The Interactive Fly
Genes involved in tissue and organ development
Maintaining transcriptional silence in pole cells, the germline cell precursors
Segmental origin of genital discs
Genes controlling germ cell migration and embryonic gonad formation
The endoderm specifies the mesodermal niche for the germline in Drosophila via Delta-Notch signaling
Genes expressed in or affecting the ovaries and testes
Maintaining transcriptional silence in pole cells, the germline cell precursors
In Drosophila, the germline precursor cells, i.e. pole cells, are formed at the posterior of the embryo. As observed for newly formed germ cells in many other eukaryotes, the pole cells are distinguished from the soma by their transcriptional quiescence. To learn more about the mechanisms involved in establishing quiescence, a potent transcriptional activator, Bicoid (Bcd), was ectopically expressed in pole cells. Bcd overrides the machinery that downregulates transcription, and activates not only its target gene hunchback but also the normally female specific Sex-lethal promoter, Sxl-Pe, in the pole cells of both sexes. Unexpectedly, the terminal pathway gene torso-like is required for Bcd-dependent transcription. However, terminal signaling is known to be attenuated in pole cells, and this raises the question of how this is accomplished. Evidence is presented indicating that polar granule component (pgc) is required to downregulate terminal signaling in early pole cells. Consistently, pole cells compromised for pgc function exhibit elevated levels of activated MAP kinase and premature transcription of the target gene tailless (tll). Furthermore, pgc is required to establish a repressive chromatin architecture in pole cells (Deshpande, 2004).
The germline of Drosophila is derived from a special group of cells called pole cells that are formed during early embryonic development. The Drosophila embryo initially develops as a syncytium of rapidly dividing nuclei that undergo multiple rounds of synchronized mitotic cycles. Prior to the tenth division cycle, several nuclei migrate into the specialized cytoplasm or pole plasm at the posterior of the embryo. These nuclei cellularize precociously and these newly formed cells divide two or three times to produce ~30-35 germline precursor cells. The remaining nuclei migrate to the surface of the embryo at nuclear division cycle 10-11. They then undergo several more synchronous divisions and cellularize at the end of nuclear cycle 14 to form the cellular blastoderm (Deshpande, 2004 and references therein).
In addition to their earlier cellularization and slower rate of mitosis, pole cells differ in their transcriptional activity. Somatic nuclei substantially upregulate RNA polymerase II transcription after they migrate to the surface of the embryo. The activation of zygotic gene expression is essential for these nuclei to respond appropriately to the maternal pathways that assign positional information along the axes of the embryo. By contrast, pole cell nuclei shut down RNA polymerase II transcription when they enter the pole plasm and they then remain transcriptionally quiescent until much later stages of embryogenesis. Transcriptional quiescence is a hallmark of germline precursor cells in many organisms. For example, in C. elegans, RNA polymerase II transcription is repressed in the germ cell lineage by the product of the pie-1 gene. Transcriptional inactivity appears to be crucial in establishing germ cell identity as mutations in pie-1 switch the fate of these cells to that of a somatic lineage (Deshpande, 2004 and references therein).
A number of maternally derived gene products are likely to contribute to transcriptional quiescence in the pole cells of Drosophila. One of these is Germ cell less (Gcl), a component of the germ plasm that is necessary for the formation of pole cells. gcl appears to be involved in the establishment of transcriptional quiescence and in embryos lacking gcl activity, newly formed pole buds are unable to silence the transcription of genes such as sisterless-a and scute. Conversely, when Gcl protein is ectopically expressed in the anterior of the embryo it can downregulate the transcription of terminal group genes such as tailless (tll) and huckebein (Leatherman, 2002). A second maternally derived gene product involved in transcriptional quiescence is Nanos. In the soma, Nanos, together with Pumilio, plays a key role in posterior determination by blocking the translation of maternally derived hunchback (hb) mRNA. Nanos (Nos) also plays a role in down-regulating transcription in pole cells, and in embryos produced by nos mutant mothers: genes that are normally active only in somatic nuclei are inappropriately transcribed in pole cells. These include the pair-rule genes fushi tarazu and even skipped, and the somatic sex determination gene Sex-lethal (Deshpande, 2004 and references therein).
The global effects of nos and gcl mutations on RNA polymerase II activity in pole cells are analogous to those seen in pie-1 mutants in C. elegans. In pie-1 mutants, genes that are normally expressed only in somatic lineages are turned on in the germ cell lineage. In wild-type C. elegans embryos, the inhibition of transcription in the germ cell lineage is correlated with a marked reduction in phosphorylation of the CTD repeats of the large subunit of RNA polymerase II (Seydoux, 1997). The CTD repeats are phosphorylated when polymerase is transcriptionally engaged. PIE-1 protein may prevent transcription by inhibiting this modification. As in C. elegans, the RNA polymerase II CTD repeats are underphosphorylated in the pole cells of wild-type Drosophila embryos. In the pole cells of gcl and nos mutant embryos, however, the level of CTD phosphorylation is elevated (Leatherman, 2002; Deshpande, 2004 and references therein).
Previous studies have shown that when a heterologous transcriptional activator, GAL4-VP16, is expressed in pole cells, it is unable to activate transcription of target gene(s) (Van Doren, 1998). This finding suggests that even if a potent activator were to be produced in pole cells, it would not be able to overcome the inhibition of the basal transcriptional machinery by gcl, nos and other factors. However, since GAL4-VP16 is a chimera of a yeast DNA-binding domain and a mammalian activation domain, an alternative possibility is that co-factors essential for its activity may be absent or inactive in Drosophila pole cells. For these reasons, tests were performed to see whether a transcription factor that is normally present and active in the somatic cells of early Drosophila embryos can promote the transcription of target genes when inappropriately expressed in pole cells. The homeodomain protein Bicoid (Bcd), which activates the zygotic transcription of hb and other genes specifying anterior development, was tested. A Bcd protein gradient is generated in precellular blastoderm embryos from the translation of maternal mRNA localized at the anterior pole. Although Bcd is not present in the posterior of wild-type embryos, increasing the bcd gene dose results in expansion of the gradient toward the posterior and a concomitant change in the pattern of zygotic gene expression. This result suggests that co-factors crucial for Bcd function are likely to be ubiquitous (Deshpande, 2004 and references therein).
Ectopic expression of Bcd in pole cells can induce the transcription of the bcd target gene hb. In addition to activating hb transcription, Bcd protein perturbs the migration of the pole cells to the primitive somatic gonad and causes abnormalities in cell cycle control. These effects on germ cell development resemble those observed in embryos from nos mutant females. Moreover, as in the case of nos- pole cells, the Sxl promoter Sxl-Pe is also turned on in pole cells by Bcd in a sex-nonspecific manner. Surprisingly, transcriptional activation in pole cells by Bcd requires the activity of the terminal signaling system. This observation is unexpected, since previous studies have established that the transcription of a downstream target gene of the terminal pathway, tailless (tll) is shut down completely in pole cells. Moreover, the doubly phosphorylated active isoform of MAP kinase ERK, which serves as a sensitive readout of the terminal pathway, is nearly absent in pole cells. Taken together, these findings argue that the activity of terminal signaling pathway in pole cells of wild-type embryos must be substantially attenuated, but not shut off completely. What mechanisms are responsible for downregulating terminal signaling in the presumptive germline? Evidence indicates that polar granule component (pgc) functions to attenuate the terminal pathway in newly formed pole cells. pgc encodes a non-translated RNA that is localized in specialized germ cell-specific structures called polar granules (Nakamura, 1996). Loss of pgc function in newly formed pole cells results in the ectopic phosphorylation of ERK and the activation of the ERK dependent target gene tll. pgc is required to block the establishment of an active chromatin architecture in pole cells (Deshpande, 2004).
Thus Bcd protein expressed from a bcd-nos3'UTR transgene (the 3' UTR of nos serves to localize the bcd message to pole cells) can activate the transcription of its target gene hb in pole cells, overcoming whatever mechanisms are responsible for transcriptional quiescence. In addition to activating transcription of hb, Bcd has other phenotypic effects. It prevents the pole cells from properly arresting their cell cycle and disrupts their migration to the somatic gonad. Because similar defects in pole cell development can be induced by the inappropriate expression of Sxl protein in these cells, one plausible hypothesis is that Bcd not only activates the hb promoter, but also turns on the Sxl establishment promoter, Sxl-Pe. Consistent with this idea, the Sxl-Pe:lacZ reporter is turned on in the pole cells of male and female bcd-nos 3' UTR embryos and Sxl protein accumulates in these cells. Although previous studies indicate that Sxl-Pe is responsive to Bcd, it is somewhat surprising that Sxl-Pe is not only inappropriately turned on in pole cells by Bcd, but that it is activated in both sexes. This suggests that Bcd activation of Sxl-Pe in pole cells must proceed by a mechanism that bypasses the X/A chromosome counting system which controls Sxl-Pe activity in the soma. It is interesting to note that the activation of Sxl-Pe in pole cells in the absence of nos function also seems to depend upon a mechanism(s) that circumvents the X/A chromosome counting system (Deshpande, 2004).
That Bcd protein depends upon other ancillary factors to turn on transcription in pole cells is demonstrated by the requirement for tsl function in the activation of both the hb and Sxl-Pe promoters. tsl is a component of the maternal terminal signaling pathway that activates the zygotic genes, tll and huckebein (hkb), at the poles of the embryo. In addition, the terminal pathway has opposing effects on the expression of bcd-dependent gap genes. At the anterior pole, where terminal signaling activity is highest, Bcd targets such as hb and orthodenticle (otd) are repressed. At a distance from the anterior pole, where both the concentration of Bcd protein and the strength of the terminal signaling cascade is much lower, the terminal pathway has an opposite, positive effect on hb and otd expression. Two mechanisms are thought to account for the positive effects of the terminal pathway on bcd target genes: (1) Bcd is a direct target for phosphorylation by the terminal signaling cascade; (2) regulatory regions of bcd target genes have sites for other transcription factors whose activity can be directly modulated by the terminal system (Deshpande, 2004).
The concentration of Bcd protein produced by the bcd-nos 3' UTR transgene in pole cells is much less than it is at the anterior pole. Similarly, the activity of the terminal signaling cascade in pole cells is much reduced compared with that in the somatic nuclei at the anterior and posterior poles. Thus, in both of these respects, the conditions in the bcd-nos 3' UTR pole cells would appear to most closely approximate those in the region of the embryo where the terminal signaling cascade potentiates rather than inhibits Bcd activity. This would explain why activation of transcription in pole cells by Bcd depends on the terminal signaling pathway and why in this particular instance this pathway does not antagonize the activity of the ectopically expressed Bcd protein (Deshpande, 2004).
The fact that the terminal pathway can function in pole cells, yet does not turn on its target gene tll indicates that the activity of this pathway is attenuated in the germline. It seems likely that several different mechanisms may be responsible for preventing pole cells from responding to the terminal pathway and turning on tll transcription. One mechanism appears to be an inhibition of the signaling cascade itself. In the posterior and anterior soma of pre-cellular blastoderm embryos, the terminal signaling cascade directs the phosphorylation of the MAP kinase ERK. While phosphorylated ERK can also be detected in wild-type pole cells, the amount of activated kinase is much less than in the surrounding soma. Consistent with this observation, potentiating the terminal system using either a gain-of-function torso receptor mutant or by expressing elevated levels of the receptor in pole cells using a torso transgene (which has the nos 3' UTR) had only a small effect on the activity of a tll-lacZ reporter in the germline. By contrast, gain-of-function torso mutation substantially upregulates the tll reporter in the soma (Deshpande, 2004).
To identify factors that could be involved in repressing the terminal pathway in pole cells, three genes, nos, gcl and pgc, were examined that are known to play an important role in the early development of the germline and have been implicated in transcriptional quiescence. Of these three, only pgc appears to have significant effects on the terminal signaling pathway in pole cells. The expression of a tll reporter is turned on in pole cells of embryos deficient in pgc activity. That this is due at least in part to a failure to properly attenuate the terminal signaling pathway in the germline is suggested by the fact that the level of activated ERK is greatly elevated in pgc pole cells compared with wild type. Although these findings implicate pgc in downregulating the terminal pathway, how this is accomplished and whether pgc has a direct rather than an indirect role in this process remains to be determined. In addition, these studies indicate that pgc has functions in addition to attenuating this signaling cascade: (1) it was found that there are abnormalities in the formation of pole cells in pgc embryos and Vasa-positive 'cells' are observed in cycle 9-10 embryos at abnormal locations; (2) the loss of pgc activity may lead to the inappropriate activation of genes in addition to tll. Two markers for global transcriptional activity, CTD phosphorylation and histone H3 K4 methylation, are present in pole cells of pgc embryos (Deshpande, 2004).
The results also suggest that multiple and interrelated levels of regulation are responsible for ensuring transcriptional quiescence in the pole cells. For example, Sxl-Pe can be upregulated by the terminal pathway in the soma and requires this pathway to be activated by Bcd in pole cells. However, this promoter is not activated in pole cells in the absence of pgc function. Thus, the activation of the terminal signaling cascade in pole cells is not sufficient in itself to induce Sxl-Pe. This suggests that mechanisms are in place in pgc pole cells that would override any effects of activated ERK on Sxl-Pe activity. Similarly, although loss of nos activity leads to the activation of Sxl-Pe in pole cells, and the upregulation of tll in the posterior soma, the tll promoter is not turned on in nos pole cells. It is presumed that tll is not activated in pole cells because it requires the terminal system that still remains attenuated in nos pole cells. Redundancy is also suggested by the finding that although the loss of gcl leads to the expression of the X chromosome counting genes sis-a and scute in pole cells (Leatherman, 2002), Sxl-Pe is not activated, suggesting that nos function is sufficient to keep Sxl-Pe off in gcl mutant pole cells even though several X chromosome counting genes are activated. Similarly, no obvious effect was observed of nos mutations on scute expression in pole cells. This implies that gcl and nos may be responsible for repressing the transcription of different sets of genes (Deshpande, 2004).
Finally, although transcription is upregulated in pgc pole cells between nuclear cycles 9/10-13, a high level of transcriptional activity is not maintained in the pole cells that are present by the time the cellular blastoderm is formed. The tll reporter is turned off, and both CTD phosphorylation and histone H3 K4 methylation disappear. One possible interpretation of this finding is that pgc has an early function in establishing transcriptional quiescence, but is not required after nuclear cycle 13 because of the activity of other factors such nos or gcl. However, since the number of pole cells at cellularization is reduced compared with the number present earlier, it also possible that the only pole cells that remain are the ones in which the amount of pgc activity is sufficient to establish some degree of transcriptional repression. Further studies with bona fide null alleles will be required to resolve this question, and to understand how pgc functions during pole cell formation and germ cell determination (Deshpande, 2004).
Segmental origin of genital discs
Each of the somatic cell types of the gonad arises from mesodermal cells that constitute the embryonic gonad. Using markers for the precursors of the somatic cells of the gonad, five discrete steps have been identified in gonadal development:
The functions of the homeotic genes abdominal A and Abdominal B are both required for the development of gonadal precursors. Each plays a distinct role. abd A activity alone specifies anterior gonadal precursor fates, whereas abd A and Abd B act together to specify a posterior subpopulation of gonadal precursors. Once specified, gonadal precursors born within posterior parasegments move to the site of gonad formation. The proper regional identities, as established by homeotic gene function, are required for the arrest of migration at the correct position. abd A is required in a population of cells within parasegments 10 and 11 that partially ensheath the coalescing gonad. Mutations in iab-4, a distal enhancer element, abolish expression of abd A within these cells, blocking the coalescence of the gonad (Boyle, 1995).
The genital disc develops from three embryonic abdominal segments: A8, A9 and A10. Male and female genital discs each contain a repressed genital primordium of the opposite sex that does not form any adult structure. The female genitalia develops from embryonic segment A8 and the male genitalia develops from embryonic segment A9. The anal primordium is developed from segment A10 (Freeland, 1996).
The female genital primordium gives rise to the 8th tergite and both the external and the internal genitalia, including the vaginal plates with three types of bristles on them (the long bristle, the thorn bristle, and the sensilla trichodea), the dorsal and ventral vulva, the uterus, the seminal receptacle the parovaria, the spermatathecae and the ovaduct. These female genital structures map to the thick ventral epithelium of the disc, except for the parovaria, which maps to the dorsal epithelium. The anal plate gives rise to the dorsal and ventral anal plates as well as the hindgut. The anal plates are derived from the thickened posterior part of the dorsal epithellium (Chen, 1997).
The male genital disc gives rise to both the external and internal genitalia, including the phragma, the genital arch, the lateral plates, the claspers, the penis apparatus, the sperm pump, the ejaculatory duct, the paragonia and the vas deferens. These male genital structures map to the anterior lobe and the ventral lateral regions of the disc. The anal plate gives right to left and right anal plates, as well as the hind gut. The anal plates are derived from the thickened posterior regions of the dorsal epithelium (Chen, 1997)
The segmental organization and gene expression of each disc is directly related to the segmental organization of the embryonic segments from which they are derived. For example, engrailed is expressed in three regions of both male and female discs, reflecting the origin of these discs from three segments (Freeland, 1996).
Genes controlling germ cell migration and embryonic gonad formation
Gonadogenesis in the Drosophila embryo is a complex process involving numerous cellular migratory steps and cell-cell interactions. The mechanisms guiding germ cells to move through, recognize and adhere to specific cell types are poorly understood. In order to identify genes that are required for these processes, extensive mutagenesis of the third chromosome was carried out; this chromosome was then screened for mutations disrupting germ cell migration at any point in embryonic development. Phenotypic analysis of these mutants demonstrates that germ cell migration can be broken down into discrete developmental steps, with each step requiring a specific set of genes. The genes serpent and huckebein are required for the initial step, migration through the midgut. The genes zfh-1, columbus (HMG Coenzyme A reductase) and heartless are required for the second step, attachment to somatic mesoderm. These genes, functioning from the mesoderm, direct the germ cells away from the endoderm and into the mesodermal regions. abdominalA, AbdominalB, trithorax, and trithoraxgleich (trg) are required for the subsequent step, alignment and maintenance of association of germ cells with gonadal mesoderm. Mutations in trg show genetic interactions with homeotic genes, including Ultrabithorax, abdA and AbdB. Flies that are transheterozygous for trg and any of these homeotic genes are semi-viable and often show thoracic abnormalities, suggesting that trg is a new member of the trithorax-group of genes. Mutations in tinman result in a disorganization of germ cell alignment (what would normally be the next developmental step); furthermore, it is known that the somatic gonadal cell precursor marker clift is drastically reduced in tin mutants. It is unclear why tin mutants show such a relatively late germ cell migration defect, given tin's striking effect on expression of gonadal mesoderm markers. Mutations in a novel gene, fear of intimacy (foi), specifically affect the ability of the germ cells and gonadal mesoderm to coalesce into the embryonic gonad, the final step in formation of the gonad. In foi mutants, somatic gonadal cell precursors appear as if they are incapable of making close contacts with one another (Moore, 1998).
Many of these third chromosome genes are involved in the development of gonadal mesoderm, the tissue that associates with germ cells to form the embryonic gonad. Isolated mutations affecting embryonic patterning as well as germ cell migration suggest that the origin of gonadal mesoderm lies within the even-skipped domain of the developing mesoderm. Of those genes located on the third chromosome, fushi-tarazu, odd-paired and hedgehog are required for development of the midgut visceral mesoderm and fat body. The origin of the midgut visceral mesoderm and fat body is to be found within the 'eve domain' of each parasegment. The genetic screen demonstrates that genes required for the development of these tissues are also required for germ cell migration. The germ cell migration defects in these mutants are most likely due to their effect on gonadal mesoderm development (Moore, 1998).
Since a thorough screening of the third chromosome for genes required zygotically for germ cell migration reveals no compelling candidates for genes that function in the germ cells for the many processes that they must execute to form a coalesced gonad, it is likely that these factors are maternally provided to the embryo, and thus could not be identified in a zygotic screen. Indeed, two proteins known to act in germ cells for proper gonad formation, nanos and Polar granule component-1, are both contributed maternally to the oocyte (Moore, 1998 and references).
Interactions between niche cells and stem cells are vital for proper control over stem cell self-renewal and differentiation. However, there are few tissues where the initial establishment of a niche has been studied. The Drosophila testis houses two stem cell populations, which each lie adjacent to somatic niche cells. Although these niche cells sustain spermatogenesis throughout life, it is not understood how their fate is established. This study shows that Notch signaling is necessary to specify niche cell fate in the developing gonad. Surprisingly, these results indicate that adjacent endoderm is the source of the Notch-activating ligand Delta. Niche cell specification occurs earlier than anticipated, well before the expression of extant markers for niche cell fate. This work further suggests that endoderm plays a dual role in germline development. The endoderm assists both in delivering germ cells to the somatic gonadal mesoderm, and in specifying the niche where these cells will subsequently develop as stem cells. Because in mammals primordial germ cells also track through endoderm on their way to the genital ridge, this work raises the possibility that conserved mechanisms are employed to regulate germline niche formation (Okegbe, 2011).
The data reveal that Notch signaling is necessary to specify hub cell fate. A similar conclusion has recently been reached by Kitadate (2010). It is interesting to note that in three well-characterized stem cell-niche systems in Drosophila, including the transient niche for adult midgut progenitors, the female gonad and now the developing male gonad, Notch signaling is directly responsible for niche cell specification. Moreover, Notch has been found to play a role in the maintenance of various mammalian stem cell populations, including neural stem cells, HSCs and hair follicle stem cells. However, owing to difficulty in performing lineage-specific knockouts in these systems, it remains unclear which cells require Notch activity. As the various cases in Drosophila all require direct Notch activation for niche cell specification, perhaps this reveals a conserved role for Notch signaling in other, more complex stem cell systems (Okegbe, 2011).
Notch signaling specifies niche cells in both the male and female Drosophila gonad; however, it is important to note that there are still some differences. For the ovary, only Delta is required to activate the Notch receptor for proper niche cell specification. For the testis, both ligands contribute to the process, although, here too, it appears that Delta is the dominant ligand employed. Interestingly, depleting Delta or (genetically) separating the endoderm from SGPs (somatic gonadal precursors) both led to a 70% reduction in hub cell number, while depleting Serrate yielded a 30% reduction. Perhaps Delta-Notch signaling from the endoderm accounts for two-thirds of hub cell specification, while Serrate-Notch signaling accounts for only one-third of this process. Although the source of Serrate could not be identifed in this study, Kitadate (2010) has shown that Serrate mRNA is expressed from SGPs after gonad coalescence. Perhaps, this late expression accounts for the modest role Serrate plays in hub specification. That study did not explore in detail a potential role for Delta in hub specification, and the current data suggests that that role is carried out at earlier stages, and from outside the gonad proper (Okegbe, 2011).
In the ovary, cells within the developing gonad appear to present the Notch-activating ligand, although it is unclear whether germ cells or somatic cells are the source of Delta. The current data suggests that cells from a distinct germ layer, the endoderm, present Delta to SGPs in the male gonad. These differences may indicate distinct evolutionary control over gonadal niche development between the sexes (Okegbe, 2011).
Although the gonad first forms during mid-embryogenesis, hub cells only become identifiable just prior to hatching of the larvae, some 6 hours later. At that time, hub cells begin to tightly pack at the anterior of the gonad, upregulate several cell adhesion and cytoskeletal molecules (Fascilin 3, Filamin, DN-Cadherin, DE-Cadherin) as well as induce Upd expression and other markers of hub fate. Surprisingly, the current data reveal that most hub cells are specified well before these overt signs of hub cell differentiation, as judged by Notch reporter activation and Notch rescue. Although it was previously thought that SGPs were equivalent at the time of gonad coalescence it is now clear that due to Notch activity, the SGPs are parsed into a group of either hub cells or cyst cells before gonad coalescence occurs (Okegbe, 2011).
Thus, it is believed that a series of steps must occur before the hub can function as a niche. First, the PMG (posterior midgut) presents Delta, leading to Notch activation in some SGPs as they are carried over these endodermal cells during germ band retraction. Activation might be dependent on, for example, length of time in contact with passing PMG cells. At the present time, it is unclear whether all SGPs are activated for Notch (Kitadate, 2010), or only some of them (this study). After gonad coalescence, activated SGPs must then migrate anteriorly. Although it is known that integrin-mediated adhesion is required to maintain the hub at the anterior (Tanentzapf, 2007), no cues have been identified that could guide the migration of the Notch-activated SGPs. Next, as the cells reach the anterior of the gonad they must execute a mesenchymal-to-epithelial transition, as evidenced by the upregulation of cell-adhesion molecules and preferential associations between hub cells. This step occurs independently of the integrin-mediated anchoring at the anterior. Finally, the hub cells must induce Upd expression and recruit neighboring cells to adopt stem cell fate. The apparent delay between the activation of the Notch pathway and the initiation of the hub cell gene expression program might suggest that initiating that hub program first requires that the cells coalesce into an epithelium. Such a mechanism would prevent precocious or erroneous stem cell specification within the gonad (Okegbe, 2011).
Although these data reveal Notch-activated SGPs at all positions within the gonad and that some of these become hub cells, it is unclear how hub cell number is tightly regulated. Potentially, SGP migration over endodermal cells could induce Notch activation among SGPs throughout the forming gonad, potentiating these cells to become hub cells. However, solely relying on that mechanism could lead to the specification of too many hub cells. It appears, though, that specification is regulated by EGFR pathway activation (Kitadate, 2010). EGFR protein is observed on most SGPs throughout the embryonic gonad, beginning at gonad coalescence (stage 13). The EGFR ligand Spitz is expressed from all germ cells during gonad coalescence and activates EGFR among posterior SGPs. This activity antagonizes Notch and that appears to regulate final hub cell number. How EGFR activation is restricted or enhanced only among posterior SGPs is at present unclear (Okegbe, 2011).
Given that this study found that hub cell specification occurs prior to gonad coalescence, it is also possible that Notch and EGFR act in a temporal sequence. In this case, early Notch-activated SGPs, perhaps even those in the posterior will adopt hub cell fate. But, as EGFR becomes activated, further induction of the Notch pathway in the posterior is antagonized, prohibiting the specification of too many hub cells. Such a temporal inhibition might be important, as Serrate is expressed on the SGPs (Kitadate, 2010) and both Delta and Serrate are robustly expressed on tracheal cells, the activity of which might otherwise lead to excess hub cell induction. Perhaps during later stages of gonadogenesis (stages 14-16) a small number of anterior SGPs become Notch activated due to the activity of Serrate-Notch signaling from other SGPs, supplementing the hub cells previously specified by Delta-Notch signaling (Okegbe, 2011).
Given that niche cells in the Drosophila ovary become activated via Delta-Notch signaling by neighboring somatic cells, it was initially expected that Notch would be activated in a subset of SGPs by ligand presented from other SGPs. However, this study could not detect Delta nor Serrate expression among SGPs. Furthermore, although nearby tracheal cells expressed both ligands robustly, that expression appears later than Notch rescue suggests would be necessary, and genetic ablation of tracheal cells did not influence hub cell number (Okegbe, 2011).
Instead, this study found that a crucial signal for niche cell specification is presented from the endoderm, as Delta is expressed robustly on posterior midgut cells, at a time consistent with the requirement for Notch function. Furthermore, these endodermal cells are close enough to SGPs for productive Delta-Notch signaling to occur. Although visceral mesodermal cells are also close to the PMG and the SGPs, this tissue does not affect hub specification, since this study found that brachyenteron mutants exhibited normal hub cell number. By contrast, in mutants that do not internalize the gut (fog), and thus would not present Delta to SGPs, a drastic reduction was seen in hub cell number (Okegbe, 2011).
Additionally, it is noted that absolute hub cell number varies among animals and according to genetic background. This is attributed to normal biological variation, just as germline stem cell number varies. Potentially, this variation could be caused by the robustness with which the Notch pathway is activated in SGPs, as they are carried over the midgut cells. It will be interesting to test this hypothesis by genetically manipulating the number of midgut cells or the time of contact between endoderm and SGPs. Additionally, the antagonistic effects of EGFR signaling might account for some of the observed variation. In fact, gonads heterozygous for Star, a component of the EGFR pathway, exhibit increased hub cell number (Okegbe, 2011).
Finally, it is interesting to consider why the endoderm would be crucial for the proper specification of the GSC niche. In Drosophila, as in many animals, there is a special relationship between the gut and the germ cells. Primordial germ cells in mammals and in Drosophila must migrate through the endoderm to reach the gonadal mesoderm. In fact, in Drosophila, the gut exercises elaborate control over germ cell migration. As the germ cells begin their transepithelial migration and exit from the midgut pocket, tight connections between midgut cells are dissolved, allowing for easy germ cell passage. Germ cells then migrate on the basal surface of endodermal cells and midgut expression of wunens (which encodes lipid phosphate phosphatases) repels germ cells, driving them into the mesoderm. Thus, the endoderm not only delivers germ cells to the somatic mesoderm, but the same endoderm specifies niche cells from among the somatic mesoderm wherein germ cells can subsequently develop into stem cells. In mammals, although the exact make-up of the spermatogonial stem cell niche has not been determined, it must (in part) derive from cells of the genital ridge. It will be interesting to determine whether proximity to the gut endoderm is important for the specification of this niche (Okegbe, 2011).
See also the oogenesis site.
References
Boyle, M. and DiNardo, S. (1995). Specification, migration and assembly of the somatic cells of the Drosophila gonad. Devlopment 121: 1815-25
Chen, E. H. and Baker, B. S. (1997). Compartmental organization of the Drosophila genital imaginal discs. Development 124: 205-218. 9006081
Freeland, D. E. and Kuhn, D. T. (1996). Expression patterns of developmental genes reveal segment and parasegment organization of D. melanogaster genital discs. Mech. Dev. 56: 61-72. 8798147
Deshpande, G., Calhoun, G. and Schedl, P. (2004). Overlapping mechanisms function to establish transcriptional quiescence in the embryonic Drosophila germline. Development 131: 1247-1257. 14960492
Kitadate, Y. and Kobayashi, S. (2010). Notch and EGFR signaling act antagonistically to regulate germ-line stem cell niche formation in Drosophila male embryonic gonads. Proc. Natl. Acad. Sci. 107: 14241-14246. PubMed Citation: 20660750
Leatherman, J., Levin, L., Boero, J. and Jongens, T. (2002). germ cell-less acts to repress transcription during the establishment of the Drosophila germ cell lineage. Curr. Biol. 12: 1681-1685. 12361572
Moore, L. A., et al. (1998). Identification of genes controlling germ cell migration and embryonic gonad formation in Drosophila. Development 125(4): 667-678. PubMed Citation: 9435287
Nakamura, A., Amikura, R., Mukai, M., Kobayashi, S. and Lasko, P. (1996). Requirement for a noncoding RNA in Drosophila polar granules for germ cell establishment. Science 274: 2075-2079. 8953037
Okegbe, T. C. and DiNardo, S. (2011). The endoderm specifies the mesodermal niche for the germline in Drosophila via Delta-Notch signaling. Development 138(7): 1259-67. PubMed Citation: 21350008
Seydoux, G. and Dunn, M. A. (1997). Transcriptionally repressed germ cells lack a subpopulation of phosphorylated RNA polymerase II in early embryos of Caenorhabditis elegans and Drosophila melanogaster. Development 124: 2191-2201. 9187145
Tanentzapf, G., et al. (2007). Integrin-dependent anchoring of a stem-cell niche. Nat. Cell Biol. 9(12): 1413-8. PubMed Citation: 17982446
Van Doren, M., Williamson, A. L. and Lehmann, R. (1998). Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr. Biol. 8: 243-246. 9501989
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