Ovaries and testis

The Interactive Fly

Genes involved in tissue and organ development

Gonads - Ovaries and testes and associated structures

  • Oogenesis
  • Spermatogenesis

    Gene expression in pole cells
  • Maintaining transcriptional silence in pole cells, the germline cell precursors
  • Drosophila germ granules are structured and contain homotypic mRNA clusters

    Germ cell migration
  • Genes controlling germ cell migration and embryonic gonad formation
  • 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
  • Collectively stabilizing and orienting posterior migratory forces disperses cell clusters in vivo
  • Migration of primordial germline cells is negatively regulated by surrounding somatic cells during early embryogenesis in Drosophila melanogaster
  • Quantifying the range of a lipid phosphate signal in vivo

    Genital disks
  • Segmental origin of genital discs

    Development of the Germline
  • The endoderm specifies the mesodermal niche for the germline in Drosophila via Delta-Notch signaling
  • Expression of the core promoter factors TBP and TRF2 in Drosophila germ cells and their distinct functions in germline development
  • Characterization of a TUTase/nuclease complex required for Drosophila gametogenesis
  • Expression of the core promoter factors TBP and TRF2 in Drosophila germ cells and their distinct functions in germline development
  • From embryo to adult: piRNA-mediated silencing throughout germline development in Drosophila
  • Maternal Piwi Regulates Primordial Germ Cell Development to Ensure the Fertility of Female Progeny 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
  • DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1
  • The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells
  • Pgc suppresses the zygotically-acting RNA decay pathway to protect germ plasm RNAs in the Drosophila embryo
  • Reactive oxygen species signaling in primordial germ cell development in Drosophila embryos
  • Maternally inherited intron coordinates primordial germ cell homeostasis during Drosophila embryogenesis
  • Nuclear lamina dysfunction triggers a germline stem cell checkpoint
  • Trajectory mapping of the early Drosophila germline reveals controls of zygotic activation and sex differentiation
  • Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching

    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).

    Expression of the core promoter factors TBP and TRF2 in Drosophila germ cells and their distinct functions in germline development

    In Drosophila, the expression of germline genes is initiated in primordial germ cells (PGCs) and its expression is known to be associated with germline establishment. However, the transcriptional regulation of germline genes remains elusive. Previous studies found that the BTB/POZ-Zn-finger protein, Mamo, is necessary for the expression of the germline gene, vasa, in PGCs. Moreover, the truncated Mamo lacking the BTB/POZ domain (MamoAF) is a potent vasa activator. This study investigated the genetic interaction between MamoAF and specific transcriptional regulators to gain insight into the transcriptional regulation of germline development. A general transcription factor, TATA box binding protein (TBP)-associated factor 3 (TAF3/BIP2), and a member of the TBP-like proteins, TBP-related factor 2 (TRF2), were found as new genetic modifiers of MamoAF. In contrast to TRF2, TBP was found to show no genetic interaction with MamoAF, suggesting that Trf2 has a selective function. Therefore, this study focused on Trf2 expression and investigated its function in germ cells. The Trf2 mRNA, rather than the Tbp mRNA, was found to be preferentially expressed in PGCs during embryogenesis. The depletion of TRF2 in PGCs resulted in decreased mRNA expression of vasa. RNA interference-mediated knockdown showed that while Trf2 is required for the maintenance of germ cells, Tbp is needed for their differentiation during oogenesis. Therefore, these results suggest that Trf2 and Tbp expression is differentially regulated in germ cells, and that these factors have distinct functions in Drosophila germline development (Nakamura, 2020).

    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).

    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).

    Collectively stabilizing and orienting posterior migratory forces disperses cell clusters in vivo

    Individual cells detach from cohesive ensembles during development and can inappropriately separate in disease. Although much is known about how cells separate from epithelia, it remains unclear how cells disperse from clusters lacking apical-basal polarity, a hallmark of advanced epithelial cancers. Using live imaging of the developmental migration program of Drosophila primordial germ cells (PGCs), this study shows that cluster dispersal is accomplished by stabilizing and orienting migratory forces. PGCs utilize a G protein coupled receptor (GPCR), Tre1, to guide front-back migratory polarity radially from the cluster toward the endoderm. Posteriorly positioned myosin-dependent contractile forces pull on cell-cell contacts until cells release. Tre1 mutant cells migrate randomly with transient enrichment of the force machinery but fail to separate, indicating a temporal contractile force threshold for detachment. E-cadherin is retained on the cell surface during cell separation and augmenting cell-cell adhesion does not impede detachment. Notably, coordinated migration improves cluster dispersal efficiency by stabilizing cell-cell interfaces and facilitating symmetric pulling. This study demonstrates that guidance of inherent migratory forces is sufficient to disperse cell clusters under physiological settings and present a paradigm for how such events could occur across development and disease (Lin, 2020).

    The developmental migration of Drosophila PGCs is an excellent model to study how actomyosin contractility is deployed to separate cells from non-epithelial clusters. PGCs are a group of 30-40 cells born at the posterior of the embryo. During gastrulation, PGCs are swept into the interior of the embryo, where they reside as a tight cluster in a rosette configuration enveloped by the endoderm. PGCs subsequently separate and individually transmigrate through the endoderm as it undergoes a developmentally programmed epithelial-to-mesenchymal transition (EMT). How cluster separation is achieved mechanistically remains elusive. Known autonomous proteins required for PGC cluster dispersal are the orphan G-protein-coupled receptor (GPCR), Tre1 and its associated Gβγ subunit, consisting of Gβ13F and Gγ1, as well as the small Rho GTPase, RhoA, suggesting the involvement of contractile forces. However, actomyosin dynamics during PGC cluster dispersal remain uncharacterized and how Tre1 influences the spatiotemporal dynamics of these networks is unknown (Lin, 2020).

    This work harnesses two-photon live imaging to provide an in vivo description of how actomyosin contractility is deployed to disperse cell clusters lacking apical-basal polarity under physiological conditions. In contrast to current models of epithelial delamination, cluster dispersal does not involve a sustained downregulation of cell-cell adhesion or augmented force production and is surprisingly robust to increased levels of adhesion. Rather, inherent migratory forces are co-opted to liberate cells. This is accomplished through the sensing of a directed migration cue via the GPCR, Tre1. Tre1 signaling stabilizes and orients migratory polarity radially from the cell cluster, thereby positioning posterior myosin II dependent contractile forces towards cell-cell interfaces in the cluster interior. This collective radial polarity stabilizes cell-cell interfaces and enables symmetric tugging, increasing the efficiency of cluster dispersal (see PGC clusters disperse by directing individual migratory polarity outward to collectively remove cell-cell adhesions). Symmetric tugging, however, is not absolutely necessary for cell separation, as individual WT PGCs can still detach from tre1 PGC clusters, albeit less efficiently. Subsequent detachment requires sustained pulling on cell-cell adhesions provided by a stable migratory polarity. Thus, randomly migrating cells, equally capable of contractile force production, are unable to separate because they do not pull on cell-cell adhesions in a given orientation for a sufficient period of time. A caveat to this model is that this study has not directly shown that migrating PGCs exert posterior pulling forces, as this is technically challenging at the depth where PGC cluster dispersal occurs. However, posterior pulling forces have been clearly demonstrated in various cell types utilizing a rearward driven 3D migration mode which closely resembles PGC migration in Drosophila (Lin, 2020).

    Mechanistically, the migration-based cluster dispersal mechanism described in this study harbors many commonalities with hepatocyte growth factor (HGF) mediated epithelial scattering. Myogenic precursors induced to delaminate by ectopic application of HGF maintain expression of N-cadherin, the cardinal adhesion molecule originally linking them to the dermomyotome. Similarly, HGF induced scattering of Madin-Darby canine kidney (MDCK) cells does not involve direct alterations in E-cadherin. Instead, HGF promotes motility and strengthens cell-ECM attachment through integrins, which in turn generate a local increase in tension on cell-cell adhesions until they are physically disrupted. PGCs, on the other hand, continue to utilize E-cadherin to adhere to another cellular substrate, the surrounding endoderm, to pull away from each other. For both PGCs and HGF stimulated MDCK cells, the absence of free space to migrate is sufficient to block dispersal (Lin, 2020).

    Cell ensembles frequently exhibit collective migration during development and disease. How is group cohesion maintained if contractile migratory forces are sufficient to disrupt cell-cell adhesion? In collectively migrating squamous cell carcinoma (SSC) cells, primary colorectal cancer explants, and Xenopus neural crest, actomyosin contractility is enriched along the group perimeter and is actively suppressed from cell to cell interfaces in SSC cells to prevent cell detachment. This suppression relies on Discoidin domain receptor 1 (DDR1), which acts in a noncanonical manner at cell-cell interfaces by recruiting Par3 and Par6 to control the localization of RhoE to antagonize RhoA activity. Depletion of DDR1 and other members of the complex result in elevated levels of active Myosin II at cell-cell margins and individual cell migration away from the group, leading to group dispersal. This is strikingly similar to the mechanism uncovered in this study during PGC cluster dispersal and that of HGF mediated cell scattering. Recent work has also revealed an alternative strategy to reduce RhoA signaling at cell-cell junctions of collectively migrating SSC cells through Snail dependent expression of claudin-11, which activates Src to recruit p190RhoGAP to cell-cell interfaces (Li, 2019). In collectively migrating Drosophila border cells, E-cadherin tension is also reduced in the interior of the migrating cell cluster. Thus, while the suppression of contractile forces at cell-cell contacts appears to be a general principle to ensure cohesion in collectively migrating cell populations, PGCs actively direct actomyosin contractility towards cell-cell interfaces to separate. It will be interesting to assess whether pathological cell aggregates can be coaxed to disperse through a similar mechanism (Lin, 2020).

    This work demonstrates that cluster dispersal can be driven by the concerted reorientation of migratory actomyosin forces towards cell-cell interfaces. For PGCs, this is accomplished by sensing a directed migration cue through a GPCR, Tre1. However, in principle, any migratory cue, such as ECM or substrate stiffness, could be sufficient to mediate this reorientation. Subsequent cell-cell separation does not require any alterations of the inherent actomyosin forces driving migration. Rather, these forces must be applied on cell-cell junctions for a sufficient period of time, highlighting a potential safeguard against erroneous cell detachment. An open question is the identity of the Tre1 ligand. Given that PGCs are directed to migrate toward the endoderm within a tightly enclosed pocket, the ligand is likely to be surface bound, which would concur with the known roles of Tre1 in orienting neuroblast division56 and immune cell extravasation. Overall, it is anticipated that directed motility-based separation will have general relevance for individual cell detachment events from clustered cell groups lacking apical-basal polarity in development and disease (Lin, 2020).

    Migration of primordial germline cells is negatively regulated by surrounding somatic cells during early embryogenesis in Drosophila melanogaster

    The initiation and maintenance of the cell movement state requires the activation of many factors involved in the regulation of transcription, signal transduction, adhesive interactions, modulation of membranes and the cytoskeleton. However, cell movement depends on the status of both migrating and surrounding cells, interacting with each other during movement. The surrounding cells or cell matrix not only form a substrate for movement, but can also participate in the spatio-temporal regulation of the migration. To determine the role of the cell environment in the regulation of individual cell migration, the migration of primordial germline cells (PGC) was studied during early embryogenesis in Drosophila melanogaster. Normally, PGC are formed at the 3rd stage of embryogenesis at the posterior pole of the embryo. During gastrulation (stages 6-7), PGC as a consolidated cell group passively transfers into the midgut primordium. Further, PGC are individualized, acquire an amoeboid form, and actively move through the midgut epithelium and migrate to the 5-6 abdominal segment of the embryo, where they form paired embryonic gonads. A screen was performed for genes expressed in the epithelium surrounding PGC during early embryogenesis and affecting their migration. The myc, Hph, stat92E, Tre-1, and hop genes, whose RNA interference leads to premature active PGC migration at stages 4-7 of embryogenesis, were identified. These genes can be divided into two groups: 1) modulators of JAK/STAT pathway activity inducing PGC migration (stat92E, Tre-1, hop), and 2) myc and Hph involved in epithelial morphogenesis and polarization, i. e. modifying the permeability of the epithelial barrier. Since a depletion of each of these gene products resulted in premature PGC migration, it can be concluded that, normally, the somatic environment negatively regulates PGC migration during early Drosophila embryogenesis (Dorogova, 2020).

    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).

    Expression of the core promoter factors TBP and TRF2 in Drosophila germ cells and their distinct functions in germline development

    In Drosophila, the expression of germline genes is initiated in primordial germ cells (PGCs) and its expression is known to be associated with germline establishment. However, the transcriptional regulation of germline genes remains elusive. Previous studies found that the BTB/POZ-Zn-finger protein, Mamo, is necessary for the expression of the germline gene, vasa, in PGCs. Moreover, the truncated Mamo lacking the BTB/POZ domain (MamoAF) is a potent vasa activator. This study investigated the genetic interaction between MamoAF and specific transcriptional regulators to gain insight into the transcriptional regulation of germline development. A general transcription factor, TATA box binding protein (TBP)-associated factor 3 (TAF3/BIP2), and a member of the TBP-like proteins, TBP-related factor 2 (TRF2), were found as new genetic modifiers of MamoAF. In contrast to TRF2, TBP was found to show no genetic interaction with MamoAF, suggesting that Trf2 has a selective function. Therefore, this study focused on Trf2 expression and investigated its function in germ cells. The Trf2 mRNA, rather than the Tbp mRNA, was found to be preferentially expressed in PGCs during embryogenesis. The depletion of TRF2 in PGCs resulted in decreased mRNA expression of vasa. RNA interference-mediated knockdown showed that while Trf2 is required for the maintenance of germ cells, Tbp is needed for their differentiation during oogenesis. Therefore, these results suggest that Trf2 and Tbp expression is differentially regulated in germ cells, and that these factors have distinct functions in Drosophila germline development (Nakamura, 2020).

    During early embryogenesis in animals, primordial germ cells (PGCs) are specified, and the expression of germline genes is initiated in these cells. The vasa (vas) gene encodes a highly conserved DEAD-box RNA helicase, and its expression is a marker of germline establishment in many animal species. Additionally, recent transcriptome analyses have revealed more novel germline genes that are preferentially expressed in PGCs. Expression of germline genes implies that a network of transcriptional regulators exists in these germline cells. However, the underlying mechanism of transcriptional regulators that control germline gene expression remains elusive (Nakamura, 2020).

    In Drosophila, maternal factors localized in the germ plasm are necessary for germ cell establishment. It was assumed that the regulation of germline gene expression requires the presence of maternal transcriptional regulators in the germ plasm. However, RNA interference (RNAi)-mediated knockdown experiments of germ plasm-enriched maternal mRNAs have demonstrated that several transcriptional regulators are required for the expression of germline genes in PGCs. Among these, the transcriptional activator, OvoB, is predominantly expressed in PGCs, and its function is essential for germline development (Nakamura, 2020).

    Previous work has shown that Mamo, a PGC-enriched maternal factor, is necessary for the expression of vas in PGCs (Mukai, 2007). Mamo protein has been identified as a zinc-finger protein that contains both a BTB/POZ domain and C2H2 Zn-finger domains (Mukai, 2007). Subsequent biochemical analyses demonstrated that the C2H2 Zn-finger domains of Mamo directly bind to a specific DNA sequence in the first intron of the vas gene. Overexpression of N-terminal truncated Mamo (MamoAF) lacking the BTB/POZ domain, but having the C2H2 Zn-finger domains, strongly promotes vas expression. Furthermore, Mamo mRNA encoding a truncated Mamo isoform, which is similar to MamoAF, is predominantly expressed in PGCs (Nakamura, 2019). Therefore, these results suggest that the short Mamo lacking the BTB/POZ domain is a potent vas activator. Moreover, this study found that MamoAF collaborates with both an epigenetic regulator, CREB-binding protein (CBP), and the germline transcriptional activator, OvoB, to activate vas expression (Nakamura, 2019). These observations imply that MamoAF acts as a hub molecule to interact with transcriptional regulators (Nakamura, 2020).

    Recently, core promoter factors and their homologs have been found to selectively regulate gene expression and control specific developmental processes. The TATA box-binding protein (TBP), which is a subunit of the TFIID complex, recognizes core promoters containing the TATA box to regulate gene expression (Haberle, 2018). TBP-associated factor 3 (TAF3/BIP2) is a general transcription factor, but exhibits selective physical interactions with transcriptional regulators such as Antennapedia, BAB1, and BAB2. TBP-related factor 2 (TRF2), a member of the TBP-like proteins, selectively regulates the expression of a subset of genes that differ from those regulated by TBP. TRF2 interacts with the transcriptional regulator, Fruitless, to masculinize neurobehavioral traits in Drosophila. Therefore, core promoter factors may mediate a new layer of transcriptional regulation that controls specific developmental processes (Nakamura, 2020).

    In this study, a genetic experiment was conducted to identify the cofactors of MamoAF to provide insights into the role of transcriptional regulators in germ cells. The Drosophila compound eye is a precise structure that sensitively reflects genetic interactions. Genetic modifier screening using the compound eyes has been performed to identify cofactors for many genes. Genetic modifier screening has also succeeded in identifying the cofactors that play a role in germ cells. Therefore, a genetic experiment was conducted on compound eyes overexpressing MamoAF to identify the genetic modifiers of MamoAF. The core promoter factors, TAF3/BIP2 and TRF2, were identified as the genetic modifiers of MamoAF. Trf2 mRNA, rather than Tbp mRNA, was preferentially expressed in PGCs. Although Trf2 is required for maintenance of germ cells, Tbp is necessary for their differentiation during oogenesis. Therefore, these results suggest that the expression of Trf2 and Tbp is differentially regulated in germ cells and these factors have distinct functions in germline development in Drosophila (Nakamura, 2020).

    Previous transcriptome analyses revealed that the genes encoding these Mamo interactors are expressed in the adult heads or the cell line derived from eye-antenna discs. Recently, Mamo has also been shown to play a role in neuronal fate specification and maintenance during brain development. Therefore, the transcriptional regulators that collaborate with Mamo can be expressed in neural cells. Moreover, genetic screening using adult compound eyes may be useful for the identification of Mamo interactors that act on germ cells (Nakamura, 2020).

    As several regulators of histone modifications are essential for germline development, this study found that both CBP and HDAC1 preferentially interacted with MamoAF. Additionally, since CBP is responsible for H3K27ac and HDAC1 is required for the deacetylation of H3K27ac, it can be said that proper regulation of H3K27ac levels may play an essential role in germline development. Therefore, future studies will focus on identifying the target genes that are epigenetically regulated by H3K27ac in germ cells (Nakamura, 2020).

    Some TFIID subunits vary with target genes and cell types. The Drosophila TAF-TRF2 complex has been proposed to perform distinct functions in regulating neural stem cell identity (Neves, 2019). Moreover, previous biochemical studies have shown that both TAF3/BIP2 and TRF2 interact with TAF6 (Weake, 2009). This study also found that MamoAF preferentially interacted with both Taf3 and Trf2. Therefore, future studies investigating the physical interaction between Mamo and these general transcription factors will provide insights into the mechanism through which Mamo collaborates with the complex containing both TAF3/BIP2 and TRF2 (Nakamura, 2020).

    Core promoter factors are considered to be universally required in all eukaryotic cells. However, recent studies suggested that some core promoter factors are developmentally regulated. This study showed that Trf2 and Tbp expression was differentially regulated in germ cells. A previous report showed that TBP protein is highly stable in insect TN-368 cells. Consistent with this report, TBP protein appeared to be stable in these experiments, as it was detected in PGCs and germline cysts where Tbp mRNA was not detected. Therefore, the stability of TBP protein may ensure the robustness of transcriptional regulation. In contrast, Trf2 mRNA was continuously expressed in germline cells, and TRF2 protein in PGCs was found to be sensitive to Trf2-RNAi. Therefore, as compared with Tbp, Trf2 activity can be transcriptionally regulated in germline cells (Nakamura, 2020).

    Trf2 may regulate the expression of target genes that differ from those of Tbp during germline development. TRF2, rather than TBP, specifically mediates the transcription of ribosomal protein genes. TRF2 selectively regulates the TATA-less histone H1 gene promoter. Moreover, TRF2 is required for piRNA expression. Combined with the current data, TRF2 may selectively support the transcription of target genes in order to maintain germline cells. This study also found that the Trf2 RNAi (BL64561)-mediated knockdown affected the formation of egg chambers. Therefore, the expression of the genes that control germ cell differentiation may be regulated by TRF2 (Nakamura, 2020).

    Drosophila Trf2 encodes TRF2S and TRF2L. TRF2S is conserved between Drosophila and mammals because TRF2S appears to be more closely related to the human TRF2 protein, which lacks the long N-terminal extension present in TRF2L. TRF2S is known to act on the DNA replication-related element, downstream promoter element (DPE), and TCT motifs. In this study, Trf2 activity was found to be required for vas expression in PGCs. The vas gene contains a DPE-like motif (+25 to +29) near the transcription start site. Whether TRF2S acts on this DPE motif is unclear, but it supports the idea that TRF2S is involved in the transcriptional regulation of vas. In contrast to Trf2 RNAi (V10443), the overexpression of TbpRNAi did not decrease vas expression. However, as it was not possible to knock down TBP in PGCs, it could not be definitively excluded that TBP mediates the transcription of vas. TBP binds to the TATA box in promoters to regulate TATA-dependent transcription. However, the vas promoter does not appear to contain a TATA box near the transcription start site. Therefore, it is concluded that vas transcription is regulated by TRF2S rather than by TBP. TRF2L may also regulate vas transcription because TRF2L can activate both DPE-dependent and TATA-dependent promoters (Nakamura, 2020).

    Previous studies using a weak Trf2 allele, polyhomeoticP1 Trf2P1, in which TRF2 expression in germ cells is decreased but TRF2 is not depleted from germ cells, have shown that Trf2 is required for germ cell differentiation and that TRF2L and TRF2S can rescue the mutant phenotypes individually. This shows that TRF2S and TRF2L have redundant functions in germ cell differentiation. This study also showed that Trf2 RNAi (V10443)-mediated knockdown resulted in an agametic phenotype and that the overexpression of Trf2L rescued the agametic phenotype, thereby suggesting that germ cell maintenance may require a TRF2L function. The Trf2L cDNA used in the rescue experiment may produce both TRF2S and TRF2L because of the presence of the IRES sequence in the Trf2L cDNA. However, this study found that Trf2S expression alone did not rescue the agametic phenotype induced by Trf2 RNAi, thereby confirming that TRF2L is required for germ cell maintenance (Nakamura, 2020).

    Previous reporter assays have shown that TRF2S preferentially activates DPE-dependent promoters, whereas TRF2L activates both DPE-dependent and TATA-dependent promoters TRF2L may regulate the expression of distinct target genes to maintain germ cells. Previous biochemical studies have shown that both TRF2S and TRF2L are present in the same protein complex that contains ISWI ATPase. However, some TRF2L proteins exhibit different chromatographic properties from those of TRF2S, thereby suggesting that TRF2L cofactors may differ from those of TRF2S. Moreover, the coiled-coil domains in the long N-terminal domains of TRF2L may mediate their interaction. Therefore, future studies on the identification of TRF2L target genes and TRF2L cofactors may provide insights into the mechanisms through which TRF2L regulates germ cell maintenance (Nakamura, 2020).

    The core promoter factors that mediate a new layer of transcriptional regulation may be involved in germline development in Drosophila. Moreover, the homologs of core promoter factors have been found to selectively regulate transcriptional programs and control specific developmental processes in Drosophila and mouse models. This study found that TRF2 and TBP had distinct functions in germline development. As Trf2 is conserved in bilaterian organisms and is required for spermiogenesis in mice, it is speculated that Trf2 might play a role in germline development in other animals as well. Hence, it is expected that the current results will facilitate the understanding of the transcriptional regulation network that controls germline development in animals (Nakamura, 2020).

    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).

    Maternal Piwi Regulates Primordial Germ Cell Development to Ensure the Fertility of Female Progeny in Drosophila

    In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. This study shows that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, this study found that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. This study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila (Gonzalez, 2021).

    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).

    Adult tissues are maintained by stem cells that self-renew and differentiate into functional cells. Stem cells reside within a specialized microenvironment known as the niche, and their self-renewal, numbers and activities are regulated by extrinsic cues from the niche. Understanding the niche structure is fundamental to harnessing stem cells in applications such as regenerative medicine. The cellular organization of the stem cell niche is complex, and it can include stem cells themselves, their progeny, nearby mesenchymal cells or stromal cells, muscles, extracellular matrix, and distant sources within or even outside the tissue. How different niche components interact with each other remains elusive (Wang, 2018).

    Studies on Drosophila ovarian germline stem cells (GSCs) have provided an archetypal example of a stem cell niche composed of adjacent support cells. In the Drosophila ovary, two or three GSCs are located at the apex of each ovariole in a structure known as the germarium. GSCs form direct contact on their anterior side with a cluster of five to seven disc-shaped cap cells via adherens junctions. This anchorage is essential for GSC self-renewal. Furthermore, cap cells secrete bone morphogenetic protein (BMP) ligands including Decapentaplegic (Dpp) and Glass bottom boat (Gbb) to repress differentiation of GSCs. As a GSC divides, it produces a self-renewing GSC daughter that remains in contact with cap cells, and a cystoblast daughter positioned away from the niche. Without continuous BMP signaling, the cystoblast differentiates into a germline cyst and eventually an egg. For these reasons, the cap cells are considered to be the GSC niche (Wang, 2018).

    Escort cells are a population of 30-40 squamous cells that line the basement membrane of the anterior half of the germarium, and they extend cytoplasmic processes to encase each GSC, cystoblast and developing germline cyst. Escort cells play an essential role in germline differentiation, as many studies have shown that escort cell disruptions result in an accumulation of undifferentiated, stem-like germline cells. Over the last decade, scattered observations have suggested a role for unspecified escort cells in maintaining GSCs, but this role has not been probed in depth (Wang, 2018).

    This study demonstrates that anterior escort cells, which contact the GSCs, are essential for GSC maintenance. Like cap cells, the most anterior escort cells anchor GSCs through DE-cadherin-based junctions, and these anterior escort cells produce Dpp ligand necessary for full-strength BMP signaling in GSCs. Furthermore, these anterior escort cells are maintained specifically by cap cell-secreted Wnt6 ligands: when Wnt6 is knocked down in cap cells, anterior escort cells frequently die and are not replaced, resulting in a loss of Dpp signaling and GSC loss from the niche. Altogether, these data provide direct evidence that anterior escort cells are an essential cell type within the stem cell niche, and they indicate that cap cells maintain anterior escort cells in the niche by promoting anterior escort cell survival through Wnt6 signaling (Wang, 2018).

    Previously, it was held that the GSC niche was composed of cap cells located at the anterior tip of the germaria. Cap cells produce BMP ligands to inhibit differentiation, and they anchor GSCs via DE-cadherin-mediated adherens junctions for continuous self-renewal. This study demonstrates that, in addition to cap cells, the anterior-most escort cells are required to maintain GSCs in the niche. Although these anterior escort cells have not been identified with a specific cell marker, multiple lines of evidence point to anterior escort cells having a crucial niche function. First, like cap cells, anterior escort cells form adherens junctions with GSCs via DE-cadherin, and when DE-cadherin is knocked down in all escort cells, GSCs are lost; this requirement suggests that anterior escort cells participate with cap cells in physically attaching GSCs in the niche. Second, when all escort cells are challenged and dying, as a result of either impaired Wnt signaling or direct killing with hid, remaining escort cells cluster in the anterior around the GSCs. GSC loss is evident only after nearly all escort cells have died, leaving visible anterior vacancies around the GSCs. Third, when all escort cells are dying, GSCs lose the full-strength BMP signaling that is necessary to maintain the stem-cell state; in control germaria, the BMP ligand Dpp is expressed exclusively in escort cells of Region 1, primarily in the anterior-most escort cells, in an apparently transient manner. Fourth, Wnt6 ligand is required specifically in cap cells and not in escort cells for maintaining anterior escort cell survival, for maintaining anterior escort cell architecture within the niche, for full-strength BMP signaling in GSCs, and for maintaining GSCs in the niche. Together, these data demonstrate that anterior escort cells are crucial components of the GSC niche. Furthermore, anterior escort cells share the niche hallmarks of dpp expression and DE-cadherin attachments to GSCs, both of which are required in escort cells as well as cap cells for GSC maintenance in the niche (Wang, 2018).

    This model of escort cell participation in the GSC niche is consistent with and extends some previous observations. One study (Wang, 2011) showed that when escort cells were knocked down for the histone modifier eggless, escort cells slowly died with a concomitant loss of GSCs, but this phenotype was not quantified or further investigated. Several labs have shown by RT-PCR or by a conventional and challenging in situ hybridization method that escort cells contribute Dpp ligand to the germarium environment. Importantly, when dpp was knocked down in all escort cells with adult-specific expression of ptc-Gal4, GSC loss was observed. These results are all consistent with the current data and model of anterior escort cell function (Wang, 2018).

    Escort cells are better known as the 'differentiation niche', because they are required for the proper differentiation of GSC progeny. Indeed, several studies have shown that escort cells, and specifically Wnt signaling in escort cells, are essential for germline differentiation. Like these groups, this study observed a germline differentiation phenotype when Wnt signaling was compromised in escort cells in addition to the GSC-loss phenotype, but, interestingly, the two phenotypes were inversely correlated: manipulations that resulted in the greatest number of undifferentiated germ cells (such as sggS9A overexpression or moderate induction of hid) were those that maintained a moderate escort cell number, and these displayed the lowest level of GSC loss; reciprocally, manipulations that resulted in the greatest loss of GSC (such as Axn overexpression or high induction of hid) were those that induced a severe loss of escort cells, and these displayed the lowest levels of undifferentiated germ cells. It is concluded that the earliest phenotype caused by escort cell death is a failure of germline differentiation, appearing as a germline tumor. The loss of GSCs from the niche is a later phenotype, appearing only after nearly all the escort cells have been lost from the germarium, which happens when Wnt signaling is strongly impaired or when hid is highly expressed. The inverse correlation makes sense because when GSCs are lost from the niche, fewer of their cystoblast progeny are born to populate a germline tumor. It is expectrf that studies analyzing the role of Wnt in germ cell differentiation might not have detected the weak loss of GSCs in their strong differentiation mutants, and further, weak GSC loss is hard to detect in the presence of many undifferentiated germ cells because of the large number of spectrosomes. These two phenotypes represent two distinct functions of escort cells: promoting germline stemness in the GSC niche at the anterior of the germarium, and promoting germline differentiation in the differentiation niche in more posterior positions. Both Wnt6 and Hh, signaling from cap cells to anterior escort cells, are positioned appropriately to signal this switch in escort cell function (Wang, 2018 and references therein).

    Intriguingly, this study found that cap cells signal via Wnt6 to anterior escort cells to promote their survival. This signaling between two different niche cell types is crucial for niche function, as without Wnt6, niche escort cells die, dpp expression in anterior escort cells is lost, BMP signaling in GSCs is decreased, and GSCs are lost. It seems likely that the loss of dpp expression is an indirect effect of losing the anterior escort cells themselves, rather than a direct effect of the loss of Wnt signaling, as it has been reported that cap cell-derived Wnt ligands limit rather than promote dpp signaling. Also, it has been previously shown that cap cell-derived Hh ligands promote dpp expression in escort cells. Thus, a model is favored in this study in which Wnt6 is important for anterior escort cell survival and recruitment. In support of this model, it was observed that in the presence of intact Wnt6 signaling, when escort cells were killed by hid, surviving escort cells routinely clustered at the GSC niche, even though escort cell death occurred evenly across the germarium. Indeed, GSCs were maintained in the niche until virtually all escort cells had died, when there were few or no remaining escort cells to fill vacancies in the niche. Escort cells behaved very differently, however, when Wnt6 was knocked down in cap cells. Without Wnt6, an increase was observed in cell death specifically in the anterior of the germarium, and lost cells were not replaced, leaving functional vacancies in the GSC niche. Thus, cap cell-produced Wnt6 seems to ensure continued occupancy of escort cells in the GSC niche. It is also possible that Wnt6 could coordinate the niche cell types during changes in niche size, as previous studies have shown that both the numbers of GSCs and cap cells decrease in response to a poor diet and increase under rich food conditions (Wang, 2018).

    Anterior escort cell replacements appear to derive from the more posterior cycling somatic cells, labeled by FUCCI (fluorescence ubiquitination-based cell cycle indicator). Based on recent work by Reilein (2017), it appears that these cycling cells are stem cells from which both follicle and escort cells derive. The anterior migration of stem cell daughters into escort cell territory has been captured by live imaging ex vivo (Reilein, 2017), strong evidence that anterior movement occurs also in vivo. Furthermore, this study observed some BrdU-labeled cells that probably migrated from this cycling area into Region 1. Thus, Wnt6 might act as a homing signal for newly born escort cells, attracting them to the anterior-most location in the GSC niche (Wang, 2018).

    It has been proposed that a stem cell niche acts as an 'interlocutor' or interpreter, relaying information about the status of the organism or tissue to the stem cells. Because of this interpreter role, it is expected that niches would be composed of multiple cell types to report different types of information. Indeed, some mammalian somatic stem cell niches are known to be composed of multiple cell types. The bone marrow niche for hematopoietic stem cells (HSCs), one of the best understood mammalian stem cell niches, is composed of multiple cell types, including different endothelial cells in the circulatory system and cells in the nervous and immune systems. Another example is the mammalian intestinal stem cell niche, composed of paneth cells, pericryptic fibroblasts and smooth muscle cells. This study has demonstrated that escort cells are an essential and non-redundant niche cell type, acting in concert with the cap cells to form the Drosophila ovarian GSC niche. Following the interlocutor model, what could each of these two cell types be communicating to the GSCs? Germline differentiation and the development of gametes need to be coordinated with at least two types of information: nutritional status of the organism, and the level of threat to the genome from transposable elements. The cap cells are known to gather information on the nutritional status of the organism, as they change their number or alter the availability of signaling ligands in response to diet. Interestingly, a recent study has shown that escort cells respond to transposable element activation by downregulating Wnt4 levels, a potentially direct mechanism by which escort cells communicate the level of transposon threat to the germline. In this scenario, increased transposon activity leads to reduced Wnt4 signaling, and the data shows that reduced Wnt4 results in potentially corrupted GSCs being lost from the perpetuity of the niche. Thus, both cap cells and escort cells are poised to transmit crucial information relevant to gamete development through the GSC niche (Wang, 2018).

    DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1

    Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. This study report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, this study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila. Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. The study shows that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis (Wong, 2017).

    Recent studies have uncovered a class of stable intronic sequence RNAs (sisRNAs) that are derived from the introns post splicing. sisRNAs are present in various organisms such as viruses, yeast, Drosophila, Xenopus, and mammals. Studies in Drosophila and mammalian cells suggest that sisRNAs function in regulating the expression of their parental genes (host genes where they are derived from) via positive or negative feedback loops. In yeast, sisRNAs are involved in promoting robustness in response to stress, while in Drosophila, sisRNAs have been shown to be important for embryonic development. However, very little is understood about the biological functions of sisRNAs in terms of regulating cellular processes such as differentiation, proliferation, and cell death (Wong, 2017).

    The Drosophila genome encodes for several double-stranded RNA (dsRNA) binding proteins that localize to the nucleus. Most of them have been found to regulate specific RNA-mediated processes such as RNA editing, X chromosome activation, and miRNA biogenesis. The Disco-interacting protein 1 (DIP1) is a relatively less characterized dsRNA binding protein that has been implicated in anti-viral defense and localizes to the nucleus as speckles. Otherwise, not much is known about the biological processes regulated by DIP1 (Wong, 2017 and references therein).

    This paper show that the regulation of a Drosophila sisRNA sisR-1 by DIP1 is important for keeping female germline stem cell homeostasis in place. DIP1 is shown to regulate INE-1 sisRNAs and localizes to a previously undescribed nuclear body around the fourth chromosomes, called the satellite body. The regulation of sisR-1, which is not on the fourth chromosome, by DIP1 presumably does not occur in the satellite bodies (Wong, 2017).

    The results reveal the importance of the regulation of sisRNA activity/expression in GSC-niche occupancy. It is proposed that the sisR-1 axis maintains GSCs in the niche, however, uncontrolled accumulation of sisR-1 due to its unusual stability can lead to increase number of GSCs at the niche. DIP1 in turn limits the build-up of sisR-1 to maintain ~2 GSCs per niche. GSC-niche occupancy is highly regulated by homeostatic mechanisms via negative feedback loops at the cellular and molecular levels. Misregulation of the niche may pose a problem as it allows for a greater chance of GSCs to accumulate mutations that may lead to tumor formation. On the other hand, mechanisms that promote GSC-niche occupancy may be important to facilitate the replenishment of GSCs during aging. Understanding the control of stem cell-niche occupancy will provide important insights to reproduction, cancer, and regenerative medicine (Wong, 2017).

    In a large-scale RNAi screen for genes that regulate GSC self-renewal and differentiation, rga was identified as a gene required for GSC differentiation. How rga regulates GSC self-renewal is currently unknown but the current data suggest that GSC-niche adhesion and Mei-P26 are involved. The rga gene encodes for the NOT2 protein in the CCR4 deadenylase complex. Surprisingly, studies have shown that other components of the CCR4 complex such as CCR4, Not1, and Not3 function in promoting GSC maintenance. Interestingly, Twin has been proposed to function with distinct partners to mediate different effects on GSC fates. This suggests that other components such as CCR4 can also have additional functions outside the CCR4-NOT deadenylase complex in mediating GSC maintenance, thus affecting GSCs in opposite ways to Rga (Wong, 2017).

    This study puts forward a proposed model for sisR-1-mediated silencing. It is hypothesized that folded sisR-1 harboring a 3' tail may form a ribonucleoprotein complex, which confers its stability, and allows scanning for its target via its 3' tail. Binding of the 3' tail to the target may promote local unwinding of sisR-1 as the 3' end of ASTR invades to form a more stable 76 nt duplex. This study shows that, in principle, it is possible to design a chimeric sisRNA to target a long ncRNA of interest such as rox1. In future, sisRNA can be potentially developed as tools to regulate nuclear RNAs of interest. Clearly, the efficiency and specificity of sisRNA-mediated silencing need to be optimized. Because sisRNA-mediated target degradation requires a more extensive base-pairing between sisRNA and the target, the chances of off-target effects ought to be lower than siRNAs and antisense oligonucleotides. In broader terms, this study provides a paradigm, which encourages exploration of whether other sisRNAs or ncRNAs utilize a similar silencing strategy as sisR-1 (Wong, 2017).

    This study describes a nuclear body (named satellite body) that associates with the fourth chromosomes. Satellite body adds to an existing group of nuclear bodies (nucleolus, HLB, and pearl) that associate with specific genomic loci. It is generally believed that formation of such nuclear bodies correlates with a high concentration of RNA transcribed from the tandemly repeated gene loci. The formation of satellite bodies around the fourth chromosomes probably reflects a high concentration of DIP1 in regulating INE-1 sisRNAs transcribed there. The formation of satellite bodies may be promoted by the high concentration of INE-1 sisRNAs transcribed on the fourth chromosomes, and may facilitate the decay of INE-1 sisRNAs . It is speculated that in the nucleoplasm, DIP1 that does not form observable satellite bodies is sufficient to regulate sisRNAs such as sisR-1 transcribed from other chromosomes. Since DIP1 is a dsRNA binding protein, it may bind to mature sisRNAs to destabilize them. It may do so by recruiting RNA degradation factors (such as nuclear exosomes) or introducing RNA modification to 'mark' sisRNAs for degradation. In future, it will be important to identify more components of the satellite bodies and their dynamics during differentiation and in response to stimuli in order to better understand the molecular mechanism of sisRNA metabolism (Wong, 2017).

    The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells

    The preservation of germ cell sexual identity is essential for gametogenesis. This study shows that H3K9me3-mediated gene silencing is integral to female fate maintenance in Drosophila germ cells. Germ cell specific loss of the H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, WDE, and HP1a, leads to ectopic expression of genes, many of which are normally expressed in testis. SETDB1 controls the accumulation of H3K9me3 over a subset of these genes without spreading into neighboring loci. At phf7, a regulator of male germ cell sexual fate, the H3K9me3 peak falls over the silenced testis-specific transcription start site. Furthermore, H3K9me3 recruitment to phf7 and repression of testis-specific transcription is dependent on the female sex determination gene Sxl. Thus, female identity is secured by an H3K9me3 epigenetic pathway in which Sxl is the upstream female-specific regulator, SETDB1 is the required chromatin writer, and phf7 is one of the critical SETDB1 target genes (Smolko, 2018).

    In metazoans, germ cell development begins early in embryogenesis when the primordial germ cells are specified as distinct from somatic cells. Specified primordial germ cells then migrate into the embryonic gonad, where they begin to exhibit sex-specific division rates and gene expression programs, ultimately leading to meiosis and differentiation into either eggs or sperm. Defects in sex-specific programming interferes with germ cell differentiation leading to infertility and germ cell tumors. Successful reproduction, therefore, depends on the capacity of germ cells to maintain their sexual identity in the form of sex-specific regulation of gene expression (Smolko, 2018).

    In Drosophila melanogaster, germ cell sexual identity is specified in embryogenesis by the sex of the developing somatic gonad. However, extrinsic control is lost after embryogenesis and sexual identity is preserved by a cell-intrinsic mechanism. The Sex-lethal (Sxl) female-specific RNA binding protein is an integral component of the cell-intrinsic mechanism, as loss of Sxl specifically in germ cells leads to a global upregulation of spermatogenesis genes and a germ cell tumor phenotype. Remarkably, sex-inappropriate transcription of a single gene, PHD finger protein 7 (phf7), a key regulator of male identity, is largely responsible for the tumor phenotype. Depletion of phf7 in mutants lacking germline Sxl suppresses the tumor phenotype and restores oogenesis. Moreover, forcing PHF7 protein expression in ovarian germ cells is sufficient to disrupt female fate and give rise to a germ cell tumor. Interestingly, sex-specific regulation of phf7 is achieved by a mechanism that relies primarily on alternative promoter choice and transcription start site (TSS) selection. Sex-specific transcription produces mRNA isoforms with different 5' untranslated regions that affect translation efficiency, such that PHF7 protein is only detectable in the male germline. Although the Sxl protein is known to control expression post-transcriptionally in other contexts the observation that germ cells lacking Sxl protein show defects in phf7 transcription argues that Sxl is likely to indirectly control phf7 promoter choice. Thus, how this sex-specific gene expression program is stably maintained remains to be determined (Smolko, 2018).

    This study reports the discovery that female germ cell fate is maintained by an epigenetic regulatory pathway in which SETDB1 (aka EGGLESS, KMT1E, and ESET) is the required chromatin writer and phf7 is one of the critical SETDB1 target genes. SETDB1 trimethylates H3K9 (H3K9me3), a feature of heterochromatin. Using tissue-specific knockdown approaches this study established that germ cell specific loss of SETDB1, its protein partner WINDEI [WDE, aka ATF7IP, MCAF1 and hAM10], and the H3K9me3 reader, HP1a, encoded by the Su(var)205 locus, leads to ectopic expression of euchromatic protein-encoding genes, many of which are normally expressed only in testis. It was further found that H3K9me3 repressive marks accumulate in a SETDB1 dependent manner at 21 of these ectopically expressed genes, including phf7. Remarkably, SETDB1 dependent H3K9me3 deposition is highly localized and does not spread into neighboring loci. Regional deposition is especially striking at the phf7 locus, where H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific TSS. Lastly, this study found that H3K9me3 accumulation at many of these genes, including phf7, is dependent on Sxl. Collectively these findings support a model in which female fate is preserved by deposition of H3K9me3 repressive marks on key spermatogenesis genes (Smolko, 2018).

    This study reveals a role for H3K9me3 chromatin, operationally defined as facultative heterochromatin, in securing female identity by silencing lineage-inappropriate transcription. H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, its binding partner WDE, and the H3K9 binding protein HP1a, are required for silencing testis gene transcription in female germ cells. These studies further suggest a mechanism in which SETDB1, in conjunction with the female fate determinant Sxl, controls transcription through deposition of highly localized H3K9me3 islands on a select subset of these genes. The male germ cell sexual identity gene phf7 is one of the key downstream SETDB1 target genes. H3K9me3 deposition on the region surrounding the testis-specific TSS guaranties that no PHF7 protein is produced in female germ cells. In this model, failure to establish silencing leads to ectopic PHF7 protein expression, which in turn drives aberrant expression of testis genes and a tumor phenotype (Smolko, 2018).

    Prior studies have established a role for SETDB1 in germline Piwi-interacting small RNA (piRNA) biogenesis and TE silencing. However, piRNAs are unlikely to contribute to sexual identity maintenance as mutations that specifically interfere with piRNA production, such as rhino, do not cause defects in germ cell differentiation. These findings, together with the observation that rhino does not control sex-specific phf7 transcription, suggests that the means by which SETDB1 methylates chromatin at testis genes is likely to be mechanistically different from what has been described for piRNA-guided H3K9me3 deposition on TEs.

    The accumulation of H3K9me3 at many of these genes, including phf7, is dependent on the presence of Sxl protein. Thus, these studies suggest that Sxl is required for female-specific SETDB1 function. Sxl encodes an RNA binding protein known to regulate its target genes at the posttranscriptional levels. Sxl control may therefore be indirect. However, studies in mammalian cells suggest that proteins with RNA binding motifs are important for H3K9me3 repression, raising the tantalizing possibility that Sxl might play a more direct role in governing testis gene silencing. Further studies will be necessary to clarify how the sex determination pathway feeds into the heterochromatin pathway (Smolko, 2018).

    phf7 stands out among the cohort of genes regulated by facultative heterochromatin because of its pivotal role in controlling germ cell sexual identity. Because ectopic protein expression is sufficient to disrupt female fate, tight control of phf7 expression is essential. phf7 regulation is complex, employing a mechanism that includes alternative promoter usage and TSS selection. This study reports that SETDB1/H3K9me3 plays a critical role in controlling phf7 transcription. In female germ cells, H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific transcription start site. Dissolution of the H3K9me3 marks via loss of Sxl or SETDB1 protein is correlated with transcription from the upstream testis-specific site and ectopic protein expression, demonstrating the functional importance of this histone modification. Together, these studies suggest that maintaining the testis phf7 promoter region in an inaccessible state is integral to securing female germ cell fate (Smolko, 2018).

    Although the loss of H3K9me3 pathway members in female germ cells leads to the ectopic, lineage-inappropriate transcription of hundreds of genes, integrative analysis identified only 21 SETDB1/H3K9me3 regulated genes. Given that one of these genes is phf7 and that ectopic PHF7 is sufficient to destabilize female fate, it is likely that inappropriate activation of a substantial number of testis genes is a direct consequence of ectopic PHF7 protein expression. How PHF7 is able to promote testis gene transcription is not yet clear. PHF7 is a PHD-finger protein that preferentially binds to H3K4me2, a mark associated with poised, but inactive genes and linked to epigenetic memory. Thus, one simple model is that ectopic PHF7 binds to H3K4me2 marked testis genes to tag them for transcriptional activation (Smolko, 2018).

    It will be interesting to explore whether any of the other 20 SETDB1/H3K9me3 regulated genes also have reprogramming activity. In fact, ectopic fate-changing activity has already been described for the homeobox transcription factor Lim1 in the eye-antenna imaginal disc. However, whether Lim1 has a similar function in germ cells is not yet known. Intriguingly, protein prediction programs identify three of the uncharacterized testis-specific genes as E3 ligases. SkpE is a member of the SKP1 gene family, which are components of the Skp1-Cullin-F-box type ubiquitin ligase. CG12477 is a RING finger domain protein, most of which are believed to have ubiquitin E3 ligases activity. CG42299 is closely related to the human small ubiquitin-like modifier (SUMO) E3 ligase NSMCE2. Given studies that have linked E3 ligases to the regulation of chromatin remodeling, it is tempting to speculate that ectopic expression of one or more of these E3 ligases will be sufficient to alter cell fate. Future studies focused on this diverse group of SETDB1/H3K9me3 regulated genes and their role in reprogramming may reveal the multiple layers of regulation required to secure cell fate (Smolko, 2018).

    The SETDB1-mediated mechanism for maintaining sexual identity uncovered in this study may not be restricted to germ cells. Recent studies have established that the preservation of sexual identity is essential in the adult somatic gut and gonadal cells for tissue homeostasis. Furthermore, the finding that loss of HP1a in adult neurons leads to masculinization of the neural circuitry and male specific behaviors suggests a connection between female identity maintenance and H3K9me3 chromatin. Thus, it is speculated that SETDB1 is more broadly involved in maintaining female identity (Smolko, 2018).

    These studies highlight an emerging role for H3K9me3 chromatin in cell fate maintenance. In the fission yeast S. pombe, discrete facultative heterochromatin islands assemble at meiotic genes that are maintained in a silent state during vegetative growth. Although less well understood, examples in mammalian cells indicate a role for SETDB1 in lineage-specific gene silencing. Thus, silencing by SETDB1 controlled H3K9 methylation may be a widespread strategy for securing cell fate. Interestingly, H3K9me3 chromatin impedes the reprogramming of somatic cells into pluripotent stem cells (iPSCs). Conversion efficiency is improved by depletion of SETDB1. If erasure of H3K9me3 via depletion of SETDB1 alters the sexually dimorphic gene expression profile in reprogrammed cells, as it does in Drosophila germ cells, the resulting gene expression differences may cause stem cell dysfunction, limiting their therapeutic utility (Smolko, 2018).

    Pgc suppresses the zygotically-acting RNA decay pathway to protect germ plasm RNAs in the Drosophila embryo

    Specification of germ cells is pivotal to ensure continuation of animal species. In many animal embryos, germ cell specification depends on maternally supplied determinants in the germ plasm. Drosophila polar granule component (pgc) mRNA is a component of the germ plasm. pgc encodes a small protein that is transiently expressed in newly formed pole cells, the germline progenitors, where it globally represses mRNA transcription. pgc is also required for pole cell survival, but the mechanism linking transcriptional repression to pole cell survival remains elusive. This study reports that pole cells lacking pgc show premature loss of germ plasm mRNAs, including the germ cell survival factor, nanos, and undergo apoptosis. pgc (-) pole cells misexpress multiple miRNA genes. Reduction of miRNA pathway activity in pgc (-) embryos partially suppressed germ plasm mRNA degradation and pole cell death, suggesting that Pgc represses zygotic miRNA transcription in pole cells to protect germ plasm mRNAs. Interestingly, germ plasm mRNAs are protected from miRNA-mediated degradation in vertebrates, albeit by a different mechanism. Thus, independently evolved mechanisms are used to silence miRNAs during germ cell specification (Hanyu-Nakamura, 2019).

    Reactive oxygen species signaling in primordial germ cell development in Drosophila embryos

    REDOX mechanisms that induce biosynthesis of the reactive oxygen species (ROS) have attracted considerable attention due to both the deleterious and beneficial responses elicited by the reactive radical. In several organisms including Drosophila melanogaster, modulation of ROS activity is thought to be crucial for the maintenance of cell fates in developmental contexts. Interestingly, REDOX mechanisms have been shown to be involved in maintaining progenitor fate of stem cells as well as their proliferation and differentiation. This study has explored the possible functions of ROS during proper specification and developmental progression of embryonic primordial germ cells (PGCs). Indicating its potential involvement in these processes, ROS can be detected in the embryonic PGCs and the surrounding somatic cells from very early stages of embryogenesis. Using both "loss" and "gain" of function mutations in two different components of the REDOX pathway, this study shows that ROS levels are likely to be critical in maintaining germ cell behavior, including their directed migration. Altering the activity of a putative regulator of ROS also adversely influences the ability of PGCs to adhere to one another in cellular blastoderm embryos, suggesting potential involvement of this pathway in orchestrating different phases of germ cell migration (Syal, 2020).

    Maternally inherited intron coordinates primordial germ cell homeostasis during Drosophila embryogenesis

    Primordial germ cells (PGCs) give rise to the germline stem cells (GSCs) in the adult Drosophila gonads. Both PGCs and GSCs need to be tightly regulated to safeguard the survival of the entire species. During larval development, a non-cell autonomous homeostatic mechanism is in place to maintain PGC number in the gonads. Whether such germline homeostasis occurs during early embryogenesis before PGCs reach the gonads remains unclear. Previous work has shown that the maternally deposited stable intronic sequence RNA (sisRNA) sisR-2 can influence GSC number in the female progeny. This study uncover the presence of a homeostatic mechanism regulating PGCs during embryogenesis. sisR-2 represses PGC number by promoting PGC death. Surprisingly, increasing maternal sisR-2 leads to an increase in PGC death, but no drop in PGC number was observed. This is due to ectopic division of PGCs via the de-repression of Cyclin B, which is governed by a genetic pathway involving sisR-2, bantam and brat. A cell autonomous model is proposed whereby germline homeostasis is achieved by preserving PGC number during embryogenesis (Osman, 2020).

    Nuclear lamina dysfunction triggers a germline stem cell checkpoint

    LEM domain (LEM-D) proteins are conserved components of the nuclear lamina (NL) that contribute to stem cell maintenance through poorly understood mechanisms. The Drosophila emerin homolog Otefin (Ote) is required for maintenance of germline stem cells (GSCs) and gametogenesis. This study shows that ote mutants carry germ cell-specific changes in nuclear architecture that are linked to GSC loss. Strikingly, both GSC death and gametogenesis are rescued by inactivation of the DNA damage response (DDR) kinases, ATR and Chk2. Whereas the germline checkpoint draws from components of the DDR pathway, genetic and cytological features of the GSC checkpoint differ from the canonical pathway. Instead, structural deformation of the NL correlates with checkpoint activation. Despite remarkably normal oogenesis, rescued oocytes do not support embryogenesis. Taken together, these data suggest that NL dysfunction caused by Otefin loss triggers a GSC-specific checkpoint that contributes to maintenance of gamete quality (Barton, 2018).

    The Drosophila emerin homolog Ote has an essential requirement for GSC survival and germ cell differentiation. This study shows that Ote loss causes GSC-specific nuclear defects that include a thickened and irregular NL and aggregation of heterochromatin. Strikingly, inactivation of two DDR kinases, ATR, and Chk2, rescues oogenesis in ote-/- females, a rescue that is cell-type specific. Genetic and cytological features of the checkpoint pathway present in ote mutant GSCs differ from those found in canonical DDR pathways. In addition, although heterochromatin coalesce is present, such defects by themselves do not trigger the checkpoint. Instead, the data correlate Chk2 activity with defects in NL structure, indicating that NL dysfunction is responsible for the activation of a checkpoint pathway in GSCs. Despite remarkably normal oogenesis, rescued oocytes do not support embryogenesis. It is suggested that this NL checkpoint pathway functions in GSCs to ensure that only healthy gametes are passed on to the next generation (Barton, 2018).

    These studies identify ATR as the critical responder kinase and Chk2 as the critical transducer kinase in the NL checkpoint. This signaling axis differs from the canonical ATM-to-Chk2 or ATR-to-Chk1 axes. Several factors might contribute to the choice of responder and transducer kinase. First, species-specific constraints might exist. ATR, but not ATM is essential in mammals, whereas ATM, but not ATR, is essential in Drosophila. Second, cell-type specific distinctions are apparent. In both the fly and mouse germline, persistent meiotic double-strand breaks activate ATR and Chk2, implying that the ATR-Chk2 axis might be dominant in germ cells. Third, the nature of the trigger might influence which proteins are involved in signaling. For example in Drosophila, ATR and Chk2 are both required for the patterning defects caused by a failure to repair meiotic double-strand breaks. However, in DNA-damaged GSCs, ATR protects against GSC death, whereas Chk2 promotes it. These data suggest that in the case of the NL checkpoint, both ATR and Chk2 promote germ cell death. These studies add to growing evidence that the DDR pathway is modular, with selective use of pathway components in response to various cellular stresses (Barton, 2018).

    Activated Chk2 is commonly associated with phosphorylation and activation of p53. Canonically, p53 activation leads to cell cycle arrest and apoptosis. These studies demonstrate that GSC loss persists in ote-/-; p53-/- females, suggesting that classical apoptosis is not responsible for GSC death. These findings are consistent with the absence of classic markers of apoptosis in ote mutants and observations that the p53 regulatory network differs in GSCs. Recently, an alternative cell death pathway was identified in spermatogonia of Drosophila testes. This pathway is responsible for spontaneous elimination of spermatogonia, using activated lysosomal and mitochondrial-associated factors. Additional studies are needed to determine whether ATR/Chk2-dependent GSC loss in ote mutants targets a similar pathway (Barton, 2018).

    The data suggest that NL dysfunction is the primary cause of the ATR/Chk2 checkpoint in GSCs. Notably, NL defects are found only in affected cells and persist in rescued chk2-/-, ote-/- double mutants. Multiple mechanisms might connect nuclear architecture changes to ATR/Chk2 activation. First, altered NL structure might change genomic contacts needed for appropriate transcriptional regulation, with resulting gene expression changes prompting activation of the checkpoint. While global transcriptional changes during oogenesis were not observed, identification of transcriptional changes specific to GSCs or early germ cells would have been masked in these studies. Second, disruptions in the NL might affect trafficking of products between the nucleus and cytoplasm. Notably, a recent study identified large ribonucleoparticles (megaRNPs) that exit the nucleus by egress or budding through the inner and outer nuclear membranes, a process disrupted by defects in the NL. As such, it remains possible that the thickened NL in ote-/- GSCs disrupts large ribonucleoprotein (megaRNP) egress, leading to cellular stress and ATR/Chk2 activation. Third, defects in the NL structure might alter scaffolding of components of the DDR pathway, leading to checkpoint activation. Indeed, proteomic studies from Drosophila cultured somatic cells found that Ote interacted with proteins involved in DNA replication and repair, implying that Ote might assemble responder and transducer kinases complexes at the NL. However, observations that the ATR/Chk2-dependent checkpoint is GSC-specific, coupled with findings that meiotic double-strand breaks are repaired appropriately in chk2-/-, ote-/- germaria, argue against this model. Fourth, structural alteration in the nuclear envelope itself might trigger ATR/Chk2 activation. Indeed, emerging evidence implicates ATR as a general sensor of the structural integrity of cellular components. Further studies are needed to identify how NL dysfunction triggers the GSC-specific checkpoint (Barton, 2018).

    Mutations in NL LEM-D proteins cause dystrophic diseases. Much evidence suggests that these diseases result from compromised stem cell populations that underlie the defects in tissue homeostasis. Indeed, a wealth of evidence links NL defects to increased DNA damage. The data are consistent with these reports, as it was shown that elevated accumulation of the commonly used DNA damage marker. However, this study found that phosphorylation of the H2A variant occurs downstream of Chk2, suggesting that accumulation of DNA damage in cells with a dysfunctional NL might be a consequence of cells dying, not the primary cause. These unexpected results suggest that caution is needed in linking causation of γH2Av/H2X accumulation to DNA damage and a failure in DNA repair. Indeed, recent studies of progerin-expressing cells indicated that the cellular defect in Hutchinson-Gilford progeria cells does not lie in defective DNA repair and DNA damage, even though these cells accumulate phosphorylated H2AX. These findings establish a new context for consideration of mechanisms of laminopathic diseases, suggesting that detrimental effects of NL dysfunction are primary events that are linked to checkpoint activation and stem cell loss (Barton, 2018).

    Trajectory mapping of the early Drosophila germline reveals controls of zygotic activation and sex differentiation

    Germ cells in Drosophila melanogaster are specified maternally shortly after fertilization and are transcriptionally quiescent until their zygotic genome is activated to sustain further development. To understand the molecular basis of this process, this study analyzed the progressing transcriptomes of early male and female germ cells at the single-cell level between germline specification and coalescence with somatic gonadal cells. The data comprehensively covered zygotic activation in the germline genome, and analyses on genes that exhibit germline-restricted expression revealed that polymerase pausing and differential RNA stability are important mechanisms that establish gene expression differences between the germline and soma. In addition, an immediate bifurcation was observed between the male and female germ cells as zygotic transcription begins. The main difference between the two sexes is an elevation in X Chromosome expression in females relative to males signifying incomplete dosage compensation with a few select genes exhibiting even higher expression increases. These indicate that the male program is the default mode in the germline that is driven to female development with a second X Chromosome (Li, 2021).

    Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching

    Ribosomes have long been thought of as homogeneous macromolecular machines, but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. This study characterised ribosomal protein heterogeneity across 4 tissues of Drosophila melanogaster. Testes and ovaries were found to contain the most heterogeneous ribosome populations, which occurs through a combination of paralog-enrichment and paralog-switching. Structures were solved of ribosomes purified from in vivo tissues by cryo-EM, revealing differences in precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-switching could alter the ribosome surface, enabling different proteins to regulate translation. One testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of ribosome heterogeneity. Overall, this work leads to a proposal that mRNA translation might be regulated in the gonads through ribosome heterogeneity, providing a potential means of ribosome specialisation (Hopes, 2021).

    See also the oogenesis site.


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