Germline/soma distinction in Drosophila embryos requires regulators of zygotic genome activation
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Curated by eLife
Evaluation Summary:
The early differentiation of germ cells, those that will form egg and sperm, is a critical and nearly universal step in animal development. This paper reveals new layers of molecular and cellular regulation that control this process in the fly, and as such be of broad interest to cell and developmental biologists, especially those interested in critical cell fate decisions. The paper contains a wealth of experimental data demonstrating that processes generally thought to be restricted to somatic cells alter the differentiation of germ cells, but provides only limited functional interpretation of the observed phenotypes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
In Drosophila melanogaster embryos, somatic versus germline identity is the first cell fate decision. Zygotic genome activation (ZGA) orchestrates regionalized gene expression, imparting specific identity on somatic cells. ZGA begins with a minor wave that commences at nuclear cycle (NC)8 under the guidance of chromatin accessibility factors (Zelda, CLAMP, GAF), followed by the major wave during NC14. By contrast, primordial germ cell (PGC) specification requires maternally deposited and posteriorly anchored germline determinants. This is accomplished by a centrosome coordinated release and sequestration of germ plasm during the precocious cellularization of PGCs in NC10. Here, we report a novel requirement for Zelda and CLAMP during the establishment of the germline/soma distinction. When their activity is compromised, PGC determinants are not properly sequestered, and specification is disrupted. Conversely, the spreading of PGC determinants from the posterior pole adversely influences transcription in the neighboring somatic nuclei. These reciprocal aberrations can be correlated with defects in centrosome duplication/separation that are known to induce inappropriate transmission of the germ plasm. Interestingly, consistent with the ability of bone morphogenetic protein (BMP) signaling to influence specification of embryonic PGCs, reduction in the transcript levels of a BMP family ligand, decapentaplegic ( dpp ), is exacerbated at the posterior pole.
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Evaluation Summary:
The early differentiation of germ cells, those that will form egg and sperm, is a critical and nearly universal step in animal development. This paper reveals new layers of molecular and cellular regulation that control this process in the fly, and as such be of broad interest to cell and developmental biologists, especially those interested in critical cell fate decisions. The paper contains a wealth of experimental data demonstrating that processes generally thought to be restricted to somatic cells alter the differentiation of germ cells, but provides only limited functional interpretation of the observed phenotypes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers …
Evaluation Summary:
The early differentiation of germ cells, those that will form egg and sperm, is a critical and nearly universal step in animal development. This paper reveals new layers of molecular and cellular regulation that control this process in the fly, and as such be of broad interest to cell and developmental biologists, especially those interested in critical cell fate decisions. The paper contains a wealth of experimental data demonstrating that processes generally thought to be restricted to somatic cells alter the differentiation of germ cells, but provides only limited functional interpretation of the observed phenotypes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Primordial germ cells are formed in the posterior pole of developing Drosophila embryo via taking up of maternally supplied germline determinants (a.k.a., germ plasm). PGC formation occurs approximately at the stage of 10th nuclear division cycle, located between minor and major ZGA waves which take place in somatic nuclei. Zelda and CLAMP are two key factors essential for global zygotic genome activation in soma. Since Zelda mutant retain apparently intact PGCs, Zelda has been thought to be dispensable for PGC formation. However, in this study, the authors identified slight loss of PGC number in both mutants lacking Zelda and CLAMP, which led authors propose a model in which somatic ZGA factors influence PGC specification.
The authors show that maternal or zygotic RNAi against Zelda or CLAMP caused …
Reviewer #1 (Public Review):
Primordial germ cells are formed in the posterior pole of developing Drosophila embryo via taking up of maternally supplied germline determinants (a.k.a., germ plasm). PGC formation occurs approximately at the stage of 10th nuclear division cycle, located between minor and major ZGA waves which take place in somatic nuclei. Zelda and CLAMP are two key factors essential for global zygotic genome activation in soma. Since Zelda mutant retain apparently intact PGCs, Zelda has been thought to be dispensable for PGC formation. However, in this study, the authors identified slight loss of PGC number in both mutants lacking Zelda and CLAMP, which led authors propose a model in which somatic ZGA factors influence PGC specification.
The authors show that maternal or zygotic RNAi against Zelda or CLAMP caused abnormally broader distribution of germ plasm and resulted in an abnormal positioning of PGCs slightly away from posterior poles. The authors suggest that germline determinants are not efficiently captured by the cellularizing PGCs. As a result, the number of specified PGCs was slightly fewer. The Authors further show abnormal segregation of centrosomes accompanied with (and may be a cause of) an abnormal germ plasm trafficking. Moreover, authors show an aberrant pattern of gene expression both in soma and PGC, such as reduction of dpp transcript in posterior region, reduction of tll in posterior, and increased slam and sxl-pe in nascent PGCs when their global transcript is normally silent, suggesting that the germline-soma distinction is compromised in these mutants.
Strengths:
Historically, PGC specification in Drosophila has been believed to occur mainly by preformation-based mechanism. However, the authors focus on extrinsic regulations, particularly, function of centrosomes and cytoskeletons in proper transport of germ plasm components. This is a certainly important aspect to understand similarity and differences of PGC specification mechanism across species. The same group has demonstrated several mutant conditions causing aberrant extrinsic regulation of PGC specification in the past, and thus they are uniquely suited to pursue this line. The authors monitor germ plasm localization and gene expression by smFISH, which enables quantitative analyses. Detection of nascent transcript also reports zygotic transcription in a highly quantitative manner.
Weaknesses:
Overall the manuscript is descriptive and does not clearly provide functional interpretations of observed phenotypes. Specifically, the authors need to consider and discuss potential mechanism of this process.
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Reviewer #2 (Public Review):
Colonetta, Schedl, and Deshpande evaluated how loss of regulators of Zygotic Genome Activation (ZGA) impacts the germ-soma decision in early Drosophila embryos. The prevailing view of that decision in fly embryos has been that ZGA regulates the turn-on of somatic genes in the syncytial embryo and that the primordial germ cells or pole cells, which are cellularized at an earlier stage than somatic cells, are not influenced by ZGA. The authors tested that by eliminating 2 ZGA regulators, Zelda and CLAMP, either maternally or zygotically (but see below), and imaging markers of pole cells and markers of somatic cells. They found that loss of ZGA regulators caused pole cells to display some somatic features and caused somatic cells to display some pole cell features. Specific pole cell defects in mutant embryos …
Reviewer #2 (Public Review):
Colonetta, Schedl, and Deshpande evaluated how loss of regulators of Zygotic Genome Activation (ZGA) impacts the germ-soma decision in early Drosophila embryos. The prevailing view of that decision in fly embryos has been that ZGA regulates the turn-on of somatic genes in the syncytial embryo and that the primordial germ cells or pole cells, which are cellularized at an earlier stage than somatic cells, are not influenced by ZGA. The authors tested that by eliminating 2 ZGA regulators, Zelda and CLAMP, either maternally or zygotically (but see below), and imaging markers of pole cells and markers of somatic cells. They found that loss of ZGA regulators caused pole cells to display some somatic features and caused somatic cells to display some pole cell features. Specific pole cell defects in mutant embryos were reduced levels of germ plasm (Vasa staining), defective concentration of germ plasm RNAs (pgc, gcl, and oskar), and failure to maintain transcriptional quiescence (slam and Sxl-Pe). Specific somatic cell defects were aberrant presence of germ plasm RNAs (pgc, gcl, and oskar) in posterior somatic cells, defects in turn-on of somatic genes in posterior somatic cells (dpp and tll), and attempted budding of somatic cells at the anterior pole similar to budding of pole cells at the posterior pole. All but the anterior budding may be a consequence of impaired delivery of germ plasm to the pole cells due to impaired centrosome duplication and separation. The authors conclude that ZGA is critical for the germ-soma distinction and for proper early development of both somatic and germline cells. This represents an important advance.
Some limitations:
The reported findings are based entirely on imaging of proteins by immunostaining (Vasa, Cnn, Pnut) and RNAs by smFISH (pgc, gcl, oskar, slam, Sxl-Pe, tll, dpp). The images for the most part present clear evidence of the defects claimed by the authors. And the distribution diagrams in Fig. 2 and 3 display the range of defects in embryos. A significant shortcoming of some of the analyses is that, although the authors reported on the percentages of embryos that displayed particular defects, it is not clearly explained how the images were quantified to arrive at those percentages.
In several places, the authors compared the defects caused by loss of maternal versus zygotic Zelda or CLAMP. Loss of maternal (m-) used mothers that expressed an RNAi or shRNA transgene during oogenesis. Loss of zygotic (z-) used fathers that delivered an RNAi transgene to embryos. I think that in general m- and z- caused similar defects, which led me to wonder if knock-down of Zelda or CLAMP in oocytes in fact knocks down both maternal and zygotic. In other words, would RNAi or shRNA driven in the maternal germline be loaded into embryos to knock down zygotic expression as well? If maternal knock-down (m-) is actually maternal plus zygotic knock-down (m-z-), then comparing the effects of m- and z- is actually comparing m-z- and z- and is not valid.
The anterior pole-cell-like budding phenomenon in Figure 9 & 10 is really interesting. It sounds as if that occurs without any of the critical pole plasm components at the anterior pole. If that is right, then the implication is that the anterior end has the potential for pole-cell-like budding and that ZGA normally turns on a gene that blocks that. One thing that is unclear is how transient this phenomenon is and whether it is efficiently captured by fixation.
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