Single-cell transcriptomes of zebrafish germline reveal progenitor types and feminization by Foxl2l
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eLife Assessment
This manuscript provides important findings for understanding the mechanisms of a major gene causing the gonad of fish and other vertebrates, including mammals, to become an ovary rather than a testis. Evidence is solid, but alternative explanations for a number of the claims must be considered and discussed. The impact of the work would benefit by placing it in a richer historical context.
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Abstract
Zebrafish is an important organism for genetic studies, but its early germ cell types and the mechanism of sex differentiation have not been fully characterized. Here, we profiled single-cell transcriptomes and charted a developmental trajectory going from germline stem cells, through early, committed, and late progenitors, to premeiotic and meiotic cells. We showed that the transcription factor Foxl2l was expressed in the progenitor, directing progenitor differentiation toward oocytes. CRISPR-Cas9-mediated mutation of foxl2l produced 100% male fish with normal fertility. Another single-cell profiling of foxl2l -/- germ cells revealed the arrest of germ cell development at the stage of progenitor commitment. Concomitantly, nanos2 transcript (germline stem cell marker) was elevated together with an increase of nanos2 + germ cells in foxl2l mutants, indicating the acquisition of a novel stem cell state. Thus, we have identified developmental stages of germ cells in juvenile zebrafish and demonstrated that zebrafish Foxl2l drives progenitor germ cells toward feminization and prevents them from expressing nanos2 .
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eLife Assessment
This manuscript provides important findings for understanding the mechanisms of a major gene causing the gonad of fish and other vertebrates, including mammals, to become an ovary rather than a testis. Evidence is solid, but alternative explanations for a number of the claims must be considered and discussed. The impact of the work would benefit by placing it in a richer historical context.
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Reviewer #1 (Public Review):
The mechanisms that regulate establishment of the germline stem cells and germline progenitors during zebrafish reproductive development are not understood. Prior single cell analysis characterized the cell types of the early zebrafish ovary during and at stages after sexual differentiation. In this work Hsu et al. took a single approach to analyze the cell types present in the early gonad during early sex determination. As expected, they identified germline stem cells (GSCs) that express canonical GSC markers and distinct populations of progenitors. Unexpectedly, they found multiple populations of transcriptionally distinct progenitor populations that the authors termed early (those lacking the differentiation marker foxl2l), committed (those expressing fox2l2 and S-phase genes) and late (those expressing …
Reviewer #1 (Public Review):
The mechanisms that regulate establishment of the germline stem cells and germline progenitors during zebrafish reproductive development are not understood. Prior single cell analysis characterized the cell types of the early zebrafish ovary during and at stages after sexual differentiation. In this work Hsu et al. took a single approach to analyze the cell types present in the early gonad during early sex determination. As expected, they identified germline stem cells (GSCs) that express canonical GSC markers and distinct populations of progenitors. Unexpectedly, they found multiple populations of transcriptionally distinct progenitor populations that the authors termed early (those lacking the differentiation marker foxl2l), committed (those expressing fox2l2 and S-phase genes) and late (those expressing fox2l2 and meiotic genes) progenitors. Comparisons of their dataset to the published zebrafish ovary datasets confirmed the presence of these distinct progenitor populations in the ovary. Further, they convincingly validated the presence of these progenitor subtypes using fluorescent in situ hybridization. To investigate the relationship between progenitor subsets and known regulators of ovary differentiation, the authors conducted single cell analysis of gonads lacking the transcription factor, Foxl2l. As previously reported, Foxl2l absence blocks ovary differentiation and all foxl2l mutants develop testes. The single cell analysis here indicates that foxl2l is inappropriately expressed in GSCs and early progenitors and that germ cell differentiation is blocked at the committed progenitor stage since few committed progenitors and no late progenitors or meiotic transcripts were detected in the single cell analysis of foxl2l mutants. Based on the coexpression of genes that are not typically expressed together in normally developing germ cells, specifically nanos2 and foxl2l, and dmrt1 and foxl2l, the authors conclude that Foxl2l is required for the committed progenitor program and that it prevents committed progenitors from returning to the GSC state.
Overall, the data provide new insights into the cell populations of the early differentiating gonad, define distinct progenitor states, pinpoint a requirement for the ovary differentiation factor Foxl2l at a specific stage of progenitor differentiation, and generate new hypotheses to be tested. Many but not all of the conclusions are supported by compelling data, and some findings and conclusions need to be clarified in the context of the published literature.
(1) The authors conclude that the committed progenitors revert to GSCs based on the coexpression of nanos2 and foxl2l nanos2 and based on expression of id1 in mutants but not in WT. Without functional data demonstrating that the progenitors revert to an earlier state, alternative interpretations should be considered. For example, it is possible that the cells initiate the committed progenitor program but continue to express the GSC program and that the coexpression of both programs blocks differentiation. Consistent with this possibility, some Fox family members, FoxL2 and FoxPs for example, are known to be both activators and repressors of transcription or act primarily as repressors. Potentially relevant to this work, repressive activity of FoxL2 has been previously reported in the mammalian ovary (Pisarska et al Endocrinology 2004, Pisarska Am J. Phys Endo. Metabolism 2010, Kuo Reproduction 2012, Kuo Endocrinology 2011, as well as more recent publications). In that context interfering with FoxL2 was proposed to cause upregulated expression of genes normally repressed by FoxL2, accelerated follicle recruitment, and premature ovarian failure.
(2) The authors conclude that the committed progenitor stage is "the gate toward female determination" and that the cells "stay at S-Phase temporarily before differentiation". This conclusion seems to be based solely on single cell RNAseq expression. In several species, including zebrafish, meiotic entry occurs earlier in females and has been correlated with ovary development. The possibility that the late progenitor stage, the stage when meiotic genes are detected in this study and a stage missing in foxl2l mutants, is actually the key stage for female determination cannot be excluded by the data provided.
(3) The authors discuss prior working showing that loss of germ cells leads to male development and that germ cells are required for female development and claim to extend that work by showing here that some progenitors are already sexually differentiated. First, the stages compared are completely different. The earlier work looks at the primordial germ cells and their loss in the first few days of development before a gonad forms. In contrast, this work examines stages well after the gonad has formed and during sex determination. The second concern is that the conclusion that the progenitors are differentiated is based solely on the expression of foxl2l, which is initially expressed in the juvenile ovary state that lab strains have been shown to develop through (Wilson et al Front Cell Dev Bio 2024). While it is fair to state that some cells express ovary markers at this stage, it is unclear that this is sufficient evidence that the cells are differentiated. For example, in the context of the foxl2l mutant, the authors observe that GSCs and early progenitors inappropriately express foxl2l, but the mutants develop as males. Thus, expression of foxl2l transcripts alone is insufficient evidence to claim that the cells are already differentiated as female.
(4) The comparison between medaka and zebrafish foxl2l mutants seems to suggest that Foxl2l is required for meiosis in medaka but has a different role in zebrafish. However, if foxl2l represses the earlier developmental programs of GSCs and early progenitors, it is possible that continued expression of these early programs interferes with activation of meiotic genes. This could account for the absence of the late progenitor stage in foxl2l mutants since the late progenitor stage is defined by and distinguished from the earlier stages by expression of foxl2l and meiotic genes. If so, foxl2l may be similarly required in both systems.
(5) The authors state that "Foxl2l may ensure female differentiation by preventing stemness and antagonizing male development." It is unclear why suppressing stemness would be necessary for female differentiation since female zebrafish have stem cells as do male zebrafish. It seems likely that turning off the GSC and early differentiation programs is important for allowing expression of meiosis and oocyte differentiation genes, and that a gene other than Foxl2l is required for differentiation from GSCs to spermatocytes.
(6) Based on its expression in mutant progenitors, p53 is proposed to assist with alternative differentiation of mutant germ cells. Although p53 transcripts are expressed, no evidence is provided that p53 is involved in differentiation of germ cells, and sex bias has not been associated with the published p53 mutants in zebrafish. Furthermore, while p53 has been shown to be important for ovary to testis transformation in mutant contexts in adults, it appears dispensable for testis development in mutants that disrupt ovary differentiation in earlier stages (Rodriguez-Mari et al PLoS Gen 2010, Shive PNAS 2010, Hartung et al Mol. Reprod. Dev 2014, Miao Development 2017, Kaufman et al PLoSGen 2018, Bertho et al Development 2021. It is possible that p53 eliminates foxl2l mutant germ cells that are simultaneously expressing multiple developmental programs, but this possibility would need to be tested.
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Reviewer #2 (Public Review):
In this manuscript, Hsu et al. used scRNA-seq to profile germ cells isolated from zebrafish ovaries. They identified the transcriptional profile of germ cells representing the early stages of oogenesis, from germline stem cells to newly formed follicle stage oocytes. They identified foxl2l as a gene expressed in probable oocyte progenitor cells, one of the least understood germ cell stages in the ovary. To understand to role of Foxl2l in oogenesis, they produced loss-of-function mutations in foxl2l using CRISPR/Cas9. They found that all foxl2l mutants are males as adults, suggesting that Foxl2l is required for oogenesis. To gain more insights, they performed scRNA-seq on cells isolated from 28 dpf foxl2l mutant ovaries and found that in the absence of foxl2l, germ cells appear to arrest as early progenitors. …
Reviewer #2 (Public Review):
In this manuscript, Hsu et al. used scRNA-seq to profile germ cells isolated from zebrafish ovaries. They identified the transcriptional profile of germ cells representing the early stages of oogenesis, from germline stem cells to newly formed follicle stage oocytes. They identified foxl2l as a gene expressed in probable oocyte progenitor cells, one of the least understood germ cell stages in the ovary. To understand to role of Foxl2l in oogenesis, they produced loss-of-function mutations in foxl2l using CRISPR/Cas9. They found that all foxl2l mutants are males as adults, suggesting that Foxl2l is required for oogenesis. To gain more insights, they performed scRNA-seq on cells isolated from 28 dpf foxl2l mutant ovaries and found that in the absence of foxl2l, germ cells appear to arrest as early progenitors. These results argue that Foxl2l, like its medaka homolog Foxl3, is necessary for promoting oocyte vs. spermatocyte differentiation during the oocyte progenitor stage.
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Reviewer #3 (Public Review):
This is the first report to show a transcriptional factor, foxl2l, is essential for the development of female germs. Without foxl2l, germ cells will be developed into sperms. The report also clearly defined the arrested stage of early germ cells in foxl2l mutants, or stages that is critical for foxl2l to play a role for the further development of female germ cells. Due to lack of cell lineage tracing, the claim of foxl2l suppression of dedifferentiate of progenitor cells to GSC based on the gene expression and cell number changes is weak. In addition, separation of early germ cell types in foxl2l mutant using marker genes from WT may not be optimal.
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Author response:
Reviewer #1 (Public Review):
(1) The authors conclude that the committed progenitors revert to GSCs based on the coexpression of nanos2 and foxl2l nanos2 and based on expression of id1 in mutants but not in WT. Without functional data demonstrating that the progenitors revert to an earlier state, alternative interpretations should be considered. For example, it is possible that the cells initiate the committed progenitor program but continue to express the GSC program and that the coexpression of both programs blocks differentiation.
Thanks for your insightful comment. We have explored possible alternative interpretations of our data. Regarding the suggested possibility of a continued GSC program in the mutant, we have examined the expression of GSC markers including nanos2 in the mutant at different stages. We found …
Author response:
Reviewer #1 (Public Review):
(1) The authors conclude that the committed progenitors revert to GSCs based on the coexpression of nanos2 and foxl2l nanos2 and based on expression of id1 in mutants but not in WT. Without functional data demonstrating that the progenitors revert to an earlier state, alternative interpretations should be considered. For example, it is possible that the cells initiate the committed progenitor program but continue to express the GSC program and that the coexpression of both programs blocks differentiation.
Thanks for your insightful comment. We have explored possible alternative interpretations of our data. Regarding the suggested possibility of a continued GSC program in the mutant, we have examined the expression of GSC markers including nanos2 in the mutant at different stages. We found that in the mutant, nanos2 or other GSC markers were not significantly upregulated in GSC-to progenitor transition (G-P) and early progenitors (Prog-E) (Fig. 4B). The expression of these GSC markers was also low in the integrated clusters I4-I6 when G-P and Prog-E stages were prominent (Fig. 3D and Fig. 3E). GSC marker nanos2 was high only in mutant Prog-C. These results argue against continued GSC programs in the foxl2l mutants. Another possible explanation is that perhaps some mutant Prog-C acquires some GSC property with the upregulation of nanos2 instead of a continuous GSC program. We have now clarified our rationale about mutant cells gaining new GSC properties and included both interpretations in the Result.
Consistent with this possibility, some Fox family members, FoxL2 and FoxPs for example, are known to be both activators and repressors of transcription or act primarily as repressors. Potentially relevant to this work, repressive activity of FoxL2 has been previously reported in the mammalian ovary (Pisarska et al Endocrinology 2004, Pisarska Am J. Phys Endo. Metabolism 2010, Kuo Reproduction 2012, Kuo Endocrinology 2011, as well as more recent publications). In that context interfering with FoxL2 was proposed to cause upregulated expression of genes normally repressed by FoxL2, accelerated follicle recruitment, and premature ovarian failure.
FoxL2 exerts both activating and repressive activities. We believe that Foxl2l can also activate and repress its target gene expression. Although its target genes have not been clearly identified, Foxl2l may activate genes involved such process as oogenic meiosis, and may also repress other genes involved in other processes, say perhaps nanos2.
(2) The authors conclude that the committed progenitor stage is "the gate toward female determination" and that the cells "stay at S-Phase temporarily before differentiation". This conclusion seems to be based solely on single cell RNAseq expression. In several species, including zebrafish, meiotic entry occurs earlier in females and has been correlated with ovary development. The possibility that the late progenitor stage, the stage when meiotic genes are detected in this study and a stage missing in foxl2l mutants, is actually the key stage for female determination cannot be excluded by the data provided.
We agree that Prog-L is important for the initiation of female meiosis. We have made revision in the text to point out the importance of Prog-L in female differentiation.
(3) The authors discuss prior working showing that loss of germ cells leads to male development and that germ cells are required for female development and claim to extend that work by showing here that some progenitors are already sexually differentiated. First, the stages compared are completely different. The earlier work looks at the primordial germ cells and their loss in the first few days of development before a gonad forms. In contrast, this work examines stages well after the gonad has formed and during sex determination.
Both previous studies and our study indicate the important role of germ cells in zebrafish sex differentiation during gonadal development. The earlier works show that the abundance of primordial germ cells contributes to sex differentiation. Our current finding further suggests the existence of female identify in some germ cells at the juvenile stage and discusses the importance of cell in sexual differentiation. We have added the developmental age in our study to emphasize the age difference.
The second concern is that the conclusion that the progenitors are differentiated is based solely on the expression of foxl2l, which is initially expressed in the juvenile ovary state that lab strains have been shown to develop through (Wilson et al Front Cell Dev Bio 2024). While it is fair to state that some cells express ovary markers at this stage, it is unclear that this is sufficient evidence that the cells are differentiated.
The conclusion about the differentiation of progenitors is not based solely on foxl2l expression; rather, it is according to the whole transcriptomic profiles of both WT (Figure 1B) and foxl2l mutant cells (Figure 3A) as well as the foxl2l mutant phenotype (Figure 2C). Three types of progenitors, Prog-E, Prog-C and Prog-L were identified by whole transcriptomic analysis in WT. In foxl2l mutants, the transcriptomic profile further shows that Prog-L and meiotic cells are completely lost, and all germ cells undergo male differentiation eventually. These results together indicate that the differentiation of Prog-C to Prog-L guides the progenitor toward female differentiation. Our result also showed that in the juvenile gonad, foxl2l expression is high in two types of progenitors, Prog-C and Prog-L, and become low after meiotic entry.
For example, in the context of the foxl2l mutant, the authors observe that GSCs and early progenitors inappropriately express foxl2l, but the mutants develop as males. Thus, expression of foxl2l transcripts alone is insufficient evidence to claim that the cells are already differentiated as female.
The foxl2l mutants develop into males because they lack functional Foxl2l. Although the mutated foxl2l transcript is present in mutant cells, these transcripts are not functional. These mutants develop into males eventually. This result is consistent with our claim that functional Foxl2l is important for the development of Prog-L and female differentiation.
(4) The comparison between medaka and zebrafish foxl2l mutants seems to suggest that Foxl2l is required for meiosis in medaka but has a different role in zebrafish. However, if foxl2l represses the earlier developmental programs of GSCs and early progenitors, it is possible that continued expression of these early programs interferes with activation of meiotic genes. This could account for the absence of the late progenitor stage in foxl2l mutants since the late progenitor stage is defined by and distinguished from the earlier stages by expression of foxl2l and meiotic genes. If so, foxl2l may be similarly required in both systems.
Medaka and zebrafish Foxl2l may share similar functions such as the stimulation of meiotic gene expression and promotion of oogenesis in the female germ cells preparing for meiotic entry. In addition, we also detected aberrant upregulation of nanos2 in some foxl2l mutant cells. The idea of “continued expression of these early programs interferes with activation of meiotic genes” is conceivable, but for now we have no evidence for it. We do not know whether the absence of meiotic genes is due to an interference caused by the activation of nanos2 or due to the complete loss of Prog-L and meiotic cells. It will also be interesting to find out whether medaka Foxl2l has a role in early progenitors
(5) The authors state that "Foxl2l may ensure female differentiation by preventing stemness and antagonizing male development." It is unclear why suppressing stemness would be necessary for female differentiation since female zebrafish have stem cells as do male zebrafish. It seems likely that turning off the GSC and early differentiation programs is important for allowing expression of meiosis and oocyte differentiation genes, and that a gene other than Foxl2l is required for differentiation from GSCs to spermatocytes.
It is true that we have not proved whether suppression of stemness is required for female differentiation. Maybe our earlier statement is a bit misleading. We agree that it is likely that turning off the GSC and early differentiation programs is important for allowing expression of meiotic and oocyte differentiation genes, and that a gene other than Foxl2l is required for differentiation from GSCs to spermatocytes. To avoid confusion, we have modified our statement in the text.
(6) Based on its expression in mutant progenitors, p53 is proposed to assist with alternative differentiation of mutant germ cells. Although p53 transcripts are expressed, no evidence is provided that p53 is involved in differentiation of germ cells, and sex bias has not been associated with the published p53 mutants in zebrafish. Furthermore, while p53 has been shown to be important for ovary to testis transformation in mutant contexts in adults, it appears dispensable for testis development in mutants that disrupt ovary differentiation in earlier stages (Rodriguez-Mari et al PLoS Gen 2010, Shive PNAS 2010, Hartung et al Mol. Reprod. Dev 2014, Miao Development 2017, Kaufman et al PLoSGen 2018, Bertho et al Development 2021. It is possible that p53 eliminates foxl2l mutant germ cells that are simultaneously expressing multiple developmental programs, but this possibility would need to be tested.
The tp53-/-foxl2l-/- double mutant cannot alleviate the all-male phenotype of foxl2l-/- mutant (Dev Biol, 517, 91-99, 2024), indicating that the male development is not due to p53-mediated germ cell apoptosis. We have cited the suggested papers and compared relation of tp53 between these mutants (fancl, zar1, etc.) mentioned in the cited papers. Since tp53 was enriched in certain foxl2l-/- mutant cell clusters, and tp53 mutation fails to rescue the all-male phenotype, it is possible that p53 expressed in these mutant cell clusters has roles other than inducing apoptosis. One assumption is that p53 may be involved in the germ cell differentiation, especially p53 is known to promote differentiation of airway epithelial progenitors, adipogenesis and embryonic stem cells. We have emphasized that the suggested role of p53 in germ cell differentiation is our assumption in the Discussion.
Reviewer #3 (Public Review):
This is the first report to show a transcriptional factor, foxl2l, is essential for the development of female germs. Without foxl2l, germ cells will be developed into sperms. The report also clearly defined the arrested stage of early germ cells in foxl2l mutants, or stages that is critical for foxl2l to play a role for the further development of female germ cells.
(1) Due to lack of cell lineage tracing, the claim of foxl2l suppression of dedifferentiate of progenitor cells to GSC based on the gene expression and cell number changes is weak.
Thanks for your comments pointing out our contribution and also weakness. We acknowledge the lack of direct evidence on the reversion of mutant Prog-C to GSC in our data. We now removed the claim about the repression of stemness by Foxl2l.
(2) In addition, separation of early germ cell types in foxl2l mutant using marker genes from WT may not be optimal.
The cell type of mutant cell is determined by two independent analyses. First is inferring the developmental stage of mutant cells. This approach assumes that mutant cells can indeed be mapped to specific WT stages through their transcriptomic profiles. However, as indicated by this reviewer’s comments, mutant cells exhibited heterogeneity and can be distinct from WT cells. Defining cell types in mutants by WT markers may not be optimal. To address this, we conducted another analysis, co-clustering. Mutant cells and WT cells at early stages (GSC , G-P, Prog-E, Prog-C(S) and Prog-C) were co-clustered. This approach does not assume a direct correspondence between mutant and WT developmental stages. Instead, it facilitates the identification of novel germ cell types in mutants while characterizing the relationship between WT and mutant cells. In some clusters, both WT and mutant cells were present, indicating high transcriptomic similarity. In other clusters, most cells are only mutant cells, indicating distinct mutant cell types (Figure 3C). We can, therefore, assign developmental properties to these mutant cells with confidence.
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