InteractiveFly: GeneBrief

germ cell-less: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs |References

BIOLOGICAL OVERVIEW

During early Drosophila and C. elegans development, the germ cell precursors undergo a period of transcriptional quiescence. Germ cell-less (Gcl), a germ plasm component necessary for the proper formation of 'pole cells', the germ cell precursors in Drosophila, is required for the establishment of this transcriptional quiescence. While control embryos silence transcription prior to pole cell formation in the pole cell-destined nuclei, this silencing does not occur in embryos that lack Gcl activity. The failure to establish quiescence is tightly correlated with failure to form the pole cells. Furthermore, Gcl can repress transcription of at least a subset of genes in an ectopic context, independent of other germ plasm components. These results place Gcl as the earliest gene known to act in the transcriptional repression of the germline. Gcl's subcellular distribution on the nucleoplasmic surface of the nuclear envelope (Jongens, 1994) and its effect on transcription suggest that it may act to repress transcription in a manner similar to that proposed for transcriptional silencing of telomeric regions (Leatherman, 2002).

A period of transcriptional quiescence in the early germ cells is not only a conserved feature in Drosophila and C. elegans, but it also appears to be important for their development, since mutations that disrupt this quiescence affect the formation of the germline. The pie1 gene encodes a protein that acts as a transcriptional repressor in the early germ cell precursors of C. elegans. Embryos that lack pie1 activity fail to repress transcription in the germ cell precursors and also fail to form a proper germline. In Drosophila, the nanos and pumilio genes are required for transcriptional quiescence of the pole cells. In embryos that lack nanos or pumilio activity, the pole cells prematurely or inappropriately express genes and fail to develop into functional germ cells (Leatherman, 2002).

The Germ cell-less protein is a germ plasm component and specifically associates with those nuclei that enter the germ plasm, induce the formation of pole buds, and then are incorporated into the resulting pole cells (Jongens, 1992). In these nuclei, Gcl is localized to the nucleoplasmic surface of the nuclear envelope (Jongens, 1994), a localization necessary for its function (Robertson, 1999). Previously, examination of the germ cell-less null phenotype revealed that most embryos lacking maternally contributed gcl (hereafter called Δgcl embryos) form no pole cells; the remaining embryos form a very small number of pole cells (Robertson, 1999). This failure to form pole cells does not appear to be due to a defect in germ plasm formation, maintenance, or levels. Analysis of known germ plasm components in Δgcl embryos has failed to reveal any defects, suggesting that the failure to form pole cells in these embryos is due to a direct requirement for Gcl in this process (Robertson, 1999; Leatherman, 2002).

To investigate whether loss of gcl activity results in a failure to establish or maintain the state of transcriptional quiescence necessary for proper germ cell development, both Δgcl and control embryos were stained with the H5 antibody. This monoclonal antibody recognizes a phosphorylated form of RNA polymerase II that is associated with active transcription, and it has been used as a reliable marker for the transcriptional quiescence of the germ cell precursors of Drosophila and C. elegans (Leatherman, 2002).

Interestingly, in the control embryos, a difference in H5 staining is observed in pole bud nuclei, and this occurs at an earlier stage than what was previously reported (Seydoux, 1997). Pole buds first appear during nuclear cycle 9 and pinch off at the end of nuclear cycle 10 to form the pole cells. In many cycle 9 embryos, a slight decrease in H5 staining was observed in the pole bud nuclei (identified by their association with Vasa staining) compared to the somatic nuclei, and by cycle 10, the pole bud nuclei display a dramatic reduction in H5 staining, indicating that a state of transcriptional quiescence is being established prior to the formation of the pole cells (Leatherman, 2002).

In Δgcl embryos, the pole bud nuclei fail to become transcriptionally quiescent. When stained with the H5 antibody, most pole bud nuclei in the Δgcl embryos stained at levels comparable to the somatic nuclei. However, in a few scattered Δgcl pole bud nuclei, a reduction was observed in H5 staining similar to that seen in wild-type. Since the few successfully formed pole cells in Δgcl embryos have dramatically reduced H5 staining similar to control embryo pole cells (Robertson, 1999), it is speculated that the few silenced pole bud nuclei observed might be those that will become part of the few pole cells seen in Δgcl embryos (Leatherman, 2002).

To further explore this possibility, this loss of transcriptional quiescence was quantitated by counting the pole bud nuclei in control and Δgcl embryos at the pole bud stage (nuclear cycle 10) and noting whether they had reduced H5 staining. Between 7 and 14 pole bud nuclei per embryo were found in both control and Δgcl embryos, as is expected. In control embryos, nearly all nuclei had reduced H5 staining. However, in 50% of the Δgcl embryos, none of the pole bud nuclei displayed reduced H5 staining. This correlates well with the observed 48% of Δgcl embryos with no pole cells at the blastoderm stage (Robertson, 1999). Of the total number of Δgcl pole bud nuclei counted, only 11.9% had reduced H5 staining, indicating transcriptionally silenced nuclei. After pole cell formation, each pole cell then divides between 0 and 2 times, so the total number of transcriptionally silenced pole bud nuclei cannot be compared directly to the number of pole cells observed in Δgcl embryos. However, since 11.9% of the pole bud nuclei are silenced, a reduction to a similar percent would be expected in the number of pole cells in Δgcl embryos compared to control embryos. Since control embryos have an average of 23.4 pole cells at the blastoderm stage (Robertson, 1999), an average of (23.4 × 0.119), or 2.8 pole cells, would be expected to be present in blastoderm-stage Δgcl embryos based on the number of silenced pole bud nuclei. This number is identical to the observed (Robertson, 1999) average of 2.8 pole cells in Δgcl embryos at the blastoderm stage (Leatherman, 2002).

This strong quantitative correlation, in addition to the failure to find any other defects, including defects in the germ plasm, suggests that those few nuclei that successfully silence transcription in the pole buds of Δgcl embryos (as shown by loss of H5 staining) are those that will form the few functional pole cells. This implies that establishing transcriptional quiescence is a necessary step for pole cell formation; however, further experiments will be necessary to prove this type of causal relationship between transcriptional silencing and pole cell formation (Leatherman, 2002).

Since the H5 stainings indicate that pole bud nuclei in Δgcl embryos fail to become transcriptionally silent, it was speculated that it should therefore be possible to see misexpression of specific gene transcripts in these nuclei. The expression of two genes that are transcribed at this time, sisterless A (sisA) and sisterless B (sisB), was examined. These transcripts are ubiquitiously expressed in nuclei as early as nuclear cycle 8 but are repressed in pole bud nuclei. By using whole-mount in situ hybridization, it was found that sisA and sisB transcripts are present not only in somatic nuclei, but also in the majority of pole bud nuclei in Δgcl embryos, and this finding independently verifies that Δgcl embryos are deficient in transcriptional silencing in the pole bud nuclei (Leatherman, 2002).

The results described above indicate that gcl is required to repress transcription during the establishment of the germ cell lineage. To determine if this activity is dependent or independent of other germ plasm components, the effect of ectopically localizing Gcl on transcription was examined. Replacement of the 3'UTR of the gcl transcript with the 3'UTR of bicoid results in the anterior localization of gcl mRNA and protein to the anterior pole of the embryo (Jongens, 1994). In these 'hgb' embryos, a slightly variable but consistent decrease was found in the intensity of H5 staining in the anterior nuclei compared to control embryos throughout the syncytial blastoderm stage, and this decrease indicates that Gcl is sufficient to repress transcription ectopically. However, the anterior expression of Gcl clearly does not lead to complete silencing of the anterior nuclei, since some H5 staining persists (Leatherman, 2002).

The reduced H5 staining observed in the anterior of the hgb embryos could be due to global partial repression of all genes, or it could result from a specific subset of genes being severely repressed while others are unaffected. To distinguish between these possibilities, the expression was examined of specific genes whose expression pattern includes the anterior of the embryo, including sisA, sisB, tailless, huckebein, hunchback, and knirps. These genes are all independently activated by maternally contributed factors, so any effects on their transcription are likely to be direct rather than a consequence of an earlier defect. By using in situ hybridization, it was found that the early anterior expression domains of sisA, sisB, tailless, and huckebein are severely repressed in all of the hgb embryos examined, but no effect was seen on hunchback and knirps expression. These data suggest that the transcriptionally repressive effect of Gcl is not global, but rather specific to a subset of genes. Gcl is also present in a variety of tissues later in development (Jongens, 1992), at times when transcription is active, which further suggests a non-global mode of silencing (Leatherman, 2002).

If Gcl silences only selected genes, then the question arises as to how the pole cells accomplish what appears to be complete repression of mRNA transcription. Reporter genes driven by the strong Gal4-VP16 activator have been shown to be unable to be activated in the early germ cells. If only specific genes are repressed, it is unlikely that a novel transgene would be repressed by the same silencing mechanism, thereby arguing that there is widespread repression of transcription in early pole cells. Furthermore, observations of the compaction of pole cell nuclei, that is mediated by Gcl, suggest a more global mode of silencing than current studies. Pole cell nuclei are normally round in shape and more compact than somatic nuclei, which is consistent with chromatin silencing. Compaction increases when Gcl is overexpressed, and ectopic compaction occurs when Gcl is ectopically localized (Jongens, 1994). It seems unlikely that Gcl could accomplish such a global nuclear structure change just through the repression of transcription of a few genes (Leatherman, 2002).

One possible explanation for this discrepancy is the existence of other as yet unidentified factors that act in transcriptional silencing during pole cell formation which allow the formation of the few pole cells that form in Δgcl embryos. These other factors, while less effective initially than Gcl, might have a more global mode of action. This argument is supported by experiments that show that, when Oskar is ectopically localized to the anterior, which causes ectopic pole cell formation by ectopic localization of essential germ plasm components, hunchback transcription is repressed. Since Gcl does not repress hunchback transcription, there must be other factors that accomplish this. Another possibility is that general repression can be accomplished by silencing only a few genes. At the time of pole cell formation, there are very few genes known to be transcribed, so it is conceivable that specific mechanisms could exist for silencing all of them. Repression of these few genes could alter the developmental program in the pole bud nuclei, thereby resulting in more widespread silencing in pole cells (Leatherman, 2002).

Data on Nanos and its binding partner Pumilio support this view. These factors are also required for transcriptional silencing in Drosophila pole cells, although at a later stage than that at which Gcl is required. The pole cell migration defect in nanos mutant embryos is partially alleviated when sex-lethal, one improperly expressed transcript, is removed. This suggests that the number of genes that nanos causes to be transcriptionally repressed is small, since removal of only one gene can cause rescue. Furthermore, transcriptional activation of sex-lethal is dependent on the two genes shown to be repressed in the germ plasm by Gcl, sisA and sisB; this finding hints that sex-lethal is a key gene that must be silenced in the early pole cells in order for them to achieve their proper developmental fate (Leatherman, 2002).

Work on a mouse homolog of Drosophila gcl further suggests a specific, rather than global, mode of repression for Gcl. mgcl-1, a functional homolog of gcl, is highly expressed in spermatocytes ( Leatherman, 2000) at a time when transcriptional activity in these cells is high. mGcl physically interacts with the DP3α subunit of E2F (de la Luna, 1999). mGcl can inhibit progression through the cell cycle by repressing the E2F transcription factor, possibly due to its sequestration at the nuclear envelope. This work demonstrates a mechanism of transcriptional repression for a specific set of genes -- those that are transcriptionally controlled by E2F. However, it is unclear at this point whether this specific role for Gcl occurs in Drosophila, since Gcl does not bind to Drosophila DP, the only apparent isoform of this protein in the Drosophila genome (Leatherman, 2002).

While the mechanism by which Gcl accomplishes transcriptional silencing is not known, interesting parallels in budding yeast, S. cerevisiae, suggest a possible mechanism whereby Gcl could be working. In yeast, the nuclear periphery has been linked to transcriptional silencing activity, largely through studies of telomeric chromatin and the subtelomeric genes that are affected. MLP1 and MLP2, which are tethered to the nuclear envelope, provide the anchor by which the Yku70/Yku80 heterodimer, which binds to chromatin, brings telomeric chromatin into the perinuclear 'silent domain'. Once chromatin is at the nuclear periphery, silent information regulators 3 and 4 (Sir3 and Sir4) can gain access to the DNA and cause transcriptional silencing. Since Gcl localizes to the nuclear envelope (Jongens, 1992), it is speculated that it could be accomplishing transcriptional repression similarly to MLP 1 and MLP2 by anchoring chromatin to the nuclear periphery through protein binding partners. This model is currently being tested by looking at the function of binding partners of Gcl (Leatherman, 2002 and references therein).

Antagonism between germ cell-less and Torso receptor regulates transcriptional quiescence underlying germline/soma distinction

Transcriptional quiescence, an evolutionarily conserved trait, distinguishes the embryonic primordial germ cells (PGCs) from their somatic neighbors. In Drosophila melanogaster, PGCs from embryos maternally compromised for germ cell-less (gcl) misexpress somatic genes, possibly resulting in PGC loss. Recent studies documented a requirement for Gcl during proteolytic degradation of the terminal patterning determinant, Torso receptor. This study demonstrates that the somatic determinant of female fate, Sex-lethal (Sxl), is a biologically relevant transcriptional target of Gcl. Underscoring the significance of transcriptional silencing mediated by Gcl, ectopic expression of a degradation-resistant form of Torso (torso(Deg)) can activate Sxl transcription in PGCs, whereas simultaneous loss of torso-like (tsl) reinstates the quiescent status of gcl PGCs. Intriguingly, like gcl mutants, embryos derived from mothers expressing torso(Deg) in the germline display aberrant spreading of pole plasm RNAs, suggesting that mutual antagonism between Gcl and Torso ensures the controlled release of germ-plasm underlying the germline/soma distinction (Colonnetta, 2021).

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. 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). 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. Unlike pole cell nuclei, somatic nuclei continue synchronous divisions after they reach the surface of the embryo until NC 14 when they cellularize (Colonnetta, 2021).

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. Transcription commences in the embryo during NC 6-7 when a select number of genes are active. Transcription is more globally upregulated when the nuclei reach the surface, and by the end of NC 14, zygotic genome activation (ZGA) is complete. 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. By contrast, in newly formed PGCs, transcription is switched off, and PGC nuclei have only residual amounts of Ser5 and Ser2 CTD phosphorylation. Moreover, and consistent with their transcriptionally quiescent status, other changes in chromatin architecture that accompany ZGA are also blocked in PGCs (Colonnetta, 2021).

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. 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. 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). pgc encodes a nuclear protein that binds to the transcriptional elongation kinase p-TEFb, blocking Ser5 CTD phosphorylation. In pgc mutant pole cells, Ser5 phosphorylation is enhanced, as is transcription of several somatic genes, including genes involved in terminal patterning (Colonnetta, 2021).

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. 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. 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. 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 (Colonnetta, 2021).

Studies by Leatherman (2002) suggested that the defects in PGC formation in gcl mutant embryos are linked to failing to inhibit somatic transcription. That study 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, 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 (Colonnetta, 2021).

The connection Leatherman 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, the PGC formation defects seen in gcl embryos are 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, it was concluded that it effectively blocked polymerase transcription. On the other hand, subsequent experiments by Pae (2017) raised the possibility that inhibiting transcription in pole cell nuclei is a critical step in PGC formation. The Pae paper 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. 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 (2017) was 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. 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 (Colonnetta, 2021).

The current study has revisited these conflicting claims by examining the role of Gcl in establishment/maintenance of transcriptional quiescence. The studies of Leatherman (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. This study shows 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 (2017), this study 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, this study found that expression of a mutant form of Torso that is resistant to Gcl-dependent degradation (hereafter referred to as torsoDeg: Pae, 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 (Colonnetta, 2021).

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. 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 (Colonnetta, 2021).

Two models have been proposed to account for the PGC defects in gcl mutants. In the first, Leatherman (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. 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? These studies have addressed these outstanding questions, leading to a resolved model of Gcl activity and function (Colonnetta, 2021).

Shutting off transcription is, in fact, a critical function of Gcl protein. As previously documented by Leatherman, this study found 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. Previous studies 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. 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) (Colonnetta, 2021).

Pae (2017) showed that mutations in the Gcl interaction domain of Torso (torsoDeg) 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 those and the current findings, ectopic expression of the TorsoDeg 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 (Colonnetta, 2021).

Since RNAi knockdowns of terminal pathway kinases downstream of torso did not rescue gcl mutants, Pae (2017) postulated that the Tsl-Torso receptor interaction triggered a novel, non-canonical signal transduction pathway that disrupted PGC development. If that suggestion is correct, then the activation of sis-b and Sxl-Pe in PBs/PGCs in gcl and torsoDeg embryos would be mediated by this novel terminal signaling pathway. The results of the current study are ambiguous. Consistent with the suggestion of Pae, 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 that was tested is likely not equivalent to the activity from the normal Torso-dependent signaling cascade. As the pole plasm contains at least two other factors that help impose transcriptional quiescence, the two GOF MEK mutants that were tested may simply not be sufficient to overcome their repressive functions. Two observations are consistent with this possibility. First, like torsoDeg, this study 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. 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 torsoDeg and the two GOF MEK proteins. For these reasons, it cannot be unambiguously determined 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 (Colonnetta, 2021).

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 torsoDeg 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. In this respect, a potential-if not likely-target is the microtubule cytoskeleton. In previous studies, it was 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. Preliminary imaging experiments indicate that centrosome distribution of torsoDeg 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 torsoDeg and GOF MEK embryos. While further experiments will be required to demonstrate microtubule and centrosomal aberrations in torsoDeg 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. 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, the current 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 (Colonnetta, 2021).

DEVELOPMENTAL BIOLOGY

Gcl protein specifically associates with those nuclei that later become the nuclei of the germ cell precursors (Jongens, 1992).

Pole cells and posterior segmentation in Drosophila are specified by maternally encoded genes whose products accumulate at the posterior pole of the oocyte. Among these genes is tudor (tud). Progeny of hypomorphic tud mothers lack pole cells and have variable posterior patterning defects. A null allele was isolated to further investigate tud function. While no pole cells are ever observed in embryos from tud-null mothers, 15% of these embryos have normal posterior patterning. Oskar and Vasa proteins, and nanos RNA, all initially localize to the pole plasm of tud-null oocytes and embryos from tud-null mothers, while localization of germ cell-less (gcl) and polar granule component (pgc), is undetectable or severely reduced. In embryos from tud-null mothers, polar granules are greatly reduced in number, size, and electron density. Thus, tud is dispensable for somatic patterning, but essential for pole cell specification and polar granule formation (Thomson, 2004).

Overlapping mechanisms function to establish transcriptional quiescence in the embryonic Drosophila germline

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).

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).

EVOLUTIONARY HOMOLOGS

Primordial germ cells (PGCs) are founder cells of all gametes. A number of genes that control PGCs development have been identified in invertebrates, whereas such genes are by and large unelucidated in mammals. Described here is the cloning, genomic structure and expression of the mouse homolog of germ cell-less gene which is required for PGCs formation in Drosophila. The mouse gcl shows 34% identity compared with Drosophila Gcl protein and contains BTB/POZ domain. The gcl gene consists of 14 exons and spans more than 50 kb. The CpG islands are found around exon 1 of the gene. Putative promoter region contains potential binding sites for various transcription factors. Northern blot analysis shows that its mRNA is highly expressed in adult testis with lower expression in ovary, ES (embryonic stem) cells, and various other organs. In situ hybridization analysis reveals strong expression of the gcl gene in the pachytene stage spermatocytes. The expression is also observed in post-migratory PGCs, but was not apparent in migratory and pre-migratory PGCs. Further studies including gene disruption analysis would provide an important insight into mammalian germ lineage development (Kimura, 1999).

Transcription factor E2F plays an important role in orchestrating early cell cycle progression through its ability to co-ordinate and integrate the cell cycle with the transcription apparatus. Physiological E2F arises when members of two distinct families of proteins interact as E2F-DP heterodimers, in which the E2F component mediates transcriptional activation and the physical interaction with pocket proteins, such as the tumour suppressor protein pRb. In contrast, a discrete role for the DP subunit has not been defined. DIP, a novel mammalian protein that can interact with the DP component of E2F, has been identified and characterized. DIP contains a BTB/POZ domain and shows significant identity with the Drosophila melanogaster germ cell-less gene product. In mammalian cells, DIP is distributed in a speckled pattern at the nuclear envelope region, and can direct certain DP subunits and the associated heterodimeric E2F partner into a similar pattern. DIP-dependent growth arrest is modulated by the expression of DP proteins, and mutant derivatives of DIP that are compromised in cell cycle arrest exhibit reduced binding to the DP subunit. This study defines a new pathway of growth control that is integrated with the E2F pathway through the DP subunit of the heterodimer (de la Luna, 1999).

LAP2beta is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, a novel LAP2beta-binding protein, mGCL, has been cloned that contains a BTB/POZ domain and is the mouse homolog of the Drosophila Germ-cell-less (Gcl) protein. In Drosophila embryos, Gcl is essential for germ cell formation and is localized to the nuclear envelope. In mammalian cells, Gcl co-localizes with LAP2beta to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. mGCL has been found to interact with the DP3alpha component of the E2F transcription factor. This interaction reduces the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. LAP2beta is also capable of reducing the transcriptional activity of the E2F-DP complex and it is more potent than mGCL in doing so. Co-expression of both LAP2beta and mGCL with the E2F-DP complex results in a reduced transcriptional activity equal to that exerted by the pRb protein (Nili, 2001).

Drosophila Germ cell-less is important in early events during the formation of pole cells, the germ cell precursors in the fly. A 524 amino acid mouse gene with 32% identity and 49% similarity to Drosophila gcl, termed mgcl-1, has been isolated. Like Drosophila Gcl, mGcl-1 localizes to the nuclear envelope. Ectopic expression of mgcl-1 in Drosophila rescues the gcl-null phenotype, indicating that mGcl-1 is a functional homolog of Gcl. mgcl-1 maps to chromosome 6 at 47.3 cM, and is expressed at low levels at all embryonic stages examined from 8.5 to 18.5 d.p.c. as well as in many adult tissues. Different from Drosophila gcl, mgcl-1 is not highly expressed at the time the primordial germ cells appear in the mouse, but high mgcl-1 expression is found in selected mouse adult male germ cells. The differences in these expression patterns in light of conserved activity between the two genes is discussed (Leatherman, 2000).

Emerin belongs to the 'LEM domain' family of nuclear proteins, which contain a characteristic approximately 40-residue LEM motif. The LEM domain mediates direct binding to barrier to autointegration factor (BAF; see Drosophila Baf), a conserved 10-kDa chromatin protein essential for embryogenesis in Caenorhabditis elegans. In mammalian cells, BAF recruits emerin to chromatin during nuclear assembly. BAF also mediates chromatin decondensation during nuclear assembly. The LEM domain and central region of emerin are essential for binding to BAF and lamin A, respectively (see Drosophila Lamin. However, two other conserved regions of emerin lacked ascribed functions, suggesting that emerin could have additional partners. These 'unascribed' domains of emerin mediate direct binding to a transcriptional repressor, germ cell-less (GCL). GCL co-immunoprecipitates with emerin from HeLa cells. The binding affinities of emerin for GCL, BAF, and lamin A have been determined and their oligomeric interactions analyzed. Emerin forms stable complexes with either lamin A plus GCL or lamin A plus BAF. Importantly, BAF competes with GCL for binding to emerin in vitro, predicting that emerin can form at least two distinct types of complexes in vivo. Loss of emerin causes Emery-Dreifuss muscular dystrophy, a tissue-specific inherited disease that affects skeletal muscles, major tendons, and the cardiac conduction system. Although GCL alone cannot explain the disease mechanism, these results strongly support gene expression models for Emery-Dreifuss muscular dystrophy by showing that emerin binds directly to a transcriptional repressor, GCL, and by suggesting that emerin-repressor complexes might be regulated by BAF. Biochemical roles for emerin in gene expression are discussed (Holaska, 2003).

A mouse homolog of the Drosophila germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2ß (lamina-associated polypeptide 2ß). To elucidate the role of mgcl-1, two mutant mouse lines were analyzed that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that are probably due to impaired nuclear envelope integrity are observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities are observed in testis in which the highest expression of mgcl-1 is detected. Fertility is significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations show insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, are aberrant. Considering that the first abnormality during the process of spermatogenesis is abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling (Kimura, 2003).

The nuclear envelope, which separates the chromosomes from the cytoplasm and organizes the nuclear architecture, is composed of inner and outer membranes, NPC, and nuclear lamina. The inner nuclear membrane contains a unique set of integral membrane proteins, which includes the lamin B receptor (LBR), LAP1, LAP2, emerin, MAN1, and nurim. Most of these proteins bind to the nuclear lamina, which is a network of polymers formed by lamins. Mammals have three lamin genes (LMNA, LMNB, and LMNB2), which encode seven alternatively spliced lamin isoforms. Lamins and other nuclear envelope proteins are involved in the organization of the nuclear architecture. From the analyses of human diseases and gene targeting mice, three genes, LMNA, emerin and LBR have been revealed to be essential for the maintenance of normal nuclear envelope integrity (Kimura, 2003).

The cells of mgcl-1-null mice show abnormal nuclear structures in several organs. Although mGCL-1 was reported to bind LAP2ß, the functions of these two proteins in nuclear envelope formation remain unknown. LBR and emerin are integral proteins of the nuclear inner membrane, and lamins A and C, which are A-type lamins and gene products of the LMNA gene, are major components of the nuclear lamin. Compared to these proteins, mGCL-1 appears to be a relatively minor component of the nuclear lamina, since mGCL-1 does not bind directly to lamins and is not an integral protein. An essential role for mGCL1 in nuclear integrity suggests that the nuclear lamina forms a more complicated structure than was previously believed; i.e., more proteins may participate as essential members. An alternative, though not mutually exclusive, interpretation is that the Drosophila GCL and mGCL-1 proteins have unidentified molecular properties in common. Although there are no genes that code for LAP proteins in Drosophila, GCL is localized to the nuclear envelope. Furthermore, mGCL-1 can rescue the Drosophila gcl mutant phenotype. In addition, the full-length mGCL-1 and various deletion mutants of mGCL-1 have been localized to the nuclear lamina. It is conceivable that mGCL-1 binds to some other structural components of the nuclear envelope in order to maintain the structure (Kimura, 2003).

Nuclear envelopes break down at the onset of mitosis and are reconstructed around the chromosomes during telophase. Similar to other nuclear lamina proteins, mGCL-1 diffuses into cytoplasm and/or ER during mitosis and reassembles around chromosomes at the end of mitosis. Immunostaining with antibodies against LAP2 and NPC shows that the nuclear envelope components assemble around chromosomes at the end of mitosis in mgcl-1^-/- embryonic fibroblasts, suggesting that reformation of nuclear envelopes is not affected by the absence of mGCL-1 (Kimura, 2003).

The most striking feature of the mgcl-1^-/- mouse phenotype is abnormal spermatogenesis. The abnormalities can be divided into three categories: defects in nuclear architecture, sperm morphology, and proteins that were involved in chromatin remodeling. Given that mGCL-1 binds directly to LAP2ß, and that mGCL-1 is expressed abundantly in spermatocytes, the abnormal nuclear envelope structure is probably due to the primary effect of the null mutation in mgcl-1. However, it is uncertain whether the other abnormalities are direct sequels of the null mutation or are due to the indirect effects of the abnormal nuclear envelope structure (Kimura, 2003).

Although the mgcl-1 gene is expressed ubiquitously, abnormal nuclear morphology was detected only in the restricted organs. The testes show very high levels of mgcl-1 mRNA. However, the mRNA levels in the exocrine pancreas and liver, both of which showed abnormal nuclear structures, are comparable to those in other organs. Thus, the abundance of mgcl-1 transcripts alone does not explain why mGCL-1 is important for the normal nuclear structure. Differential expression of lamins may be one reason for the tissue-specific nuclear architecture abnormalities. The nuclear envelope of spermatocytes contains the short meiosis-specific lamin isoforms C2 and B3, which are A- and B-type lamins, respectively. Due to the presence of these spermatocyte-specific lamins, the stability of the nuclear envelope of spermatocytes is considered to be lower than that of somatic cells. Abnormal nuclear morphology was found not only in spermatocytes but also in the later stages of spermiogenesis. It seems reasonable to consider that the vulnerable nuclear structure originates in spermatocytes and persists until the later phases of spermatogenesis (Kimura, 2003).

Experimental and genetic studies suggest that the nuclear lamina is involved in a number of nuclear functions, such as nuclear envelope assembly, DNA synthesis, transcription, and apoptosis. The roles of the nuclear lamina in replication and transcription are putatively related to interactions between the nuclear lamina and chromatin. From this point of view, an intriguing phenotype of the mgcl-1-null mouse is the abnormal expression of transition proteins and protamines, both of which are involved in chromatin remodeling during spermatogenesis. It is unclear whether this abnormality is a direct consequence or an indirect downstream effect of nuclear envelope abnormality. In any case, this abnormality leads to insufficient chromatin condensation in the sperm heads of mgcl-1^-/- mice. Perturbed chromatin remodeling caused by the impaired expression of transition proteins and protamines may be one of the reasons why mature mgcl-1mutant sperm have abnormal morphologies (Kimura, 2003).

mGCL-1 has been shown to interfer with the transcriptional functions of the E2F-DP complex. An independent explanation for the abnormality in mgcl-1^-/- testis is that abnormal gene expression arose from the altered transcriptional activities of E2F-DP. However, this mechanism is unlikely to be true, since the expression levels of genes whose transcription was driven by E2F-DP are not altered significantly in the mgcl-1^-/- testis (Kimura, 2003).

Interestingly, perturbation of the nuclear envelope structure is known to cause laminopathy diseases, such as Emery-Dreifuss muscular dystrophy, in which the lamin A/C or the emerin genes for nuclear-lamina components are mutated. Mice that lacked the A-type lamins had abnormal nuclear envelope integrity and suffered from muscular dystrophy. Mutations in the LBR-encoding gene alter the nuclear morphology of granulocytes. These laminopathies are defined as diseases that are caused by mutations in genes that encode either lamins or proteins that bind to lamins. The loss of mGCL-1 may not in itself constitute a laminopathy, since mGCL-1 does not bind directly to lamins. However, mGCL-1 deficiency may be considered a laminopathy in a broader interpretation of this disease. In terms of both the human diseases and the mutant mouse strains, it has been proposed that defects in the nuclear lamina eliminate the specific interaction between the nuclear matrix and chromatin, which leads to disorganized chromatin architectures and abnormal gene expression. However, the molecular mechanisms that link abnormal nuclear envelope integrity and these diseases have not been elucidated. It remains to be determined whether the pathophysiology reflects the primary molecular defect in the nuclear envelope, or is caused by downstream effects on chromatin structure and gene expression (Kimura, 2003).

Accordingly, it is difficult to ascertain how the mgcl-1-null mutation causes abnormal spermatogenesis. However, it is speculated that the LAP2-BAF association may provide a clue to the pathogenesis. Chromatin regions appear to be anchored to the nuclear lamina, and LAP2 is an important component for this binding, since two isoforms of LAP2, LAP2alpha and LAP2ß, bind to chromatin. Stable binding requires both the chromatin-binding domain and the LEM domain, which is a conserved region of ~43 amino acids that is also found in emerin and MAN1. In addition, LAP2 isoforms bind to the DNA-binding protein BAF via their LEM domains. BAF, which is a ubiquitous and highly conserved protein, colocalizes with chromatin during interphase and mitosis and probably plays a fundamental role in chromosome architecture. A null mutation in the mgcl-1 gene may affect chromatin organization and subsequent gene expression by inducing an abnormal LAP2ß-BAF association (Kimura, 2003).

The Drosophila gcl gene is required for the formation of germ cell precursors but not for germ cell development within the gonads. In contrast, mgcl-1 is dispensable for PGC formation but is important for spermatogenesis. Thus, despite the substitutable molecular function of mouse gcl in Drosophila, the physiological functions of the Drosophila and mouse gcl genes are distinct during germ cell development. One of the abnormal features of the Drosophila gcl mutant is the disordered budding of germ lineage cells. The gigantic sperm in mgcl-1^-/- mice, which were presumably caused by unsuccessful cell separation at the final stage of spermiogenesis, are reminiscent of abnormal budding in Drosophila. It is believed that comparative studies in different taxa of the functions of the Drosophila GCL and its mouse homolog will provide phylogenetic clues as to germ cell specification and differentiation (Kimura, 2003).

The gene of germ cell-less (gcl) has been shown to be important in early differentiation of germ cells in Drosophila. Although the gcl homologue genes have been identified in some organisms, there is little data on the expression pattern and functional analysis of the gcl gene in zebrafish. In this research, real-time quantitative RTPCR showed that the level of gcl mRNA expression rapidly decreases from the 4-cell stage to the sphere stage at which it reaches a minimum, gradually increases from the 50%-epiboly stage, and then remains stable during the posterior stages. Results of in situ hybridization indicated that the transcripts of zebrafish gcl are evenly distributed in all blastomeres from the 2-cell stage to the blastula period, different from that of vasa, nonas1 and dead end mRNA, and condense into some clusters of cells located along the blastoderm margin from the gastrulation period. During subsequent development, the transcripts are segregated as subcellular clumps to a small number of cells that would migrate to the position of the gonad in the dorsal side. In the adult, gcl mRNA is widely expressed in developing germ cells of both ovary and testis. These data suggest that zebrafish gcl has potentially important roles in the formation of primordial germ cells (Li, 2006).

Double abdomen in a short-germ insect: Zygotic control of axis formation revealed in the beetle Tribolium castaneum

The distinction of anterior versus posterior is a crucial first step in animal embryogenesis. In Drosophila, this axis is established by morphogenetic gradients contributed by the mother that regulate zygotic target genes. This principle has been considered to hold true for insects in general. This study investigated symmetry breaking in the beetle Tribolium castaneum, which among insects represents the more ancestral short-germ embryogenesis. Maternal Tc-germ cell-less is required for anterior localization of maternal Tc-axin, which represses Wnt signaling and promotes expression of anterior zygotic genes. Both RNAi targeting Tc-germ cell-less or double RNAi knocking down the zygotic genes Tc-homeobrain and Tc-zen1 led to the formation of a second growth zone at the anterior, which resulted in double-abdomen phenotypes. Conversely, interfering with two posterior factors, Tc-caudal and Wnt, caused double-anterior phenotypes. These findings reveal that maternal and zygotic mechanisms, including Wnt signaling, are required for establishing embryo polarity and induce the segmentation clock in a short-germ insect (Ansari, 2018).

REFERENCES

Search PubMed for articles about Drosophila germ cell-less

Amikura, R., Sato, K. and Kobayashi, S. (2005). Role of mitochondrial ribosome-dependent translation in germline formation in Drosophila embryos. Mech. Dev. 122(10): 1087-93. 16125913

Ansari, S., Troelenberg, N., Dao, V. A., Richter, T., Bucher, G. and Klingler, M. (2018). Double abdomen in a short-germ insect: Zygotic control of axis formation revealed in the beetle Tribolium castaneum. Proc Natl Acad Sci U S A 115(8): 1819-1824. PubMed ID: 29432152

Barnes, A. I., Boone, J. M., Jacobson, J., Partridge, L. and Chapman, T. (2006). No extension of lifespan by ablation of germ line in Drosophila. Proc. Biol. Sci. 273(1589): 939-47. 16627279

Colonnetta, M. M., Lym, L. R., Wilkins, L., Kappes, G., Castro, E. A., Ryder, P. V., Schedl, P., Lerit, D. A. and Deshpande, G. (2021). Antagonism between germ cell-less and Torso receptor regulates transcriptional quiescence underlying germline/soma distinction. Elife 10. PubMed ID: 33459591

de la Luna, S., Allen, K. E., Mason, S. L. and La Thangue, N. B. (1999). Integration of a growth-suppressing BTB/POZ domain protein with the DP component of the E2F transcription factor. EMBO J. 18: 212-228. 9878064

Deshpande, G., Calhoun, G. and Schedl, P. (2004). Overlapping mechanisms function to establish transcriptional quiescence in the embryonic Drosophila germline. Development 131: 1247-1257. 14960492

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Holaska J. M., et al. (2003). Transcriptional repressor germ cell-less (Gcl) and barrier to autointegration factor (BAF) compete for binding to emerin in vitro. J. Biol. Chem. 278: 6969-6975. 12493765

Jongens, T. A., Hay, B., Jan, L. Y. and Jan, Y. N. (1992). The germ cell-less gene product: A posteriorly localized component necessary for germ cell development in Drosophila. Cell 70: 569-584. 1380406

Jongens, T. A., Ackerman, L. D., Swedlow, J. R., Jan, L. Y., and Jan, Y. N. (1994). Germ cell-less encodes a cell type-specific nuclear pore-associated protein and functions early in the germ-cell specification pathway of Drosophila. Genes Dev. 8: 2123-2136. 7958883

Kimura, T., et al. (1999). Molecular cloning and genomic organization of mouse homologue of Drosophila germ cell-less and its expression in germ lineage cells. Biochem. Biophys. Res. Commun. 262: 223-230. 10448096

Kimura, T., et al. (2003). Mouse germ cell-less as an essential component for nuclear integrity. Mol. Cell Biol. 23: 1304-1315. 12556490

Leatherman, J. L., et al. (2000). Identification of a mouse germ cell-less homologue with conserved activity in Drosophila. Mech. Dev. 92: 145-153. 10727854

Leatherman, J., Levin, L., Boero, J. and Jongens, T. (2002). germ cell-less acts to repress transcription during the establishment of the Drosophila germ cell lineage. Curr. Biol. 12: 1681-1685. 12361572

Li, W., Deng, F., Wang, H., Zhen, Y., Xiang, F., Sui, Y. and Li, J. (2006). Germ cell-less expression in zebrafish embryos. Dev. Growth Differ. 48(5): 333-8. 16759283

Nakamura, A., Amikura, R., Mukai, M., Kobayashi, S. and Lasko, P. (1996). Requirement for a noncoding RNA in Drosophila polar granules for germ cell establishment. Science 274: 2075-2079. 8953037

Nili, E., et al. (2001). Nuclear membrane protein LAP2beta mediates transcriptional repression alone and together with its binding partner Gcl (germ-cell-less). J. Cell Sci. 114: 3297-3307. 11591818

Pae, J., Cinalli, R. M., Marzio, A., Pagano, M. and Lehmann, R. (2017). GCL and CUL3 control the switch between cell lineages by mediating localized degradation of an RTK. Dev Cell 42(2): 130-142 e137. PubMed ID: 28743001

Robertson, S. E., Dockendorff, T. C., Leatherman, J. L., Faulkner, D. L. and Jongens, T. A. (1999). germ cell-less is required only during the establishment of the germ cell lineage of Drosophila and has activities which are dependent and independent of its localization to the nuclear envelope. Dev. Biol. 215: 288-297. 10545238

Seydoux, G. and Dunn, M. A. (1997). Transcriptionally repressed germ cells lack a subpopulation of phosphorylated RNA polymerase II in early embryos of Caenorhabditis elegans and Drosophila melanogaster. Development 124: 2191-2201. 9187145

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Thomson, T. and Lasko, P. (2004). Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning. Genesis 40(3): 164-70. 15495201

Trcek, T., Grosch, M., York, A., Shroff, H., Lionnet, T. and Lehmann, R. (2015). Drosophila germ granules are structured and contain homotypic mRNA clusters. Nat Commun 6: 7962. PubMed ID: 26242323

Van Doren, M., Williamson, A. L. and Lehmann, R. (1998). Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr. Biol. 8: 243-246. 9501989

date revised: 12 April 2018

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