Drosophila tissue and organ development: Ovaries and testis

The Interactive Fly

Genes involved in tissue and organ development

Gonads - Ovaries and testes


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
Quantifying the range of a lipid phosphate signal in vivo
NR5A nuclear receptor Hr39 controls three-cell secretory unit formation in Drosophila female reproductive glands
Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects
Drosophila germ granules are structured and contain homotypic mRNA clusters
Functioning of an ABC transporter, Mdr49, in Hh signaling and germ cell migration
Cells on the move: Modulation of guidance cues during germ cell migration
Drosophila CG2469 encodes a homolog of human CTR9 and is essential for development
Characterization of a TUTase/nuclease complex required for Drosophila gametogenesis
From embryo to adult: piRNA-mediated silencing throughout germline development in Drosophila
Escort cells generate a dynamic compartment for germline stem cell differentiation via combined Stat and Erk signalling
Wnt6 maintains anterior escort cells as an integral component of the germline stem cell niche

Genes expressed in or affecting the ovaries and testes


Ovaries and testes







Ovaries





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:

  1. First, somatic gonadal precursor cells are specified within the mesoderm in parasegments 10 through 12.
  2. After pole cells traverse and exit the midgut they recognize and associate primarily with specific mesodermal cells laterally positioned in the mesoderm of parasegments 11 and 12. These are the migratory gonadal precursors that delaminate from the mesodermal cell sheet.
  3. In a third step, gonadal precursors and pole cells migrate anteriorly, where they contact cells in parasegment 10.
  4. Next, gonadal precursors and pole cells arrest migration at parasegment 10.
  5. Finally, the mesodermal cells partially ensheath the arriving cells, and the cluster coalesces into the gonad.

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).

The endoderm specifies the mesodermal niche for the germline in Drosophila via Delta-Notch signaling

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).

Quantifying the range of a lipid phosphate signal in vivo

Quantitative information about the range of influence of extracellular signalling molecules is critical for understanding their effects, but is difficult to determine in the complex and dynamic 3 dimensional environment of a living embryo. Drosophila germ cells migrate during embryogenesis and use spatial information provided by expression of lipid phosphate phosphatases called Wunens to reach the somatic gonad. However whether guidance requires cell contact or involves a diffusible signal is not known. This study substituted wild type Wunen expression for various segmentally repeated ectodermal and parasegmental patterns and used germ cell behavior to show that the signal is diffusible and to define its range. This was correlated back to the wild type scenario, and it was found that the germ cell migratory path can be primarily accounted for by Wunen expression. This approach provides the first quantitative information of the effective range of a lipid phosphate in vivo and has implications for the migration of other cell types that respond to lipid phosphates (Mukherjee, 2013).

This paper has explored the nature of the signal that regulates germ cell migration and survival in Drosophila embryos. Germ cells are excluded from Wunen expressing somatic domains in wild type embryos suggesting Wunen destroys an attractive signal. Ectopic mis-expression of Wunen in embryos otherwise somatically null for Wunens was used to show that Wunens are instructive for dictating the germ cell migration path. In particular Wunen expression in en or h ectodermal stripes causes germ cells to align parallel to and between the stripes. In such embryos, germ cells have an equidistance ratio closer to 1 as compared to control embryos indicating they are likely integrating a signal from both sides. By live imaging it was observed that germ cells reach and maintain these parallel positions without making any direct contacts with the stripes. Taken together these data strongly argue that the signal modulated by Wunen positive somatic cells is cell contact independent (Mukherjee, 2013).

The exclusion of germ cells from the entire ectoderm when Wun is expressed using the en or h drivers might result from germ cells being repelled by the ectodermal stripes or from death of germ cells that have entered the ectoderm. The former is favored because from live imaging germ cells were not seen to enter the ectoderm, and ectodermal Vasa positive cell remnants, which are indicative of germ cell death. were not seen. In both scenarios however, the conclusion that the mechanism is cell-contact independent remains valid (Mukherjee, 2013).

When presented with a single Wun stripe, using the NP5141 driver, the germ cells were repelled up to 33 μm. This represents an estimate of the maximal effective range of the Wun signal. This distance is comparable to assessments of the effective range of Wingless (Wg) and Hedgehog (Hh), which can form gradients over at least 50 μm in wing imaginal discs. When germ cells are faced with multiple wunen domains, such as wun expressing en stripes, then germ cells can tolerate being much closer, but make avoidance turns when 15 μm away. In wild-type 99% of germ cells are located up to 33 &mu:m from a wun2 expressing domain. Therefore although germ cells are likely influenced by Wunen expression for the entire duration of their migration, 59% of them are closer than 15 μm. It is postulated that the small patches of wild type wun2 positive cells in the lateral mesoderm and ectoderm might not repel as far as the much larger ectopic wunen expressing en stripes (Mukherjee, 2013).

Membrane bound ligands can also effect long-range signals via their presence on long actin rich cytoplasmic extensions. For example, lateral inhibition of Drosophila sensory organ precursor (SOP) fate is mediated by the transmembrane Notch ligand Delta which can signal 3 to 5 cell diameters away via cytoplasmic extensions of up to 20 μm. Therefore it was considered whether germ cells could use cytoplasmic projections to make direct contact with Wun expressing stripes. Germ cell filopodia at their leading edge and often a longer lagging tail (or uropod) are seen by both live and fixed tissue analysis. The filopodia are generally no longer than 2 μm whilst the later can be up to 8 μm. Therefore these projections are not sufficiently long to make contact with the stripes. Furthermore a constitutively active form of Moesin, MoeT559D, was used that disrupts the actin cytoskeleton in Drosophila photoreceptors and suppresses filopodia from the leading edge during dorsal closure. Expression of this construct in germ cells caused only minor defects in germ cell survival indicating that germ cells do not rely on Moesin- dependent filopodia for migration in wild-type embryos (Mukherjee, 2013).

wun2 over-expression in its endogenous pattern was used to evaluate the relative importance of somatic Wun expression levels versus their spatial distribution in permitting germ cell survival. The data show that the level of Wunen protein in endogenously Wunen expressing cells is not critical to regulate germ cell survival. Ectopic expression can be tolerated if it is spatially separated from the germ cells. Therefore it is the location of the Wunen expression, and not its overall level, which is critical for germ cells. This is similar for other signaling systems, for example mis-expression of Hh ubiquitously leads to patterning defects but over-expression in its normal locations has little effect on segmentation. Insensitivity to over-expression in endogenous domains may well be a common feature of signaling molecules (Mukherjee, 2013).

This data is consistent with a model in which the somatic Wunen expression sets up relatively short range repulsion zones within the embryo. These result were envisioned from a local depletion of a lipid phosphate substrate. This could take the form of a discrete change in levels ('all or nothing') or a gradient. The ability of cells to find equidistant positions between Wunen stripes and to be repelled over many cell diameters when faced with a single Wunen expressing stripe strongly favors a gradient (Mukherjee, 2013).

The canonical model of morphogen gradient formation is that the morphogen is secreted locally and diffuses to create a gradient. However morphogens may also be restricted to a narrower area by localized depletion accomplished by receptor-mediated endocytosis or sequestration. Whilst the source of the ligand in the case of germ cells is not known, the data is consistent with gradient formation based on dephosphorylation of an extracellular lipid phosphate by a LPP (Mukherjee, 2013).

What would be the nature of the lipid gradient? S1P is present in human plasma and serum bound to low- and high-density lipoprotein and albumin. LPA is also found in human serum bound to albumin. It is possible that a similar protein binding partner occurs in Drosophila embryos. The extracellular movement of morphogens, such as Hh and Wg, has been proposed to occur on membranous vesicles (also called argosomes or exosomes), or lipoprotein particles. It is possible that similar particles are important for the formation of a lipid gradient that affects germ cell migration (Mukherjee, 2013).

Dictyostelium cells can respond at a distance of approximately 70 μm away from a micropipette containing LPA. S1P is important for the movement of heart progenitor cells from bilateral positions to the midline in zebrafish which involves a distance of approximately 100 μm. S1P also regulates the circulation of T-lymphocytes in mouse, in particular allowing T-cells to exit from lymph nodes which are several millimeters in length. In spite of these essential roles it is not always clear whether absolute levels or gradients of S1P are required. Drug treatments that increase the S1P levels of the lymphoid organs but not the circulatory system (effectively reversing the normal difference in S1P levels between these locations) are sufficient to block T-cell exit, is suggestive that a gradient is required in this circumstance however its contour is not known. This work has shown that lipid gradients in a Drosophila embryo can exist over distances comparable to their protein counterparts. Whether such distances are scaled up in the larger embryos and tissues of other species remains an open question (Mukherjee, 2013).

NR5A nuclear receptor Hr39 controls three-cell secretory unit formation in Drosophila female reproductive glands

Secretions within the adult female reproductive tract mediate sperm survival, storage, activation, and selection. Drosophila female reproductive gland secretory cells reside within the adult spermathecae and parovaria, but their development remains poorly characterized. With cell-lineage tracing, this study found that precursor cells downregulate lozenge and divide stereotypically to generate three-cell secretory units during pupal development. The NR5A-class nuclear hormone receptor Hr 39 is essential for precursor cell division and secretory unit formation. Moreover, ectopic Hr39 in multiple tissues generates reproductive gland-like primordia. Rarely, in male genital discs these primordia can develop into sperm-filled testicular spermathecae. Drosophila spermathecae provide a powerful model for studying gland development. It is concluded that Hr39 functions as a master regulator of a program that may have been conserved throughout animal evolution for the production of female reproductive glands and other secretory tissues (Sun, 2012).

In species where fertilization takes place internally, including mammals and insects, a sperm's long and obstacle-filled journey through the female reproductive tract culminates in the penetration of the egg. Prior to reaching its target, both paternal and maternal reproductive tissues deploy mechanisms that strongly influence an individual sperm's chances for success. In particular, specialized glands in female reproductive tracts produce mucus-rich secretions that capacitate sperm to fertilize successfully, inhibit infection, and provide nutritional, maintenance, and storage factors. The interactions of sperm and seminal fluid with the female reproductive tract and its secretions in Drosophila offer an opportunity to genetically analyze these complex processes (Sun, 2012).

Two paired glands, spermathecae (SPs) and parovaria (POs), are the primary sources of secretions encountered by sperm within the Drosophila female reproductive tract (see Structure and origin of Drosophila female reproductive glands). Messenger RNAs (mRNAs) encoding serine proteases, serpins, antioxidants, immune proteins, and enzymes involved in mucus production are found in SPs. Whereas two SPs arise from the engrailed (en) and en+ domains of the A8 segment, both POs originate in the en+ domain of the A9 segment in the female genital disc during pupal development. Both types of mature gland contain large, polyploid secretory cells (SCs). Each SC connects with the gland lumen via a specialized cuticular canal equipped with a secretion-collecting 'end apparatus'. Anatomically related secretory units are found in SPs from other species and in insect epidermal glands that produce pheromones, venoms, and many other products. Despite their ubiquity, insect epidermal gland development has not been well characterized at the molecular genetic level (Sun, 2012).

Studies of genital disc development and patterning have identified multiple genes important for reproductive gland formation. lozenge (lz), encoding a runt-domain transcription factor, is essential for both SP and PO formation and may be directly regulated by the sex determination pathway. Homologous to mammalian AML-1, Lz also supports developing blood precursors and prepatterns ommatidial cells in the developing eye. The dachshund (dac) gene also acts in multiple imaginal discs and is specifically needed for spermathecal duct development. Mutations that disrupt sphingolipid metabolism also cause abnormalities in spermathecal number and structure (Sun, 2012).

One of the most interesting genes needed to form reproductive glands encodes the nuclear hormone receptor Hr39, an early ecdysone-response gene. Hr39 and Ftz-f1 are the only two NR5A class nuclear hormone receptors in Drosophila, a class that in mammals includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1). All four of these proteins share 60%-90% sequence identity within their DNA binding domains and bind in vitro to identical sequences. SF-1 is a master regulator of steroidogenesis and sex hormone production, whereas LRH-1 is required in the ovary for female fertility, in embryonic stem cells for pluripotency and in endodermal tissues for metabolic homeostasis. Weak Hr39 mutations alter the production of some SP gene products, whereas LRH-1 directly controls major secretory proteins of the exocrine pancreas. Thus, NR5A class hormone receptors may play a conserved role controlling secretions from certain tissues, including female reproductive glands (Sun, 2012).

This study characterized the cell lineage of developing reproductive glands and clarify the roles of lz and Hr39. Hr39 is expressed sex-specifically in lz-positive female gland primordia beginning shortly after the ecdysone pulse that initiates prepupal development. When levels of Hr39 are reduced, lz-expressing precursors fail to protrude, divide, or remain viable, suggesting that Hr39 expression orchestrates reproductive gland development. Mouse LRH-1, but not SF-1, can partially replace Hr39 function in gland formation. Ectopic expression of Hr39 in male larvae can induce a pigmented SP-like structure containing sperm to develop in the male reproductive tract. It is proposed that Hr39 acts as a master regulator of reproductive gland development and that the production of sperm-interacting proteins in the female reproductive tract under the control of NR5A proteins has been conserved during evolution. These findings suggest new targets for controlling agriculture pests and human-disease vectors (Sun, 2012).

These studies reveal that lz and Hr39, despite their nearly identical loss-of-function phenotypes, have distinctive expression patterns during gland development. All gland precursors express both genes following puparium formation, but within 24 hr divide to produce lz+ epithelial precursors apically and lz SUPs basally. SUPs then differentiate according to a stereotyped program involving production of two transient accessory cells and a single polyploid secretory cell (Sun, 2012).

Reproductive secretory cells arise in a superficially similar manner to sensory bristles and multiple classes of mechanosensory and chemosensory sensilla. Both utilize short fixed-cell lineages that employ transient accessory cells to generate permanent extracellular structures (secretory canal, sensory bristle, etc.), but the three-cell secretory lineage analyzed in this study differs from the four asymmetric divisions producing five different cells typical of PNS differentiation (see Lineage Analysis of Secretory Unit Formation). Many other insect epidermal glands probably develop in a generally similar manner, but the precise cell lineages and mechanisms documented in this study for Drosophila reproductive glands (three cells, absence of ciliary involvement) differ from previous models (Sun, 2012).

Drosophila secretory units provide a powerful system for analyzing insect gland development. Studies in other insects suggested that an accessory cell utilizes a ciliary process to prevent the SCs from being sealed off by cuticle-secreting epithelial cells. This study found no morphological or genetic evidence that cilia are involved in forming Drosophila secretory units. However, the apical cell (AC) may fulfill this same role using normal microtubules, in much the same way that the anterior polar cells in egg chambers template the micropyle channel during oogenesis. Membranes from the basal cell (BC) likely surround this AC process, secrete the cuticular canal, and join it to the luminal cuticle. Concomitantly, the BC likely secretes the end apparatus around a large apical segment of the SC, which it surrounds (Sun, 2012).

The NR5A hormone receptor Hr39 plays multiple roles in reproductive gland development. Initially, Hr39 orchestrates gland protrusion and in the absence of Hr39 protrusion fails to occur. Among Drosophila imaginal discs, gland protrusion in genital discs is a unique process that leads to the differentiation of a gland capsule connected to the nascent reproductive tract by a tubular duct. When Hr39 is misexpressed, patches of cells within multiple imaginal discs that do not normally express Hr39 undergo changes reminiscent of early protrusion (Sun, 2012).

Hr39, a known member of the ecdysone response pathway, is likely to time reproductive gland cell divisions during pupal development. The initial Hr39 expression we observed in the genital disc was detected shortly after the prepupal ecdysone pulse. Several additional peaks of ecdysone titer during pupal development correspond closely with the timing this study measured of the secretory cell divisions. These observations suggest that external hormonal signals rather than internal autonomous mechanisms sometimes drive precise cell lineages. In addition to its requirement within cellular precursors, Hr39 mutations alter SP secretory gene mRNA levels (Allen, 2008), suggesting that Hr39 also regulates secretory gene expression within SCs (Sun, 2012).

Finally, Hr39 acts as a high level 'master regulator' by integrating individual pathways to elicit the production of an entire gland. Most cells expressing ectopic Hr39 could not progress past the initial stage of eversion, but in male genital discs Hr39-positive clones sometimes generated integrated structures that strongly resembled small spermathecae. They contained round heads with lumens, a pigmented layer, and rarely were connected to the male reproductive tract by ducts through which sperm were taken up. Thus, Hr39 (but not lz) can reprogram male genital cells to generate ectopic spermathecae that likely synthesize and secrete products attractive to sperm (Sun, 2012).

Drosophila reproductive gland development is unusually susceptible to perturbation. Rare adults in some wild strains contain an extra spermatheca, and females bearing weak alleles of either lz or Hr39 lose parovaria (POs) entirely and produce fewer spermathecae (SPs), which vary dramatically in size and cellular content. These effects probably result from the disparate sizes of the precursor pools for individual organs. PO pools are very small, whereas the exceptionally large posterior SP primordium may easily split in two under conditions where precursor proliferation is perturbed. The effects of dac mutations on duct structure are probably also due to altered precursor pools. Sphingolipids may affect gland development by serving as endogenous Hr39 ligands, consistent with reports that SF-1 can bind sphingolipids (Sun, 2012 and references therein).

In mammals, sperm interact with female secretory products at multiple locations. Glands within the uterine endometrium are hypothesized to govern selective passage through the cervix, uterus, and subsequently, the uterotubal junction. Following entry into the oviduct, sperm induce and interact with the products of specialized tubal secretory cells that likely mediate capacitation. In some species, these products also allow sperm to be stored in the oviduct while retaining their ability to fertilize an egg. Mammalian female reproductive glands continue to nurture preimplantation embryos and are likely essential for successful pregnancy (Sun, 2012).

Drosophila is emerging as a valuable model with which to study multiple aspects of reproductive physiology, some of which may have been conserved during evolution. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle (Simeone, 1995), but its role in fertility is unknown. The murine Hr39 homolog LRH-1 is required for female fertility, but whether it plays a role in reproductive gland secretion has yet to be tested. However, LRH-1 is required for the development of several exocrine tissues and in the pancreas is directly involved in the transcription of major secretory products. Thus, LRH-1 and Hr39 may both govern the formation and secretory function of exocrine tissue (Sun, 2012).

These study studies provide further support for the idea that an NR5a-dependent program of secretory cell development has been conserved in evolution. Murine LRH-1 can partially replace Hr39 function in Drosophila reproductive gland formation. Similar rescue with two other NR5A members (mammalian SF-1 or Drosophila Ftz-F1) failed and instead suppressed all gland formation. This is consistent with previous findings that Hr39 and Ftz-F1 have opposing roles in alcohol dehydrogenase and EcR expression. Antagonistic roles in gene regulation by the two NR5A family members may be evolutionarily conserved. Further study of the roles of Hr39 and LRH-1 should help define a fundamental program of secretory cell development that may be widely used (Sun, 2012).

Drosophila germ granules are structured and contain homotypic mRNA clusters

Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. This study, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, shows that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into 'homotypic clusters' that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules (Trcek, 2015).

This study combined single-molecule FISH with structured illumination microscopy (SIM), a super-resolution technique, to gain a high-resolution view of the mRNA-bound germ granule. This combinatorial approach allowed the determination that germ granule-localized mRNAs occupy distinct positions within the granule and relative to each other, while germ granule proteins are homogeneously distributed within the granular space. Multiple localized mRNAs group to form homotypic cluster, which gives the germ granule its structure. This structure does not change through early embryonic development and does not correlate with the translational onset of localized mRNAs or with the ability of germ granules to protect bound mRNAs from decay (Trcek, 2015).

This analysis of the organizational structure of germ plasm focused on core germ granule protein components, Vas, Osk, Tud and Aub, and on cycB, nos, pgc, gcl and osk mRNA. cycB, nos, pgc, gcl and osk serve as prototypes for mRNA localization to the germ granules because their localization to the germ plasm, their regulation in the germ plasm and biological significance for germ cell biology are understood best. While mRNA localization studies suggest that up to 200 mRNAs may be localized to the posterior pole of the early embryo, it is assumed that regulatory mechanisms revealed by the study of cycB, nos, pgc and gcl are shared among other germ plasm-localized mRNAs. The study of germ plasm-localized mRNA regulation revealed that only localized mRNAs translate, while their unlocalized counterparts are translationally silent, that localized mRNAs are protected from mRNA decay, and that the 3′ UTRs of localized mRNAs are necessary and often sufficient to localize mRNAs to the posterior and render them translationally competent. The experiments demonstrate that cycB, nos, pgc and gcl mRNAs concentrate in homotypic clusters, assume specific positions within the germ granules, and can organize into separate granules. The results make it unlikely that cycB, nos, pgc and gcl clusters contain more than one type of mRNA. If clustering between heterotypic mRNAs was a common organizational strategy, the pairwise analysis with cycB, nos, pgc and gcl would have not yielded the distinct volumes observed. Thus, despite the fact that only a limited number of localized RNAs were sampled, it is anticipated that germ granule organization observed for cycB, nos, pgc and gcl is also shared by other germ granule-localized mRNAs, which are similarly regulated (Trcek, 2015).

Given that the core germ plasm proteins Osk, Vasa, Aub and Tud recruit other germ granule components and are themselves homogeneously distributed within the granule, it is unlikely that the germ granule structure is dictated by proteins alone. Homotypic clustering could also be driven by intramolecular RNA–RNA interactions, similar to those found in the localized bicoid mRNA at the anterior pole and in the co-packaged osk mRNA during transport to the oocyte posterior. The dramatic increase in mRNA concentration in the granule compared with rest of the embryo may raise the likelihood for two mRNAs to interact or even induce RNA–RNA interactions by altering mRNA conformation thus driving homotypic clustering (Trcek, 2015).

In yeast, the movement of mRNAs in and out of stress granules and processing bodies determines their translatability and stability and in Drosophila oocytes the position of bicoid and gurken mRNA within the sponge body correlates with their translational activity. This study found, however, that the mRNA position within the germ granule is independent of translational or degradation activity of localized mRNAs. Some translational and decay regulators found in germ granules are also found in sponge bodies, stress granules and processing bodies. Thus the data imply that in germ granules these proteins may regulate transcripts differently to allow for the dynamic regulation of different mRNAs. Alternatively, sorting of mRNAs into distinct granules could specify their activity. For example, pgc co-localizes with core germ-granule components as well as with osk mRNA. Thus, the pool of pgc associated with osk could be functionally different from the one that associates with Vasa, Osk, cycB, nos and gcl. Indeed, in older embryos just before pgc becomes translated, pgc moves away from osk, but not from VasaGFP, cycB, nos and gcl. Speculatively, this could be the mechanism that determines the onset of pgc translation (Trcek, 2015).

mRNA clustering could also enhance biochemical reactions locally either by enabling protein complex formation, by quick re-binding of a regulator to a neighbouring mRNA or by increasing the concentration of a regulator of the cluster RNAs. For example, it has been proposed that the repression of cycB translation by Nanos protein (Nos) depends on a high local concentration of Nos in the germ plasm. Multiple nos mRNAs within the cluster could increase the local concentration of Nos thus counteracting the loss of the unbound Nos due to diffusion into the embryo. Once bound to cycB, Nos could also be quickly re-bound by the neighbouring cycB mRNAs thus maintaining high Nos concentration and ensuring efficient cycB repression. In this way each mRNA cluster in the granule would resemble a biochemical territory, consistent with the recent observations showing that germ granules in Caenorhabditis elegans, which behave like liquid droplets, are also not homogeneous. It is proposed that an mRNA-protein granule organization similar to the one described in this study for Drosophila germ granules could be a conserved feature of larger ribonucleoprotein granules (Trcek, 2015).

Functioning of an ABC transporter, Mdr49, in Hh signaling and germ cell migration

Coalescence of the embryonic gonad in Drosophila melanogaster requires directed migration of the primordial germ cells (PGCs) towards the somatic gonadal precursor cells (SGPs). It has been recently proposed that an ATP-Binding Cassette (ABC) transporter, Mdr49, functions in the embryonic mesoderm to facilitate the transmission of the PGC attractant from the SGPs; however, the precise molecular identity of the mdr49 dependent guidance signal remains elusive. Employing the 'loss' and 'gain' of function strategies, this study shows that mdr49 is a component of the Hedgehog pathway and it potentiates the signaling activity. This function is direct as, in mdr49 mutant embryos, Hh ligand is inappropriately sequestered in the hh expressing cells. Data also suggest that role of Mdr49 is to provide cholesterol for the correct processing of the Hh precursor protein. Supporting this conclusion, PGC migration defects in mdr49 embryos are substantially ameliorated by a cholesterol-rich diet (Deshpande, 2016).

Cells on the move: Modulation of guidance cues during germ cell migration

In Drosophila melanogaster the progenitors of the germ-line stem cells, the primordial germ cells (PGCs) are formed on the outside surface of the early embryo, while the somatic gonadal precursor cells (SGPs) are specified during mid-embryogenesis. To form the primitive embryonic gonad, the PGCs travel from outside of the embryo, across the mid-gut and then migrate through the mesoderm to the SGPs. The migratory path of PGCs is dictated by a series of attractive and repulsive cues. Studies have shown that one of the key chemoattractants is the Hedgehog (Hh) ligand. Although, Hh is expressed in other cell types, the long-distance transmission of this ligand is specifically potentiated in the SGPs by the hmgcr isoprenoid biosynthetic pathway. The distant transmission of the Hh ligand is gated by restricting expression of hmgcr to the SGPs. This is particularly relevant in light of the recent findings that an ABC transporter, mdr49 also acts in a mesoderm specific manner to release the germ cell attractant. These studies have demonstrated that mdr49 functions in hh signaling likely via its role in the transport of cholesterol. Given the importance of cholesterol in the processing and long distance transmission of the Hh ligand, this observation has opened up an exciting avenue concerning the possible role of components of the sterol transport machinery in PGC migration (Deshpande, 2017).

Drosophila CG2469 encodes a homolog of human CTR9 and is essential for development

Conserved from yeast to humans, the Paf1 complex participates in a number of diverse processes including transcriptional initiation and polyadenylation. This complex typically includes 5 proteins: Paf1, Rtf1, Cdc73, Leo1 and Ctr9. Previous efforts have identified clear Drosophila homologs of Paf1, Rtf1 and Cdc73 based on sequence similarity. Further work has showed that these proteins help to regulate gene expression and are required for viability. To date, a Drosophila homolog of Ctr9 has remained uncharacterized. This study shows that the gene CG2469 encodes a functional Drosophila Ctr9 homolog. Both human and Drosophila Ctr9 localize to the nuclei of Drosophila cells and appear enriched in histone locus bodies. RNAi knock-down of Drosophila Ctr9 results in a germline stem cell loss phenotype marked by defects in the morphology of germ cell nuclei. A molecular null mutation of Drosophila Ctr9 results in lethality and a human cDNA Ctr9 transgene rescues this phenotype. Clonal analysis in the ovary using this null allele reveals that loss of Drosophila Ctr9 results in a reduction of global levels of histone H3 trimethylation of lysine 4 (H3K4me3) but does not compromise the maintenance of stem cells in ovaries. Given the differences between the null mutant and RNAi knockdown phenotypes, the germ cell defects caused by RNAi likely result from the combined loss of Drosophila Ctr9 and other unidentified genes. These data provide further evidence that the function of this Paf1 complex component is conserved across species (Chaturvedi, 2016).

Characterization of a TUTase/nuclease complex required for Drosophila gametogenesis

The 3' exoribonuclease Dis3L2 metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of non-coding RNAs. This study performed a structure-function analysis of the Drosophila TUTase Tailor, which inhibits biogenesis of splicing-derived miRNA hairpins. Tailor/Dis3L2 forms a complex via N-terminal domains in the respective proteins that are distinct from their catalytic domains. In vitro, Dis3L2 has nuclease activity but substrate oligouridylation by Tailor stimulates their degradation by Dis3L2, especially for structured substrates. Mutants of Tailor and Dis3L2 are viable and lack overt morphological defects. Instead, these mutants exhibit defects in female and male fertility, implying specific requirements in the germline. Dis3L2 defects are more severe than Tailor, and their requirements appear stronger in males than in females. In particular, loss of Dis3L2 completely blocks productive spermatogenesis causing complete male sterility. RNA-seq analysis from single and double mutant testes reveal aberrant gene expression programs, and suggest that non-coding RNAs may be preferentially affected by Dis3L2. Overall, these studies of a new tailing/trimming complex reveals unexpectedly specific requirements during gametogenesis (Bejarano, 2016).

From embryo to adult: piRNA-mediated silencing throughout germline development in Drosophila

In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development, has not been addressed. This study shows that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, rhino-dependent piRNAs are detected indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the "ping-pong" amplification step. Moreover, Heterochromatin Protein 1a (HP1a) is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, these data show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development (Marie, 2016).

Escort cells generate a dynamic compartment for germline stem cell differentiation via combined Stat and Erk signalling

Two different compartments support germline stem cell (GSC) self-renewal and their timely differentiation: the classical niche provides maintenance cues, while a differentiation compartment, formed by somatic escort cells (ECs), is required for proper GSC differentiation. ECs extend long protrusions that invade between tightly packed germ cells, and alternate between encapsulating and releasing them. How ECs achieve this dynamic balance has not been resolved. By combining live imaging and genetic analyses in Drosophila, this study has characterised EC shapes and their dynamic changes. Germ cell encapsulation by ECs is shown to be a communal phenomenon, whereby EC-EC contacts stabilise an extensive meshwork of protrusions. It was further shown that Signal Transducer and Activator of Transcription (Stat) and Epidermal Growth Factor Receptor (Egfr) signalling sustain EC protrusiveness and flexibility by combinatorially affecting the activity of different RhoGTPases. The results reveal how a complex signalling network can determine the shape of a cell and its dynamic behaviour. It also explains how the differentiation compartment can establish extensive contacts with germ cells, while allowing a continual posterior movement of differentiating GSC daughters (Banisch, 2017).

Wnt6 maintains anterior escort cells as an integral component of the germline stem cell niche

Stem cells reside in a niche, a complex cellular and molecular environment. In Drosophila ovaries, germline stem cells depend on cap cells for self-renewing signals and physical attachment. Germline stem cells also contact the anterior escort cells, and this study reports that anterior escort cells are absolutely required for germline stem cell maintenance. When escort cells die from impaired Wnt signaling or hid expression, the loss of anterior escort cells causes consequent loss of germline stem cells. Anterior escort cells function as an integral niche component by promoting DE-cadherin anchorage and by transiently expressing the Dpp ligand to promote full-strength BMP signaling in germline stem cells. Anterior escort cells are maintained by Wnt6 ligands produced by cap cells; without Wnt6 signaling, anterior escort cells die leaving vacancies in the niche, leading to loss of germline stem cells. These data identify anterior escort cells as constituents of the germline stem cell niche, maintained by a cap-cell produced Wnt6 survival signal (Wang, 2018).

See also the oogenesis site.


References

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