genes, such as diptericin, in the absence of immune challenge. totA is not constitutively expressed in the corresponding flies, indicating that Relish activation is required in fat body but is not sufficient to activate totA expression (Agaisse, 2003).
Altogether, these results demonstrate that TotA qualifies as a bona fide acute phase protein. In addition, totC and totM are also controlled by the JAK/STAT pathway upon septic injury, a characteristic that is probably shared by all the members of the tot family. Moreover, the tot family members are not the sole target of the JAK/STAT pathway upon septic injury. Expression of CG11501, a cysteine-rich polypeptide related to scorpion toxin, is also controlled by the JAK/STAT pathway. These data indicate that the JAK/STAT pathway contributes to a global response upon immune challenge by controlling the expression of several acute phase proteins. As for most of their mammalian counterparts, the function of these Drosophila acute phase proteins is unclear. totA overexpression does not appear to protect NF-kappaB mutants, such as kenny, from gram-negative bacteria infection, indicating that TotA, unlike antimicrobial peptides such as Diptericin and Drosomycin, does not prevent bacterial growth. Altogether, these observations suggest that TotA is probably a general stress response factor involved in homeostasis of (damaged) tissues. Accordingly, it has been shown that TotA overexpression confers extended survival to flies subjected to heat stress, a treatment that certainly leads to disturbances of physiological homeostasis. Further in vivo characterization of acute phase protein function, such as TotA, using Drosophila as a model system will help gain an understanding of the overall physiology of the acute phase response in insects and mammals (Agaisse, 2003).
Upd was first identified as a secreted molecule that activates the JAK/STAT pathway during Drosophila embryogenesis. Evidence is provided for the existence of a component of the JAK/STAT pathway: Upd3 that is produced in hemocytes in response to immune challenge. Although cytokine-like activities, such as IL1 and TNFα, have been previously reported as being produced by hemocytes from Lepidopteran larvae in response to LPS stimulation, none of these activities have been shown to have a physiological function in vivo. upd3 is thus the first example of a gene coding for a cytokine that is expressed in hemocytes and is required for signaling in fat body. This study therefore constitutes the first demonstration that sentinel cells, such as hemocytes, play a signaling role in the Drosophila immune response. The nature of the signals that are detected by hemocytes and the signaling pathway(s) that trigger upd3 activation in response to septic injury remain to be determined. Preliminary experiments indicate that upd3 expression is severely impaired in TAK1 flies after septic injury, suggesting that components of the Relish pathway (as defined in fat body cells) might be involved in upd3 activation in hemocytes in response to bacterial infection. However, further analysis in PGRP-LC and relish mutant backgrounds was not consistent with this hypothesis. Clearly, the mechanisms involved in upd3 regulation potentially constitute a new paradigm for studying the signals and the transduction machinery involved in the control of gene expression in activated hemocytes (Agaisse, 2003).
The characterisation of ligands that activate the JAK/STAT pathway has the potential to throw light onto a comparatively poorly understood aspect of this important signal transduction cascade. This study describes an analysis of the only invertebrate JAK/STAT pathway ligands identified to date, the Drosophila unpaired-like family. upd2 is expressed in a pattern essentially identical to that of upd and the proteins encoded by this region activate JAK/STAT pathway signalling. Mutational analysis demonstrates a mutual semi-redundancy that can be visualised in multiple tissues known to require JAK/STAT signalling. In order to better characterise the in vivo function of these ligands, a reporter based on a natural JAK/STAT pathway responsive enhancer was developed, and ectopic upd2 expression was shown to effectively activate the JAK/STAT pathway. While both Upd and Upd2 are secreted JAK/STAT pathway agonists, tissue culture assays show that the signal-sequences of Upd and Upd2 confer distinct properties, with Upd associated primarily with the extracellular matrix and Upd2 secreted into the media. The differing biophysical characteristics identified for Upd-like molecules have implications for their function in vivo and adds another aspect to understanding of cytokine signalling in Drosophila (Hombria, 2005).
Three unpaired-like genes have been identified by sequence homology searches within the 17A interval of the Drosophila X-chromosome. The founding family member upd has been molecularly characterised and its activation of the JAK/STAT signal transduction pathway is required for multiple developmental processes. In addition, a recent report has identified Upd3 as an infection specific cytokine produced by haemocytes in response to septic injury (Agaisse, 2003). However, no function has been proposed for upd2 and no analysis of the upd locus as a whole has been undertaken (Hombria, 2005).
To investigate the potential developmental roles of upd2 and upd3, their embryonic expression was analyzed. Although adult haemocytes have been shown to express Upd3 in response to bacterial challenge the only detectable expression of upd3 during embryogenesis is observed in the gonads from embryonic stages. By contrast, upd2 is expressed in the central region of the blastoderm, segmentally repeated stripes at stage 9, in the tracheal placodes at stage 10, and in a region within the hindgut and posterior spiracles from stage 11. Given the almost complete overlap between the upd2 expression pattern and that previously described for upd these results suggest that both upd2 and upd could be regulating embryonic development (Hombria, 2005).
Conceptual translation of the upd-like genes present in Drosophila melanogaster identifies three related proteins. Each of these proteins contains an N-terminal region representing a predicted signal- or anchor-sequence and multiple potential N-linked glycosylation sites. Glycosylation of these sites has been suggested to mediate the binding of Upd to the extracellular matrix (ECM). Analysis of Upd2 shows that it contains a strongly hydrophobic region from amino acid 30 to 55. However, using the SignalP 3.0 server, a hidden Markov model predicts the likelihood of Upd2 containing a signal sequence at only 28%, with a 25% confidence level of cleavage between positions 55 and 56. The same model also predicts an anchor sequence (at a 71% confidence level), a motif required to insert Type II, III and IV trans-membrane proteins into the endoplasmic reticulum (ER). By contrast, when Upd is analysed using the same hidden Markov model, a signal peptide is predicted (at 100% confidence level) with an 89% probability of cleavage between position 27 and 28. Strikingly, the Upd2-like molecules present in D. melanogaster, simulans and yakuba contain predicted anchor-sequences while more distantly related Upd2-like molecules include N-termini predicted to act as signal-sequences. This prediction therefore suggests that D. melanogaster Upd2 cannot be secreted by the 'classical' Golgi/ER-based secretion machinery and may be trapped as a trans-membrane protein within the ER (Hombria, 2005).
All related Drosophilid species for which genome sequence is available, encode clear homologues of all three upd-like genes. However, no unambiguous upd-like homologues are identifiable in more distantly related species including the fellow dipteran Anopheles gambiae. It therefore appears that the upd gene family is evolving rapidly in the Drosophilids (Hombria, 2005).
Although the N-terminus of D. melanogaster Upd2 is predicted to function as an anchor-sequence, the true nature of this signal ultimately requires experimental validation. Therefore C-terminal fusions of both Upd and Upd2 to enhanced GFP were generated to allow the direct visualisation of the resulting proteins. When expressed in S2 Drosophila tissue culture cells, UpdGFP appears to be present extracellularly around transfected cells (identified by their co-expression of nuclear localised mRFP). This extracellular GFP is only detectable in the most basal confocal sections and appears to be associated with the substrate on which the cells grow. This is consistent with previous results that identify Upd as an ECM-associated protein. By contrast, fluorescence associated with Upd2GFP expressing cells appears to be intracellular with particular accumulations surrounding the nucleus in structures that may represent the endoplasmic reticulum. Although no pattern comparable to the surrounding ECM-associated halo of UpdGFP protein is detected, very low levels of extracellular Upd2GFP are occasionally detected adjacent to Upd2GFP transfected cells. It therefore appears that Upd2 cannot associate with the ECM in a manner comparable to Upd (Hombria, 2005).
Whether the dissimilar signal-/anchor-sequences could explain the observed differences of Upd and Upd2 secretion was examined. Domain swap experiments were undertaken to determine the individual contributions of the signal-/anchor-sequences for the activity of Upd and Upd2. GFP-tagged fusion molecules consisting of the secreted portion of Upd2 joined to the signal sequence of Upd (termed Upd2SS1) and another comprising the Upd2 anchor sequence attached to the secreted portion of Upd (called Upd1SS2) were constructed. Expression in S2 cells showed that Upd2SS1 can now be visualised as an ECM-associated halo surrounding transfected cells while Upd1SS2 appears to be located exclusively intracellularly. These results appear to indicate that the nature of the signal-/anchor-sequences present is responsible for the differential ECM interactions of UpdGFP and the low levels of extracellular Upd2GFP detected. In addition, the extracellular halo of Upd2SS1 indicates that the secreted region of Upd2 is capable of associating with the ECM under these tissue culture conditions (Hombria, 2005).
Upd and Upd2 induced JAK/STAT activation can be assayed using transcriptional reporter systems in tissue culture cells. To undertake such experiments, a 6x2xDrafLuc reporter transgene containing twelve STAT92E binding sites located upstream of the gene encoding firefly Luciferase was used in the haemocyte-like Kc167 cell line. Stimulation of endogenous JAK/STAT pathway activity by ectopic expression of Upd has been shown to result in a strong 6x2xDrafLuc response dependent on endogenously expressed pathway components. This study showed that Upd2 can act as a potent activator of JAK/STAT signalling. Additional studies showed that Upd2 is unlikely to be ECM associated and is probably a freely diffusible pathway ligand (Hombria, 2005).
To test if upd2 can activate the JAK/STAT pathway in vivo, reporters of JAK/STAT activation were sought. The only rigorously analysed target of STAT, the even-skipped stripe 3 enhancer, is not useful to test induced STAT activation, as it is under negative regulation by gap genes. While searching for an alternative reporter it was observed that the dome gene might contain enhancers regulated by STAT. (1) dome mRNA expression increases after stage 11 in areas where upd is expressed: the pharynx, the hindgut, the tracheae and the posterior spiracles. (2) lacZ expressed by heterozygous P-element enhancer traps inserted in the dome 5'UTR show the same areas of elevated expression. (3) When hemizygous (and therefore causing a dome mutation), these same P-elements no longer up-regulate expression in these areas indicating that JAK/STAT activation through dome is required for dome up-regulation. Consistent with these observations, potential STAT binding sites were found in the 5'UTR and first intron of dome. Reporter constructs made using a 2.8-kb genomic fragment containing part of the first exon and most of the first intron activate lacZ expression in the pharynx and hindgut mesoderm, two of the areas expressing increased dome mRNA levels. Double RNA in situ/antibody staining shows that upd RNA is expressed in the ectoderm of the pharynx and in the hindgut, in a region adjacent to the mesoderm cells expressing the reporter construct. Given its mesoderm specific expression, the 2.8-kb construct will be referred to as dome-MESO (Hombria, 2005).
To determine if dome-MESO is regulated by JAK/STAT signalling, its expression was studied in different JAK/STAT mutant backgrounds. In dome9 mutant embryos, dome-MESO is not activated in either the pharynx or the hindgut, showing that dome-MESO regulation is comparable to that of the endogenous dome. The same result is observed in Df(1)os1A embryos lacking all Upd-like ligands. Whether pathway activation is sufficient to drive dome-MESO expression was tested. Gal4 mediated expression of upd or the activated form of the JAK kinase HopTuml using the 24B-Gal4 or the twi-Gal4 mesodermal drivers results in ectopic dome-MESO expression both in the visceral and somatic mesoderm. Also, whether signalling from the ectoderm could non-autonomously activate mesoderm specific expression of dome-MESO was tested by using the ectodermal specific line 69B-Gal4 to express upd. Under these conditions, dome-MESO was activated in the mesoderm, with the salivary glands being the only non-mesodermal tissue induced. As a control HopTuml was also expressed with 69B-Gal4. In these conditions dome-MESO was not ectopically activated in the mesoderm, confirming the ectodermal specific expression of this Gal4 line. These results show that the dome-MESO enhancer is a mesodermal specific reporter for JAK/STAT activation that mimics the behaviour of endogenous dome. These results also provide evidence of JAK/STAT signalling across germ layers and demonstrates the existence of a positive feed back loop in dome regulation (Hombria, 2005).
Having established the dome-MESO reporter as a useful tool to show the status of JAK/STAT pathway activity in mesodermal cells, whether both Upd and Upd2 have similar functions was tested in vivo. As would be expected of a bona fide pathway ligand, Upd2 expression in the mesoderm using 24B-Gal4 or in the ectoderm using 69B-Gal4 results in strong ectopic mesodermal dome-MESO activation. Upd2 is therefore able to non-autonomously activate JAK/STAT signalling in vivo (Hombria, 2005).
The expression of vvl, a gene whose expression in the anterior part of the hindgut ectoderm has been shown to depend on JAK/STAT pathway activity, was tested. Pathway induction driven by expression of either Upd or Upd2 with the 69B-Gal4 line, is sufficient to expand the domain of vvl within the hindgut. The morphological effect of Upd2 activation on germ band movements was studied; as reported for Upd, Upd2 is also sufficient to block germ band retraction (Hombria, 2005).
Finally, whether upd and upd2 expression can exert the same effects when expressed in the imaginal discs was tested. JAK/STAT pathway activation is sufficient to modulate cellular proliferation during the development of the wing imaginal disc. Using the Bx-Gal4 (also known as 1096-Gal4) driver line, either upd or upd2 was expressed in wing imaginal discs. In both cases, the resulting adults developed reduced size wings (Hombria, 2005).
All these studies taken together indicate that ectopic expression of Upd2 is sufficient to reproduce all effects caused by misexpression of Upd and suggest that both molecules are likely to activate the same pathway (Hombria, 2005).
To analyse if both ligands activate the JAK/STAT pathway through the Dome receptor, whether the wing defects caused by the ectopic misexpression of Upd and Upd2 could be rescued by simultaneous expression of dominant negative versions of the Dome receptor was tested. Having established that misexpression of dominant negative Dome alone has only mild effects, Upd or Upd2 and the dominant negative receptors were coexpressed. Expression of both transgenes resulted in an almost complete rescue of the Upd/Upd2 mediated wing defects, indicating that both Upd and Upd2 induce their effect via Dome. A similar blockage of Upd2-induced signalling is also observed in tissue culture-based assays by RNAi-induced knock down of dome mRNA (Hombria, 2005).
Since both the loss-of-function analysis and the ectopic expression tests indicate that Upd2 and Upd can act as partially redundant JAK/STAT ligands, the capacity of each ligand to rescue the absence of all other upd-like ligands was tested. For that purpose, focus was placed on the requirement of the JAK/STAT pathway for spiracle morphogenesis. Mutations in any of the components of the JAK/STAT pathway result in a highly abnormal posterior spiracle. The Klu-Gal4 spiracle driver line was used to test rescue of the Df(1)os1A spiracle defect. As a positive control, the expression of the activated JAK kinase was induced; under these conditions, UAS-hopTuml results in a partial rescue of the spiracle phenotype. Very similar rescues where obtained when expressing either UAS-upd or UAS-upd2 thus confirming that Upd and Upd2 represent redundant ligands in vivo (Hombria, 2005).
Given the sequence similarity of the upd-like genes and their clustering within the genome, it appears that this region has undergone two genomic duplication events; a similar scenario has been proposed to explain the functional redundancy of the proneural ac and sc genes. Since all available Drosophilid genomes encode all three upd-like genes, it is likely that the duplication events occurred before the radiation of the Drosophila family. However, no plausible upd-like genes were identified in the genome of the fellow dipteran Anopheles gambiae, suggesting that the Upd ligand is undergoing a phase of rapid evolutionary change. This conjecture is supported by the restriction of the upd3 expression pattern and its apparent specialisation to roles in immune signalling as well as the apparent divergence of the N-terminal signal-/anchor-sequences of Upd2 molecules in the closely related D. melanogaster, simulans and yakuba (Hombria, 2005).
Although negative autoregulation of dome in the follicle cells has been suggested to occur through two STAT binding sites 12 kb upstream of dome, this study shows that positive dome autoregulation in the mesoderm is driven by intronic regulatory sequences used to generate the dome-MESO reporter gene. Expression of dome-MESO requires the function of the JAK/STAT pathway and can be induced by its ectopic activation. The reporter includes several putative Drosophila STAT binding sites and future experiments should confirm molecularly if the autoregulation is direct. In any case, these results show that dome-MESO is a useful tool to test the state of activity of the pathway in embryos and that, in the mesoderm, JAK/STAT induces positive autoregulation of dome. In addition to the mesodermal up-regulation, dome mRNA expression also increases in the posterior spiracles and in the ectoderm of the pharynx and hindgut close to where upd is expressed, suggesting the existence of another ectoderm-specific positive autoregulatory element. It is interesting to speculate that several dome tissue-specific enhancers exist to modulate the strength of JAK/STAT signalling by increasing or decreasing the amount of receptor. These changes in Dome levels would either amplify the reception of ligands in the embryonic mesoderm and ectoderm (pharynx and hindgut), or act to down-regulate the signal in cases where only transient pathway activity is desired (such as in the follicle cells) (Hombria, 2005).
Although tissue culture-based secretion assays indicate that Upd2 is only weakly associated with the ECM immediately surrounding expressing cells, the ability of Upd2 to condition media indicates that the molecule is secreted and active under these conditions. This result is further supported by the mesodermal induction of the dome-MESO reporter following ectoderm specific expression of Upd2 (Hombria, 2005).
Striking similarities continue to emerge between the mammalian and Drosophila JAK/STAT signaling pathway. However, until now there has not been the ability to monitor global pathway activity during development. A transgenic animal was generated with a JAK/STAT responsive reporter gene that can be used to monitor pathway activation in whole Drosophila embryos. Expression of the lacZ reporter regulated by STAT92E binding sites can be detected throughout embryogenesis, and is responsive to the Janus Kinase hopscotch and the ligand Upd. The system has enabled identification of the effect of a predicted gene related to upd, designated upd2, whose expression initiates during germ band extension. The stimulatory effect of upd2 on the JAK/STAT reporter can also be demonstrated in Drosophila tissue culture cells. This reporter system will benefit future investigations of JAK/STAT signaling modulators both in whole animals and tissue culture (Gilbert, 2005; full text of article).
To visualize global JAK/STAT pathway activation in the whole animal a transgenic Drosophila line containing the lacZ gene regulated by STAT DNA binding sequences. This construct included three STAT target sites (GAS) upstream of a minimal Drosophila heat shock promoter in the pCaSpeR.hsp.bas vector. This reporter gene and the Drosophila line is referred to as (GAS)3-lacZ. Expression during embryogenesis is strikingly dynamic (Gilbert, 2005).
To evaluate expression of the reporter gene, in situ hybridization was performed to detect the lacZ mRNA transcript in homozygous (GAS)3-lacZ embryos during various stages of development. Expression of the lacZ gene is not detectable in syncytial blastoderm embryos. However, at the onset of cellularization, lacZ mRNA is detected throughout the embryo with strongest expression in the ventral region. Just prior to gastrulation, expression becomes more spatially restricted. At the onset of gastrulation, an intense lacZ signal is detected in a broad region anterior to the presumptive cephalic furrow and invaginating presumptive mesoderm. As germ band extension proceeds, expression is reduced, but reappears by early stage 9 in the head region and as a weak 14 stripe pattern. By stage 10 this pattern resolves into strong expression in 14 parasegments. The lacZ signal then recedes and is detected in small clusters of segmentally repeated cells. These data indicate that STAT binding sites within a promoter context can drive expression of a reporter gene in the Drosophila embryo. Homozygous Drosophila were also generated that contain a lacZ transgene regulated by a single GAS element. The (GAS)1-lacZ embryos exhibited a similar pattern of gene expression but with significantly weaker intensity (Gilbert, 2005).
To ensure that expression of the reporter system was in fact responsive to JAK/STAT activity, (GAS)3-lacZ expression was evaluated in embryos lacking maternal hop. These hop embryos have also been shown to exhibit reduced STAT92E protein levels. Maternal hop was removed using the FLP-DFS technique to generate females with hopC111 homozygous germ cells. These females were crossed to (GAS)3-lacZ males to generate embryos that lack maternal hop and carry a single copy of the (GAS)3-lacZ transgene. Embryos were evaluated for expression of lacZ transcript by in situ hybridization. Expression was found to be dramatically reduced in all stages examined. During early cellularization only a weak anterior signal is detected with little or no signal in the remainder of the embryo. At the onset of gastrulation, the embryos exhibited reduced expression throughout, particularly in the area corresponding to mesoderm. During germ-band extension, the expression in 14 parasegments was reduced with residual signals in small clusters of cells at the midline of the original 14 stripes. The residual activity detected may be hop independent. stat92E null alleles were also tested and showed residual activity. These results indicate that the (GAS)3-lacZ reporter system is responsive to a reduction of in vivo levels of JAK/STAT pathway components, and it was confirmed that early JAK/STAT signaling is established in cellularizing embryos in a ubiquitous manner (Gilbert, 2005).
The result suggests that the presence of Upd ligand alone is not sufficient to activate the pathway. In addition, when Upd was ectopically expressed with the paired-Gal4 driver, (GAS)3-lacZ was hyperactivated, but only in a subset of cells. These data support the finding of the inability of ectopic ligand to induce Domeless dimerization (Brown, 2003), and hence pathway activation in early embryos (Gilbert, 2005).
The removal of maternal hop by the production of germline clones had a clear inhibitory effect on the establishment of (GAS)3-lacZ expression in early cellularized embryos, gastrulation, and the maintenance of reporter expression during germ-band extension. Since loss of maternal hop has been shown to negatively regulate STAT92E protein levels, it was expected that this loss of function allele would cause the strongest loss of (GAS)3-lacZ expression. However, all three stages displayed some residual expression. Given that the hopC111 mutation in the germ line clone corresponds to a small internal deletion, it is possible that the residual reporter expression is due to low activity of a mutant Hop protein. The possiblility that residual expression is due to the activity of an unknown DNA binding factor that can interact with the reporter gene cannot be ruled out. It is also possible that there is minimal but constitutive activity of the promoter used in the construction of the (GAS)3-lacZ gene. The presence of a CAAT box and a TATA box could facilitate a low level of expression by the basal transcriptional machinery (Gilbert, 2005).
The contribution of upd, upd3 and upd2 to JAK/STAT signaling during embryo development was also evaluated. The removal of upd and upd3 or upd, upd3 and upd2 significantly decreased pathway activity. The effect was similar to the removal of maternal hop during the establishment of (GAS)3-lacZ expression in cellularized embryos. The earliest developmental stage examined for both deficiencies is slightly later than that examined for hop embryos. Given the slight difference in staging, the residual expression is similar, consisting of a weak head stripe and 2 weak stripes in the trunk. The maintenance of (GAS)3-lacZ expression in germ-band extended embryos was also analyzed. The removal of both upd, upd3 and upd2 had a more severe effect on reporter gene expression than removal of upd and upd3 alone. This increase in severity was manifest as a reduction in the number of expressing cells within the segmentally repeated cell clusters (Gilbert, 2005).
These studies performed both in vivo and in Drosophila tissue culture cells provide evidence that Upd2 can act to stimulate activation of the JAK/STAT pathway. Since the expression pattern of both upd and upd2 is similar during germ-band extension, it is possible that they serve certain biologically redundant functions similar to the manner in which IL-6 cytokines function in mammalian systems. These are pleiotropic cytokines that share structural similarity and functional redundancies in part due to the fact that they share a common receptor subunit. Alternatively, signaling by Upd and Upd2 may serve specific functions either in the embryo or during other stages of larval, pupal, or adult development. In monitoring upd2 expression by Western blot analysis, multiple isoforms were detected that may indicate post-translational modifications. The nature of the Upd2 proteins remains to be characterized and could provide insight on additional levels of signaling specificity and receptor binding. Upd2 may not be associated with the extracellular matrix like Upd and thereby able to act at a greater distance from its production to influence gene expression. The in vitro studies in Drosophila S2 cells clearly demonstrated the ability of Upd2 to stimulate specific expression of (GAS)3-lacZ (Gilbert, 2005).
This report provides evidence that JAK/STAT signaling can be monitored in vivo using the lacZ reporter gene regulated by STAT DNA binding elements. A complementary assay has recently been developed to monitor the pattern of STAT92E phosphorylation in the embryo, however this method of detecting lacZ by in situ hybridization provides a highly sensitive assay with little background to detect pathway activation from the cell surface receptor to gene expression in the nucleus. In addition, dynamic expression of the reporter can be visualized by a simple X-gal staining of whole embryos and remains sensitive enough to allow detection of changes in reporter activity in response to the removal and ectopic expression of pathway components. This capability could facilitate a genetic screen for enhancers or suppressors of JAK/STAT pathway activity during specific developmental stages. In addition, since (GAS)3-lacZ can be used to monitor JAK/STAT activation in tissue culture cells, this could facilitate a screen in tissue culture cells as well as providing a method of verifying screen-based genetic interactions. The reporter line should also be useful to characterize JAK/STAT function during later developmental stages. Preliminary experiments with X-gal staining of (GAS)3-lacZ third instar larval structures revealed β-galactosidase activity in a subset of structures known to require or possess competence for JAK/STAT signaling (Gilbert, 2005).
Until now direct monitoring of JAK/STAT pathway activation has only been possible in tissue culture cells. The establishment of an in vivo monitor of JAK/STAT pathway activation will provide an indispensable tool for the discovery of interacting proteins and tissue-specific requirements during Drosophila development. This assay can be used to visualize pathway activation and identify novel regulators of JAK/STAT signaling during embryogenesis and the isolation of a novel gene, upd2, which bears sequence homology to upd and encodes a functional ligand of the JAK/STAT pathway is specifically described. These data add to the mounting evidence that suggests the Drosophila JAK/STAT pathway is not simple, but contains multiple ligands that may act to elicit tissue and gene-specific responses (Gilbert, 2005).
A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. A new negative regulator of JAK/STAT signaling is described and a potential mechanism identified by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, Ken & Barbie (Ken), which is an ortholog of the mammalian proto-oncogene BCL6, has been identified as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, endogenous Ken is shown specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo (Arbouzova, 2006).
Analysis of phenotypes associated with mutations in Drosophila JAK/STAT pathway components have identified a wide variety of requirements for the pathway during embryonic development and in adults. What is less clear is how the repeated stimulation of a single pathway is able to generate this pleiotropy of developmental functions. In order to identify modulators of JAK/STAT signaling that may be involved in this process, a genetic screen was undertaken for modifiers of the dominant phenotype caused by the ectopic expression of the pathway ligand Unpaired (Upd) in the developing eye imaginal disc. Such misexpression by GMR-updΔ3′ results in overgrowth of the adult eye, a phenotype sensitive to the strength of pathway signaling activity. With this assay, one genomic region, defined by Df(2R)Chig320, was found to enhance the GMR-updΔ3′-induced eye overgrowth phenotype. Of the genes deleted by Df(2R)Chig320, only mutations in ken showed consistent and reproducible enhancement of the phenotype. In addition, other dominant phenotypes induced by transgene expression from the GMR promoter are not modulated by ken mutations, indicating that Ken is unlikely to interact with the misexpression construct used (Arbouzova, 2006).
The enhancement of the GMR-updΔ3′ phenotype after removal of one copy of ken implies that Ken normally functions antagonistically to JAK/STAT signaling. Therefore phenotypes associated with mutations in other pathway components were tested to establish the reliability of this initial observation. Consistent with this, genetic interaction assays between ken mutations and the hypomorphic loss-of-function allele stat92EHJ show a reduction in the frequency of wing vein defects normally associated with this stat92E allele. Moreover, the degree of suppression is consistent with the strength of ken alleles tested. Similarly, the frequency of “strong” posterior spiracle phenotypes caused by the dome367 allele of the pathway receptor is also reduced when crossed to ken alleles or the Df(2R)Chig320 deficiency, with a concomitant increase in “weak” phenotypes (Arbouzova, 2006).
Thus, multiple independent ken alleles all modify diverse phenotypes caused by both gain- and loss-of-function mutations in multiple JAK/STAT pathway components. Each of these components acts at different levels of the signaling cascade and show interactions indicating that Ken consistently acts as an antagonist of the pathway (Arbouzova, 2006).
The ken locus contains three exons encoding a 601 aa protein. Ken possesses an N-terminal BTB/POZ domain between aa 17 and 131 and three C-terminal C2H2 zinc finger motifs from aa 502 to 590. Strikingly, a number of Zn finger-containing proteins that also contain BTB/POZ domains have also been shown to function as transcriptional repressors—often via the recruitment of corepressors such as SMRT, mSIN3A, N-CoR, and HDAC-1 (Arbouzova, 2006).
Searches for proteins similar to Ken identified homologs in Drosophila pseudoobscura and the mosquito Anopheles gambiae. In vertebrates, human B-Cell Lymphoma 6 (BCL6) was the closest full-length homolog. Drosophila Ken and human BCL6 share the same domain structure and show 20.3% overall identity. Proteins listed as potential vertebrate homologs of Ken in Flybase are more distantly related (Arbouzova, 2006).
Expression of ken was also examined during development, where it is detected in a dynamic pattern from newly laid eggs, throughout embryogenesis, and in imaginal discs. As such, endogenous Ken is present in all tissues and stages in which genetic interactions were observed (Arbouzova, 2006).
Given the presence of potentially DNA binding Zn finger domains and the nuclear localization of GFPKen, the DNA binding properties of Ken was determined by using an in vitro selection technique termed SELEX (systematic evolution of ligands by exponential enrichment). With a GST-tagged Ken Zn finger domain and a randomized oligonucleotide library, ten successive rounds of selection were undertaken. Sequencing of the resulting oligonucleotide pool and alignment of 43 independent clones showed that all recovered plasmids were unique and each contained one, or occasionally two, copies of the motif GNGAAAK (K = G/T) (Arbouzova, 2006).
To confirm the SELEX results, GFPKen was expressed in tissue culture cells and these were used for electromobility shift assays (EMSA). A radioactively labeled probe containing the wild-type (wt) consensus binding site GAGAAAG gives a specific band, which can be supershifted by an anti-GFP antibody and therefore represents a GFPKen/DNA complex. In order to identify positions essential for binding, a competition assay was used in which unlabeled oligonucleotides containing single substitutions in each position from 1 to 7 were added to binding reactions. 10-fold excess of unlabeled wild-type consensus oligonucleotide greatly diminished the intensity of the GFPKen band, while 50- and 100-fold excess totally blocked the original signal. By contrast, competition with unlabeled m3 oligonucleotides containing a G to A substitution at position 3 failed to significantly reduce the intensity of the band even at 100-fold excess. With this approach, the positions 1 and 7 are found dispensable for DNA binding, whereas the central GAAA core is absolutely required. Similar results were obtained with the converse experiment with labeled mutant probes, although in this case the wt probe produces a stronger signal than the m1 and m7 mutant oligonucleotides. Taken together, these experiments not only define the core sequence for Ken binding, but also demonstrate the specificity of Ken as a site-specific DNA binding molecule. Interestingly, the core consensus bound by Ken is very similar to that identified for human BCL6, with the Zn fingers of the latter binding to a DNA sequence containing a core GAAAG motif
(Arbouzova, 2006).
One initial observation made is that the core GAAA essential for Ken binding overlaps the sequence recognized by STAT92E. Consistent with this overlap, a 100-fold excess of unlabeled oligonucleotide containing the STAT92E consensus is sufficient to fully compete for Ken in EMSA assays. Given this finding, it is hypothesized that the negative regulation of JAK/STAT signaling by Ken observed in genetic interaction assays may occur via a mechanism of competitive DNA binding site occupation. Due to the incomplete overlap between the STAT92E and Ken core sequences, this hypothesis also implies the existence of STAT92E DNA binding sites to which both STAT92E and Ken could bind (STAT+/Ken+) as well as sites with which Ken cannot associate (STAT+/Ken−) (Arbouzova, 2006).
To test this hypothesis, a cell culture-based assay was set up by using a luciferase-expressing reporter containing four STAT92E binding sites originally identified in the promoter of the Draf locus. In addition to this STAT+/Ken+ wild-type reporter, STAT+/Ken− and STAT−/Ken− variants identical but for the binding sequences were generated. When transfected into the hemocyte-like Kc167 Drosophila cell line, both STAT+/Ken+ and STAT+/Ken− reporters showed strong stimulation upon coexpression with the pathway ligand Upd, an assay previously shown to require an intact JAK/STAT cascade. When cotransfected with KenGFP, the activity of the STAT+/Ken+ reporter was reduced, an effect reproduced in three independent experiments with both KenGFP and Ken. While the reduction in reporter activity for the STAT+/Ken+ assay shown is statistically significant, the STAT+/Ken− reporter was unaffected by the coexpression of Ken. Reporters containing binding sites mutated to prevent binding of both STAT92E and Ken (STAT−/Ken−) showed no activation after pathway stimulation and did not respond to Ken (Arbouzova, 2006).
These results indicate that Ken functions as a transcriptional repressor in this cell-culture system and shows that this effect is specific to the DNA sequence determined by SELEX and EMSA. This result is also consistent with a recent whole-genome RNAi-based screen, which used a reporter containing STAT+/Ken+ binding sites and includes Ken among the list of JAK/STAT regulators identified. In addition, recent reports have also demonstrated BCL6 binding to STAT6 sites in vitro and have shown that BCL6 can act as a repressor of STAT6-dependent target gene expression in cell culture. Although this repression is mediated by the binding to corepressors to the BTB/POZ domain of BCL6, no link between BCL6 and STAT activity has been demonstrated in vivo (Arbouzova, 2006).
Finally, it should also be noted that both the STAT+/Ken+ and STAT+/Ken− reporters contain additional GAAA sequences that are not part of the characterized STAT92E binding sequences. However, despite the presence of these potential Ken binding sites within 15 bp of the STAT92E site, Ken expression did not affect the STAT+/Ken− reporter, suggesting that Ken may require STAT92E to influence gene expression. Although no direct association between Ken and STAT92E has been demonstrated, this possibility cannot be excluded, and further analysis remains to be undertaken (Arbouzova, 2006).
Having established that Ken functions at the level of DNA binding in cell culture, it was asked whether Ken also acts as a transcriptional repressor of JAK/STAT pathway target genes in vivo. For this, the effect of ectopically expressed Ken on the expression of putative JAK/STAT pathway target genes was examined and, given the high levels of maternally loaded STAT92E present at blastoderm stage, focus was placed on targets expressed later in embryogenesis. These include the hindgut-specific expression of vvl, the expression of trachealess (trh) and knirps (kni) in the tracheal placodes, and the dynamic expression of socs36E throughout the embryo (Arbouzova, 2006).
First, the effect of Ken was addressed on trh, whose expression precedes the formation of the tracheal pits in the embryonic segments T2 to A8. Levels of trh are greatly reduced in embryos uniformly misexpressing Ken driven by the daughterless-GAL4 (da-GAL4) line. Many tracheal placodes express little or no trh, and tracheal pits fail to form even in the presence of residual trh. Similar effects are seen in updOS1A mutant embryos lacking all pathway activity. Likewise, downregulation of Kni expression is also observed in embryos misexpressing ken. These results show that both endogenous trh and kni are downregulated by ectopically expressed Ken (Arbouzova, 2006).
Whether Ken can modulate the expression of socs36E, a Drosophila homolog of mouse SOCS-5, was tested. socs36E expression closely mirrors that of upd, showing JAK/STAT pathway-dependent upregulation in segmentally repeated stripes, tracheal pits, and the hindgut. By contrast to trh and kni, ectopically expressed Ken does not affect any aspect of socs36E transcription. However, controls expressing a dominant-negative form of the pathway receptor DomeΔCyt, using the same Gal4 driver line, show a strong downregulation of socs36E, an effect reproduced by the complete removal of all JAK/STAT pathway activity by the updOS1A allele. Taken together, these results illustrate that ectopic expression of Ken during Drosophila development is sufficient to downregulate the expression of only a subset of putative JAK/STAT pathway target genes (Arbouzova, 2006).
As part of this analysis, modulation of vvl by Ken was tested. In wild-type embryos, vvl is expressed in the developing trachea and lateral ectoderm (in a JAK/STAT-independent manner) and in the hindgut of stage 12–14 embryos, where it requires JAK/STAT signaling. In updOS1A mutants, no vvl expression in the hindgut can be detected, indicating that this locus is a target of pathway activation. When Ken is uniformly misexpressed throughout the embryo, vvl expression is no longer detectable in the hindgut. Thus vvl, like trh and kni, can be a target of Ken-mediated repression (Arbouzova, 2006).
Having established that ectopic Ken is sufficient to downregulate vvl in the hindgut, whether endogenous Ken performs a similar role was determined. One overlap between ken expression and regions known to require JAK/STAT signaling are the developing posterior spiracles, structures in which both the pathway ligand upd and ken are simultaneously expressed. However, vvl is never detected in the posterior spiracle primordia in wild-type embryos, despite JAK/STAT pathway activity induced by upd expression in these tissues. Intriguingly, in a heteroallelic combination of the strongest kenk11035 allele and Df(2R)Chig320, vvl transcript was detected not only in its normal expression domain within the hindgut but also in the posterior spiracles. This ectopic expression is initially detected from late stage 13 and rapidly strengthens during stage 14–15. When kenk11035/Df(2R)Chig320 embryos simultaneously mutant for the amorphic updOS1A allele were analyzed, upregulation of vvl in the presumptive posterior spiracles was never observed at the stage by which ectopic vvl expression was first detected in the ken mutant embryos. At later stages, JAK/STAT pathway activity is required for posterior spiracle morphogenesis, posterior spiracles do not form, and upregulated vvl is not present (Arbouzova, 2006).
These results demonstrate that Ken is not only sufficient to downregulate the JAK/STAT pathway-dependent expression of vvl in the hindgut, but its endogenous expression is also necessary for vvl repression in the posterior spiracles. In ken mutants, ectopic vvl expression in the posterior spiracles results from a derepression of endogenous STAT92E activity (Arbouzova, 2006).
The overlap between the consensus sequences bound by STAT92E and Ken, together with the analysis of reporters containing STAT+/Ken+ and STAT+/Ken− binding sites, indicate that Ken is likely to selectively regulate only a subset of JAK/STAT target genes. In this model, some target genes are regulated by binding sites compatible with both STAT92E and Ken, while others contain sequences to which only STAT92E can associate. While the DNA binding site is critical in cell-culture systems, similar proof is more difficult to establish in vivo. In particular, only a limited number of JAK/STAT pathway target genes have been rigorously demonstrated to require STAT92E binding in vivo (Arbouzova, 2006).
Although studied in some detail, the regulatory domains controlling vvl expression in the developing hindgut have not been identified. Therefore, although these results predict that such a domain would contain STAT+/Ken+ binding sequences, further analysis is required to confirm this hypothesis. By contrast, the regulatory domain of socs36E required to drive gene expression in the blastoderm, tracheal pits, and hindgut comprises a 350 bp region containing three STAT+/Ken+ and two STAT+/Ken− binding sites. Although not conclusive, the presence of STAT92E-exclusive sites in this region may explain the inability of Ken to downregulate socs36E in vivo (Arbouzova, 2006).
The findings also draw a parallel between Drosophila Ken and BCL6. The data presented demonstrate that both proteins show similar abilities to bind DNA and to mediate transcriptional repression with some evidence also linking BCL6 to JAK/STAT signaling as described here. Taken together, these similarities suggest that Ken and BCL6 represent functional orthologs of one another. Given this evolutionary conservation, it is tempting to speculate that the selective regulation of JAK/STAT pathway target genes is also conserved and may represent a general mechanism by which the pathway is modulated to elicit diverse developmental roles in vivo. Although many STAT targets undoubtedly remain to be identified, it will be intriguing to see which may also be coregulated by Ken/BCL6-dependent mechanisms (Arbouzova, 2006).
The JAK/STAT pathway was first identified in mammals as a signaling mechanism central to hematopoiesis and has since been shown to exert a wide range of pleiotropic effects on multiple developmental processes. Its inappropriate activation is also implicated in the development of numerous human malignancies, especially those derived from hematopoietic lineages. The JAK/STAT signaling cascade has been conserved through evolution and although the pathway identified in Drosophila has been closely examined, the full complement of genes required to correctly transduce signaling in vivo remains to be identified. A dosage-sensitive dominant eye overgrowth phenotype caused by ectopic activation of the JAK/STAT pathway was used to screen 2267 independent, newly generated mutagenic P-element insertions. After multiple rounds of retesting, 23 interacting loci that represent genes not previously known to interact with JAK/STAT signaling have been identified. Analysis of these genes has identified three signal transduction pathways, seven potential components of the pathway itself, and six putative downstream pathway target genes. The use of forward genetics to identify loci and reverse genetic approaches to characterize them has allowed us to assemble a collection of genes whose products represent novel components and regulators of this important signal transduction cascade (Mukherjee, 2006).
Cell cycle proteins: The screen identified genes responsible for the modification of the overgrown eye phenotype associated with P{w+, GMR-updδ3'}.
The eye overgrowth induced by P{w+, GMR-updδ3'} results from additional rounds of mitosis in eye-imaginal disc cells anterior to the morphogenetic furrow. Despite the ectopic JAK/STAT pathway activation caused by the misexpression of upd, these cells are patterned essentially normally and go on to form an increased number of ommatidia in the P{w+, GMR-updδ3'} eye disc. Despite this proliferation-dependent phenotype, core cell cycle regulatory proteins failed to show consistent interactions when assayed as part of a candidate approach. While unexpected, this result suggests that the core cell cycle regulatory proteins do not represent components that become rate limiting in the proliferative environment tested (Mukherjee, 2006).
Despite the lack of interaction with core cell cycle components, alleles of did, trbls, and Mob1 were identified as modifiers of the overgrown eye phenotype. Indeed, homozygous did mutants have been described as having small imaginal discs, and a phenotype similar to that is observed in hopM13 mutant third instar larval discs. While not central to cell cycle progression, these loci appear to be involved in its regulation and may imply that the interaction between JAK/STAT signaling and cellular proliferation is indirect (Mukherjee, 2006).
Of particular interest are the inconsistent interactions observed between Cdk4 alleles. Although cdk4 represents the only Drosophila component of the cell cycle machinery proposed to interact with the JAK/STAT pathway, the assay identified only one of the three alleles tested as a weak suppressor of the eye overgrowth phenotype. Previous studies did not utilize loss-of-function experiments but rather utilized the converse approach. When misexpressed by a P{w+, GMR-Gal4} driver, the coexpression of P{w+, UAS-CycD}, P{w+, UAS-Cdk4}, and P{w+, UAS-upd} dramatically enhanced the eye overgrowth phenotype over that mediated by P{w+, UAS-upd} or P{w+, UAS-CycD} and P{w+, UAS-Cdk4} alone. Although it is possible that loss of a single copy of the cdk4 locus does not reduce protein levels below a rate-limiting threshold, the inconsistency of interactions produced by multiple cdk4 alleles is puzzling and true existence or nature of any potential interaction between JAK/STAT signaling and endogenous Cdk4 remains to be established (Mukherjee, 2006).
Transcription factors and coregulators: A number of transcription factors were identified as interacting loci in the screen. One of these is the Drosophila homolog of the nuclear factor of activated T-cells (NFAT), a locus originally identified as an inducer of cytokine gene expression. Intriguingly, it has been shown that human NFAT, in conjunction with NF-kappaB, AP-1, and STATs, represents factors involved in mediating cytokine and T-cell-receptor-induced interferon-γ signaling. Intriguingly, activation of these transcription factors results in the production of numerous intrinsic antiviral factors in the vertebrate system, a role that has also been shown to depend on JAK/STAT signaling within Drosophila fat-body cells. Although further analysis of this interaction is required, this is the first report that suggests an evolutionarily conserved link between NFAT and JAK/STAT signaling in Drosophila (Mukherjee, 2006).
C-terminal binding protein (CtBP), a transcriptional corepressor previously characterized as an enhancer of the Drosophila JAK/STAT pathway, was also identified in the screen. While not all alleles of CtBP show consistent interaction with P{w+, GMR-updδ3'}, cell culture assays utilizing dsRNA-mediated knockdown imply that CtBP is a component of the JAK/STAT pathway, which acts as a positive regulator of signaling. In addition, an independent genomewide RNAi-based screen for JAK/STAT pathway interactors also identified dsRNAs targeting CtBP as a suppressor of pathway signaling. Finally, an upregulation of CtBP transcript is observed in P{w+, GMR-updδ3'} eye discs compared to wild-type eyes. Given the results from cell-based assays and in situ analysis, it appears most likely that CtBP does indeed represent a positive regulator of JAK/STAT pathway activity. This finding is particularly surprising, given the previously identified role for CtBP as a transcriptional repressor, which, in combination with the Groucho corepressor, is involved in repressing Su(H)-mediating expression of Notch pathway target genes. The significance of this result, however, remains to be determined and it is conceivable that the observed interaction with the eye overgrowth phenotype represents an indirect effect, possibly via interaction with Notch pathway signaling activity (Mukherjee, 2006).
Extracellular proteins: One aspect of the screen undertaken is the paracrine mode of Upd signaling required for cellular overproliferation. In the P{w+, GMR-updδ3'} eye, the region of upd expression is spatially separate from the domain in which increased levels of cellular proliferation are observed and the ligand must therefore be able to move to and activate the pathway in neighboring cells. Although it has been shown that Unpaired represents a secreted extracellular signaling molecule that is both post-translationally glycosylated and able to associate with the extracellular matrix (ECM), very little is known regarding the mechanisms regulating these processes (Mukherjee, 2006).
One class of molecules previously shown to be involved in the extracellular trapping and movement of signaling ligands is the heparan sulfate proteoglycans (HSPGs) Dally, Dally-like, Perlecan, and Syndecan. These molecules, and their extensive post-translational modifications, not only play important roles in providing shape and biomechanical strength to organs and tissues, but also have been shown to be required for the transduction of signaling by the Wingless, Hedgehog, and the FGF-like ligands Heartless and Breathless. Despite the significance of HSPGs for the transduction of these ligands, mutations in the HSPGs themselves, as well as mutations in the HSPG-modifying enzymes sugarless and sulphateless, do not appear to interact with the eye overgrowth phenotypes associated with P{w+, GMR-updδ3'} and suggest that Upd is likely to interact with the ECM via different mechanisms. One potential component of this alternative mechanism identified in the screen is Tenascin-major (Ten-m). Ten-M, also known as odd Oz, encodes an extracellular adhesion molecule that was also classified as a component of the JAK/STAT pathway in the tissue-culture-based paracrine signaling assay. Although the tissue culture results imply a direct function of the molecule in pathway signaling, further analysis of the role of Ten-m in controlling the secretion and/or movement of Upd remains to be determined in vivo (Mukherjee, 2006).
Signaling pathways: The Drosophila eye is dispensable in a laboratory environment and sensitized genetic screens that compromise its function have proven to be powerful tools for the identification of signal transduction pathway components. Drosophila eye development is, however, a complex process involving multiple signal transduction pathways including EGFR, Hh, Notch, Dpp, and Wingless. A number of examples of interactions between these pathways and JAK/STAT signaling have been described. For example, a gradient of four-jointed in the developing eye disc is determined by the coordinated activities of Notch, Wingless, and JAK/STAT pathways. Also, at the posterior dorso/ventral border of the eye, Notch and eye gone (eyg) have been shown to cooperatively induce expression of upd, which then acts to promote cell proliferation. Consistent with these complex interactions, the screen identified Bunched (bun), a member of the Dpp signal transduction pathway, and Bearded (brd), a member of the Notch signaling pathway. bunched is a transcription factor that genetically interacts with dpp. Strikingly, Dpp pathway components have previously been reported as modulators of the P{w+, GMR-updδ3'} eye phenotype, with hypomorphic alleles of dpp and Mothers against dpp (Mad) representing strong suppressors of eye overgrowth. Similar interactions in mammalian systems have identified the synergistic activity of STAT3 and Smad1 in the differentiation of astrocytes from their progenitor cells. These proteins, however, do not physically interact, but bind to p300/CBP to promote the transactivation of target genes (Mukherjee, 2006).
The screen also identified mth-like8, a seven-pass trans-membrane protein with predicted G-protein-coupled receptor activity. Although expression of mth-like8 changes in response to JAK/STAT pathway activation, an in-depth analysis of its interaction remains to be undertaken (Mukherjee, 2006).
Neuroblasts (NBs) generate a variety of neuronal and glial cells in the central nervous system of the Drosophila embryo. These NBs, few in number, are selected from a field of neuroepithelial (NE) cells. In the optic lobe of the third instar larva, all NE cells of the outer optic anlage (OOA) develop into either NBs that generate the medulla neurons or lamina neuron precursors of the adult visual system. The number of lamina and medulla neurons must be precisely regulated because photoreceptor neurons project their axons directly to corresponding lamina or medulla neurons. This study shows that expression of the proneural protein Lethal of scute [L(1)sc] signals the transition of NE cells to NBs in the OOA. L(1)sc expression is transient, progressing in a synchronized and ordered 'proneural wave' that sweeps toward more lateral NEs. l(1)sc expression is sufficient to induce NBs and is necessary for timely onset of NB differentiation. Thus, proneural wave precedes and induces transition of NE cells to NBs. Unpaired (Upd), the ligand for the JAK/STAT signaling pathway, is expressed in the most lateral NE cells. JAK/STAT signaling negatively regulates proneural wave progression and controls the number of NBs in the optic lobe. These findings suggest that NBs might be balanced with the number of lamina neurons by JAK/STAT regulation of proneural wave progression, thereby providing the developmental basis for the formation of a precise topographic map in the visual center (Yasugi, 2008).
NE cells are programmed to differentiate into NBs from the medial edge of
the developing optic lobe. The wave of differentiation progresses
synchronously in a row of cells from medial to lateral optic lobe sweeping
across the entire NE sheet; it is preceded by the transient expression of the
proneural gene l(1)sc. As the NBs at the medial edge are oldest and
the more lateral ones are youngest, developmental process of medulla neurons
can be viewed as an array of progressively aged cells across optic lobe
mediolaterally. This contrasts with NB formation in the embryonic CNS in which
a small number of cells are selected from NE cells to become NBs, leaving the
majority of NE cells to develop into non-neural cells. The optic lobe
proneural wave is reminiscent of the morphogenetic furrow that moves across
the developing eye imaginal disc. The morphogenetic furrow is the site where
differentiation from neuroepithelium to photoreceptor neurons is initiated. The
progression is driven by the secreted Hh expressed in the differentiated
photoreceptor cells. By contrast, the proneural wave still progresses even when
NB differentiation is impaired, suggesting that its progression is not driven
by a factor emanating from differentiated NBs. No
progression-defective phenotypes were observed when Hh or Decapentaplegic (Dpp) signaling was reduced. The model is favored that the proneural wave
progression is driven by an intrinsic mechanism such as a segmentation clock
and is negatively regulated by JAK/STAT pathway. As the JAK/STAT
ligand Upd is expressed only by the most lateral NE cells, proliferation of
the NE cells moves the source of ligand laterally and as a consequence
releases more medial NE cells from negative regulation and allows the
proneural wave to progress laterally. Alternatively, distribution of the Upd
ligand and/or the response to Upd changes as the NE cells age as graded
10xSTAT-GFP activities are more prominent in the early stage. Non-autonomous
action of JAK/STAT signal indicates that it does not directly regulate L(1)sc
expression and there are second signal(s) that regulate the expression of
L(1)sc under the control of JAK/STAT signal (Yasugi, 2008).
Three out of the four AS-C genes [sc, l(1)sc and
ase] are expressed during medulla neurogenesis. l(1)sc is
expressed in NE cells and ase in NBs, while sc is expressed
both in NE cells and NBs. Deleting all AS-C genes causes as significant
delay as da in NB formation but does not completely eliminate NB
formation, suggesting that Da-dependent proneural gene activities are required
for timely onset of NB formation. Mutation for sc or ase
alone does not affect NB formation, but the simultaneous deletion of
sc and l(1)sc causes the delay in NB formation and the
additional deletion of ase further delays NB formation. ase
expression is not altered in the absence of l(1)sc and
l(1)sc is not altered in the absence of ase, indicating that
l(1)sc and ase both contribute to the differentiation from
NE cells to NBs. Although the contribution of Sc cannot be formally excluded,
the highly specific expression pattern led to the inference that L(1)sc plays a
major role in the proneural wave (Yasugi, 2008).
JAK/STAT signaling is known to regulate stem cell maintenance in the adult
germline of Drosophila. In the male testis, germline stem cells (GSCs)
attach to a cluster of somatic support cells at the tip (hub) of the testis.
When a GSC divides, the daughter retaining contact with the hub maintains
self-renewing GSC identity, while the other daughter differentiates into
gonialblast. Upd is specifically expressed in the hub cells and activates
JAK/STAT signal in the GSCs to maintain stem cell state. In
the female ovary, JAK/STAT signaling is required in the somatic escort stem
cells whose daughters encase developing cysts.
This study shows that in the optic lobe development, JAK/STAT signaling maintains
NE cells in an undifferentiated state. It is suggested that a common mechanism
operates in both these developmental systems. Loss of Hop or Stat92E function
decreases number of stem cells and ectopic expression of Upd results in over
proliferation of undifferentiated cells. The cell fate may be determined by
the distance of the cells from the source of ligand; the cells farther from
the source commence to differentiate (Yasugi, 2008).
In the vertebrate CNS, NE cells first proliferate by symmetric cell
divisions and differentiate into neurons and glia in later developmental
stages. JAK/STAT
signaling has been implicated in maintenance of neural precursor cells, but
there is no clear evidence that those cells are in the same developmental
stage as described in this study for Drosophila. Further study of JAK/STAT
signaling will reveal whether a common mechanism underlies stem cell
development in both Drosophila and vertebrates, and should give new
insights into vertebrate CNS neurogenesis (Yasugi, 2008).
Development of a precise topographic map (retinotopic map) in
Drosophila is known to involve regulation of lamina neuron
development with respect to the incoming R axons.
The lateral NE sheet is continuous with a groove called the lamina furrow
where NE cells are arrested at G1/S phase. The
arriving R axons deliver Hh and liberate the arrested NE cells to proliferate
and develop into lamina neuron precursors. And,
thus, R axons can induce the development of their synaptic partners in their
vicinity to balance the number of R axonal termini and lamina neurons.
However, medulla development does not depend on inputs from the R axons in the
early phase. This study shows that both lamina and medulla neurons are
derived from the continuous NE sheet. Large clones of cells mutant for the
JAK/STAT signaling cause immature proliferation of medulla NBs at the expense
of lamina neurons, suggesting that the number of NE cells serves as the
limiting factor to generate precursors for lamina and medulla neurons. Thus,
the number of medulla neurons is roughly regulated at the level of NBs whose
generation might be balanced indirectly with the number of lamina neurons
through regulating proneural wave progression by JAK/STAT signaling. JAK/STAT
signaling therefore plays an important role in the formation of a precise
retinotopic map in the visual center (Yasugi, 2008).
Aging is characterized by compromised organ and tissue function. A decrease in stem cell number and/or activity could lead to the aging-related decline in tissue homeostasis. This study analyzed how the process of aging affects germ line stem cell (GSC) behavior in the Drosophila testis; significant changes within the stem cell microenvironment, or niche, occur that contribute to a decline in stem cell number over time. Specifically, somatic niche cells in testes from older males display reduced expression of the cell adhesion molecule DE-cadherin and a key self-renewal signal unpaired (upd). Loss of upd correlates with an overall decrease in stem cells residing within the niche. Conversely, forced expression of upd within niche cells maintains GSCs in older males. Therefore, these data indicate that age-related changes within stem cell niches may be a significant contributing factor to reduced tissue homeostasis and regeneration in older individuals (Boyle, 2007. Full text of article).
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date revised: 20 December 2009
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