Following fertilization, a Drosophila embryo undergoes 14 consecutive nuclear divisions to give rise to the cellular blastoderm. While the initial nuclear divisions take place in the center of the embryo, the nuclei begin to migrate toward the periphery around nuclear cycle (NC) 4–6 and reach the cortex at NC9/10 (Farrell and O'Farrell, 2014). Even before bulk nuclear migration commences, a few nuclei move toward the posterior of the embryo, enter a specialized, maternally derived cytoplasm known as the pole plasm, and induce the formation of pole buds (PBs) (Williamson and Lehmann, 1996; Wilson and Macdonald, 1993; Wylie, 1999). The centrosomes associated with these nuclei trigger the release of pole plasm constituents from the posterior cortex and orchestrate precocious cellularization to form the primordial germ cells (PGCs), the progenitors of the germline stem cells in adult gonads (Lerit and Gavis, 2011; Raff and Glover, 1989). Unlike pole cell nuclei, somatic nuclei continue synchronous divisions after they reach the surface of the embryo until NC 14 when they cellularize (Blythe and Wieschaus, 2015).

The timing of cellularization is not the only difference between the soma and PGCs. Although newly formed PGCs divide after they are formed, they undergo only one or two asynchronous divisions before exiting the cell cycle. Another key difference is in transcriptional activity (Nakamura and Seydoux, 2008). Transcription commences in the embryo during NC 6–7 when a select number of genes are active (Ali-Murthy et al., 2013). Transcription is more globally upregulated when the nuclei reach the surface, and by the end of NC 14, zygotic genome activation (ZGA) is complete (Ali-Murthy et al., 2013; Harrison and Eisen, 2015). This transition is marked by high levels of phosphorylation of residues Serine 5 (Ser5) and Serine 2 (Ser2) in the C-terminal domain (CTD) of RNA polymerase II (Schaner et al., 2003; Seydoux and Dunn, 1997). By contrast, in newly formed PGCs, transcription is switched off, and PGC nuclei have only residual amounts of Ser5 and Ser2 CTD phosphorylation (Deshpande, 2004; Martinho et al., 2004; Seydoux and Dunn, 1997). Moreover, and consistent with their transcriptionally quiescent status, other changes in chromatin architecture that accompany ZGA are also blocked in PGCs (Schaner et al., 2003).

Three different genes, nanos (nos), polar granule component (pgc), and germ cell-less (gcl), are known to be required for establishing transcriptional quiescence in newly formed PGCs (Deshpande et al., 2005; Deshpande, 2004; Deshpande et al., 1999; Hanyu-Nakamura et al., 2008; Kobayashi et al., 1996; Leatherman et al., 2002; Martinho et al., 2004). The PGCs in embryos derived from mothers carrying mutations in these genes fail to inhibit transcription, and this compromises germ cell specification and disrupts germ cell migration. (As these are maternal effect genes, embryos derived from nos/pgc/gcl mothers display the resulting mutant phenotypes and will be referred to as nos/pgc/gcl here onwards.) Interestingly, these three genes share only a few targets, suggesting overlapping yet distinct mechanisms of action. Nos is a translation factor and thus must block transcription indirectly. Together with the RNA-binding protein Pumilio (Pum), Nos interacts with recognition sequences in the 3’-untranslated regions (3’UTRs) of mRNAs and inhibits their translation (Asaoka et al., 2019; Sonoda and Wharton, 1999; Wharton and Struhl, 1991). Currently, the key mRNA target(s) that Nos-Pum repress to block transcription is unknown; however, in nos and pum mutants, PGC nuclei display high levels of Ser5 and Ser2 CTD phosphorylation and activate transcription of gap and pair-rule patterning genes and the sex determination gene Sex-lethal (Sxl) (Deshpande et al., 2005; Deshpande et al., 1999). pgc encodes a nuclear protein that binds to the transcriptional elongation kinase p-TEFb, blocking Ser5 CTD phosphorylation (Hanyu-Nakamura et al., 2008). In pgc mutant pole cells, Ser5 phosphorylation is enhanced, as is transcription of several somatic genes, including genes involved in terminal patterning (Deshpande, 2004; Martinho et al., 2004).

While the primary function of nos and pgc appears to be blocking ZGA in PGCs, gcl has an earlier function, which is to turn off transcription of genes activated in somatic nuclei prior to nuclear migration (Leatherman et al., 2002). Targets of gcl include two X-chromosome counting elements (XCEs), scute (sc/sis-b) and sisterless-a (sis-a), that function to turn on the sex determination gene, Sxl, in female soma (Cline and Meyer, 1996; Salz and Erickson, 2010). gcl embryos not only fail to shut off sis-a and sis-b transcription in PBs, but also show disrupted PGC formation. In some gcl embryos, PGC formation fails completely, while in other embryos only a few PGCs are formed (Cinalli and Lehmann, 2013; Jongens et al., 1992; Lerit et al., 2017; Robertson et al., 1999). In this respect, gcl differs from nos and pgc, which have no effect on the process of PGC formation, but instead interfere with the specification of PGC identity.

Studies by Leatherman et al., 2002 suggested that the defects in PGC formation in gcl mutant embryos are linked to failing to inhibit somatic transcription. They found that when PBs first form during NC 9 in wild-type (WT) embryos, levels of CTD phosphorylation PB are only marginally less than in nuclei elsewhere in the embryo. However, by NC 10, there was a dramatic reduction in CTD phosphorylation even before PBs cellularize. By contrast, in gcl mutant embryos, about 90% of the NC 10 PB nuclei had CTD phosphorylation levels approaching that of somatic nuclei. Moreover, this number showed an inverse correlation with the number of PGCs in blastoderm stage gcl embryos. Whereas WT blastoderm embryos have >20 PGCs per embryo, gcl embryos had on average just three PGCs under their culturing conditions. Interestingly, expression of the mouse homologue of Gcl protein, mGcl-1, can rescue the gcl phenotype in Drosophila (Leatherman et al., 2000). Supporting the conserved nature of the involvement of Gcl during transcriptional suppression, a protein complex between mGcl-1 and the inner nuclear membrane protein LAP2β is thought to sequester E2F:D1 to reduce transcriptional activity of E2F:D1 (Nili et al., 2001).

The connection Leatherman et al. postulated between failing to turn off ongoing transcription and defects in PGC formation in gcl mutants is controversial and unresolved. This model predicts that a non-specific inhibition of polymerase II should be sufficient to rescue PGC formation in gcl embryos. However, Cinalli and Lehmann, 2013 found that the PGC formation defects seen in gcl embryos were not rescued after injection of the RNA polymerase inhibitor, α-amanitin. Since α-amanitin treatment disrupted somatic cellularization without impacting PGC formation in WT embryos, they concluded that it effectively blocked polymerase transcription. On the other hand, subsequent experiments by Pae et al., 2017 raised the possibility that inhibiting transcription in pole cell nuclei is a critical step in PGC formation. These authors showed that Gcl is a substrate-specific adaptor for a Cullin3-RING ubiquitin ligase that targets the terminal pathway receptor tyrosine kinase, Torso, for degradation. The degradation of Torso would be expected to prevent activation of the terminal signaling cascade in PGCs. In the soma, Torso-dependent signaling activates the transcription of several patterning genes, including tailless, that are important for forming terminal structures at the anterior and posterior of the embryo (Casanova and Struhl, 1989; Klingler et al., 1988; Martinho et al., 2004; Pignoni et al., 1992; Strecker et al., 1989). Thus, by targeting Torso for degradation, Gcl would prevent the transcriptional activation of terminal pathway genes by the MAPK/ERK kinase cascade in PGCs. Consistent with this possibility, simultaneous removal of gcl and either the Torso ligand modifier, torso-like (tsl) or torso resulted in rescue of germ cell loss induced by gcl. Surprisingly, however, Pae et al., 2017 were unable to observe a similar rescue of gcl phenotype when they used RNAi knockdown to compromise components of the MAP kinase cascade known to act downstream of the Torso receptor (Ambrosio et al., 1989; Duffy and Perrimon, 1994; Furriols and Casanova, 2003). Based on these findings, they proposed that activated Torso must inhibit PGC formation via a distinct non-canonical mechanism that is both independent of the standard signal transduction pathway and does not involve transcriptional activation.

In the studies reported here, we have revisited these conflicting claims by examining the role of Gcl in establishment/maintenance of transcriptional quiescence. The studies of Leatherman et al., 2002 indicated that two of the key X chromosomal counting elements, sis-a and sis-b, were inappropriately expressed in gcl PBs and PGCs. Since transcription factors encoded by these two genes function to activate the Sxl establishment promoter, Sxl-Pe, in somatic nuclei of female embryos, their findings raised the possibility that Sxl might be ectopically expressed in PBs/PGCs of gcl embryos. Here we show that in gcl embryos, Sxl transcription is indeed inappropriately activated in PBs and newly formed PGCs. Moreover, ectopic expression of Sxl in early embryos disrupts PGC formation similar to gcl. Supporting the conclusion that Sxl is a biologically relevant transcriptional target of Gcl, PGC formation defects in gcl embryos can be suppressed either by knocking down Sxl expression using RNAi or by loss-of-function mutations. As reported by Pae et al., 2017, we found that loss of torso-like (tsl) in gcl embryos suppresses PGC formation defects. However, consistent with a mechanism that is tied to transcriptional misregulation, rescue is accompanied by the reestablishment of transcriptional silencing in gcl PGCs. Lending further credence to the idea that transcription misregulation plays an important role in disrupting PGC development in gcl embryos, we found that expression of a mutant form of Torso that is resistant to Gcl-dependent degradation (hereafter referred to as torso^Deg Pae et al., 2017) ectopically activates transcription of two Gcl targets, sis-b and Sxl, in PB and PGC nuclei. In addition, stabilization of Torso in early PGCs also mimics another gcl phenotype, the failure to properly sequester key PGC determinants in PBs and newly formed PGCs.

gcl differs from other known maternally deposited germline determinants in that it is required for the formation of PBs and PGCs. gcl PGCs exhibit a variety of defects during the earliest steps in PGC development. Unlike WT, gcl PGCs fail to properly establish transcriptional quiescence. While other genes like nos and pgc are required to keep transcription shut down in PGCs, their functions only come into play after PGC cellularization (Deshpande, 2004; Deshpande et al., 1999; Martinho et al., 2004). By contrast, gcl acts at an earlier stage beginning shortly after nuclei first migrate into the posterior pole plasm and initiate PB formation. In gcl PBs, ongoing transcription of genes that are active beginning around nuclear cycle 5–6 is not properly turned off. This is not the only defect in germline formation and specification. As in WT, the incoming nuclei (and the centrosomes associated with the nuclei) trigger the release of the pole plasm from the posterior cortex. However, instead of sequestering the germline determinants in PBs so that they are incorporated into PGCs during cellularization, the determinants disperse into the soma where they become associated with the cytoplasmic territories of nearby somatic nuclei. There are also defects in bud formation and cellularization. Like the release and sequestration of germline determinants, these defects have been linked to the actin cytoskeleton and centrosomes (Cinalli and Lehmann, 2013; Lerit et al., 2017).

Two models have been proposed to account for the PGC defects in gcl mutants. In the first, Leatherman et al., 2002 attributed the disruptions in PGC development to a failure to turn off ongoing transcription. The second argues that the role of gcl in imposing transcriptional quiescence is irrelevant (Cinalli and Lehmann, 2013; Pae et al., 2017). Instead, the defects are proposed to arise from a failure to degrade the Torso receptor. In the absence of Gcl-dependent proteolysis, high local concentrations of the Tsl ligand modifier at the posterior pole would activate the Torso receptor. According to this model, the ligand–receptor interaction would then trigger a novel, transcription-independent signal transduction pathway in PBs and PGCs that disrupts their development. These conflicting models raise several questions. Does gcl actually have a role in establishing transcriptional quiescence in PBs and PGCs? If so, is this activity relevant for PB and PGC formation? Is the stabilization of Torso in gcl mutants responsible for the failure to shut down transcription in PBs and PGCs? If not, does gcl target a novel, transcription-independent but Torso-dependent signaling pathway? Is the stabilization of Torso responsible for some of the other phenotypes that are observed in gcl mutants? In the studies reported here we have addressed these outstanding questions, leading to a resolved model of Gcl activity and function.

We show that shutting off transcription is, in fact, a critical function of Gcl protein. As previously documented by Leatherman et al., we find that several of the key X-linked transcriptional activators of Sxl-Pe are not repressed in newly formed PBs and early PGC nuclei, and Sxl-Pe transcription is inappropriately activated in the presumptive germline. In previous studies, we found that ectopic expression of Sxl in nos mutants disrupts PGC specification. In this case, the specification defects in nos embryos can be partially rescued by eliminating Sxl activity (Deshpande et al., 1999). The same is true for gcl mutants: elimination or reduction in Sxl function ameliorates the gcl defects in PGC formation/specification. Conversely ectopic expression of Sxl early in embryogenesis mimics the effects of gcl loss on PGC formation. Importantly, the role of Gcl in inhibiting Sxl-Pe transcription is not dependent upon other constituents of the pole plasm. When Gcl is ectopically expressed at the anterior of the embryo, it can repress Sxl. This observation is consistent with the effects of ectopic Gcl on the transcription of other genes reported by Leatherman et al., 2002. Since the rescue of gcl by eliminating the Sxl gene or reducing its activity is not complete, one would expect that there must be other important gcl targets. These targets could correspond to one or more of the other genes that are misexpressed in gcl PB/PGCs. Consistent with this possibility, transcriptional silencing in gcl PBs/PGCs is reestablished when terminal signaling is disrupted by mutations in the tsl gene. On the other hand, it is possible that excessive activity of the terminal signaling pathway also adversely impacts some non-transcriptional targets that are important for PB/PGC formation and that transcriptional silencing in only part of the story (see below).

Pae et al., 2017 showed that mutations in the Gcl interaction domain of Torso (torso^Deg) stabilize the receptor and disrupt PGC formation. Consistent with the notion that Torso receptor is the primary, if not the only, direct target of gcl, they found that mutations in the Torso ligand modifier, tsl, or RNAi knockdown of torso rescued the PGC formation defects in gcl embryos. As would be predicted from their findings and ours, ectopic expression of the Torso^Deg protein induces the inappropriate transcription of sis-b and Sxl-Pe in PBs and newly formed PGCs. Thus, the failure to shut down ongoing transcription in gcl PBs and PGCs must be due (at least in part) to the persistence of the Torso receptor in the absence of Gcl-mediated degradation. Corroborating this idea, the ectopic activation of transcription in gcl PGCs is no longer observed when the terminal signaling pathway is disrupted by the removal of tsl. Taken together, these data strongly suggest that the establishment/maintenance of transcriptional silencing in PBs is a critical function of Gcl.

Since RNAi knockdowns of terminal pathway kinases downstream of torso did not rescue gcl mutants, Pae et al., 2017 postulated that the Tsl-Torso receptor interaction triggered a novel, non-canonical signal transduction pathway that disrupted PGC development. If their suggestion is correct, then the activation of sis-b and Sxl-Pe in PBs/PGCs in gcl and torso^Deg embryos would be mediated by this novel terminal signaling pathway. Here, our results are ambiguous. Consistent with the suggestion of Pae et al., 2017, GOF mutations in MEK, a downstream kinase in the Torso signaling pathway, did not activate Sxl-Pe transcription in pole cells. However, an important caveat is that the GOF activity of MEK variants we tested is likely not equivalent to the activity from the normal Torso-dependent signaling cascade (Goyal et al., 2017). As the pole plasm contains at least two other factors that help impose transcriptional quiescence, the two GOF MEK mutants we tested may simply not be sufficient to overcome their repressive functions. Two observations are consistent with this possibility. First, like torso^Deg, we found that MEK^E203K induces Sxl-Pe expression in male somatic nuclei. The same is true for a viable GOF mutation in Torso: it can induce ectopic activation of Sxl-Pe in male somatic nuclei, but is unable to activate Sxl-Pe in PGCs (Deshpande, 2004). Second, a key terminal pathway transcription target tailless is not expressed in gcl mutant PBs/PGSs even though the terminal pathway should be fully active. This is also true for embryos expressing torso^Deg and the two GOF MEK proteins. For these reasons, we cannot unambiguously determine if it is the canonical terminal signaling pathway or another, noncanonical signaling pathway downstream of Torso that is responsible for the expression of sis-b, Sxl-Pe, and other genes in gcl mutant PB/PGCs.

There are also reasons to think that the canonical Torso signal transduction cascade must be inhibited for proper PGC formation. One of the more striking phenotypes in gcl mutants is the dispersal of key germline mRNA and protein determinants into the surrounding soma. A similar disruption in the sequestration of pole plasm components is observed not only in torso^Deg embryos but also in MEK^E203K and MEK^F53S embryos. Thus, this gcl phenotype would appear to arise from the deployment of the canonical Torso receptor signal transduction cascade, at least up to the MEK kinase. However, this result does not exclude the possibility that the Tsl→Torso→ERK pathway has other non-transcriptional targets that, like Sxl-Pe expression, can also interfere with PB/PGC formation. If this was the case, it could potentially explain why global transcriptional inhibition failed to rescue the PGC defects in gcl embryos (Cinalli and Lehmann, 2013). In this respect, a potential—if not likely—target is the microtubule cytoskeleton. In previous studies, we found that the PB and PGC formation defects as well as the failure to properly sequester critical germline determinants in gcl arise from abnormalities in microtubule/centrosome organization (Lerit et al., 2017). Preliminary imaging experiments indicate that centrosome distribution of torso^Deg PBs is also abnormal, suggesting that inappropriate activation of the terminal signaling pathway perturbs the organization or functioning of the microtubule cytoskeleton and/or centrosomes. Such a mechanism would also be consistent with the dispersal of germline mRNA and protein determinants in torso^Deg and GOF MEK embryos. While further experiments will be required to demonstrate microtubule and centrosomal aberrations in torso^Deg and GOF MEK embryos, a role for a receptor-dependent MEK/ERK signaling cascade in promoting centrosome accumulation of γ-tubulin and microtubule nucleation has been documented in mammalian tissue culture cells (Colello et al., 2012). It is thus conceivable that MEK/ERK signaling has a similar role in Drosophila PB nuclei and PGCs. It will be important to determine if Torso-dependent activation of MEK/ERK can perturb the behavior or organization of centrosomes and/or microtubules in early embryos, and, if so, whether the influence can alter the pole plasm RNA anchoring and/or transmission. Taken together, our data reveal a mutual antagonism between the determinants that specify germline versus somatic identity. Future studies will focus on how and when during early embryogenesis such feedback mechanisms are activated and calibrated to establish and/or maintain germline/soma distinction.

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 3,5,6, and 8.

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Author details

1. Megan M Colonnetta

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5685-1670

2. Lauren R Lym

   Department of Cell Biology, Emory University School of Medicine, Atlanta, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5039-2303

3. Lillian Wilkins

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. Gretchen Kappes

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Elias A Castro

   Department of Cell Biology, Emory University School of Medicine, Atlanta, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1439-5918

6. Pearl V Ryder

   Department of Cell Biology, Emory University School of Medicine, Atlanta, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   Additional information

   ORCID: 0000-0003-3699-3633

7. Paul Schedl

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

8. Dorothy A Lerit

   Department of Cell Biology, Emory University School of Medicine, Atlanta, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Writing - original draft, Writing - review and editing

   For correspondence

   dlerit@emory.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3362-8078

9. Girish Deshpande

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Writing - original draft, Writing - review and editing

   For correspondence

   gdeshpan@princeton.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5200-7090

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