Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary
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Curated by eLife
Evaluation Summary:
This single cell transcriptomic analysis of young adult zebrafish ovaries provides important new data to understand gene expression patterns in numerous ovarian cell types that lead to insights into how ovary development works, and most of the principles will likely apply across vertebrates. The work will interest researchers who study gonad development, sex determination, differences (or 'disorders') in sex development, and impacts of the environment (including toxic pollutants) on gonad development and function.
(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. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage (≥5 days post-fertilization). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA-sequencing studies that identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in the development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells, and ovarian stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, we used gene knockout experiments to determine the roles of foxl2l and wnt9b for oocyte development and sex determination and/or differentiation, respectively. Our results reveal novel insights into zebrafish ovarian development and function, and the transcriptome profiles will provide a valuable resource for future studies.
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Evaluation Summary:
This single cell transcriptomic analysis of young adult zebrafish ovaries provides important new data to understand gene expression patterns in numerous ovarian cell types that lead to insights into how ovary development works, and most of the principles will likely apply across vertebrates. The work will interest researchers who study gonad development, sex determination, differences (or 'disorders') in sex development, and impacts of the environment (including toxic pollutants) on gonad development and function.
(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. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors sought to achieve an understanding of the cell types present in the ovaries of a young mature zebrafish adult, define the set of genes each cell type expresses, to identify cell types in histological sections, and to identify function for two genes by induced mutations.
The work has several major strengths. The methodologies were appropriate for the questions posed. The results were analyzed in depth, looking at the expression patterns in the scRNA-seq data for a substantial number of genes with a focus on those genes' likely functions. The single molecule RNA in situ hybridization experiments tied the dots-on-a-sheet expressions to cells in actual animals. The identification of subpopulations of cell types is useful to understand development moving forward. The estrogen story is nice as is …
Reviewer #1 (Public Review):
The authors sought to achieve an understanding of the cell types present in the ovaries of a young mature zebrafish adult, define the set of genes each cell type expresses, to identify cell types in histological sections, and to identify function for two genes by induced mutations.
The work has several major strengths. The methodologies were appropriate for the questions posed. The results were analyzed in depth, looking at the expression patterns in the scRNA-seq data for a substantial number of genes with a focus on those genes' likely functions. The single molecule RNA in situ hybridization experiments tied the dots-on-a-sheet expressions to cells in actual animals. The identification of subpopulations of cell types is useful to understand development moving forward. The estrogen story is nice as is straightening out the foxl2 paralog naming system. The work has few detracting weaknesses.
The authors' datasets and analysis achieved their stated aims and results support conclusions.
The work will have a positive impact on the field, having identified many gene sets to be explored by the community by mutation and other types of experiments.
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Reviewer #2 (Public Review):
The goal of this study was to describe the cell types and hierarchies present in the juvenile zebrafish ovary using single-cell RNA sequencing. The authors were successful in describing unexpected complexities in the various cell types of the ovaries including follicle cells, theca cells, and interstitial stromal cells. For germ cells, they were able to provide molecular profiles of pre-meiotic, meiotic and early-stage oocytes. For the ovarian mesenchyme, they describe follicle cells and pre-follicle cell subtypes, theca cell subpopulations, and other stromal cell subpopulations, including a putative stromal progenitor. Many of these cell subtypes were validated by their expression of molecular markers and their histologies. The authors also produced reverse genetic evidence of the role of newly identified …
Reviewer #2 (Public Review):
The goal of this study was to describe the cell types and hierarchies present in the juvenile zebrafish ovary using single-cell RNA sequencing. The authors were successful in describing unexpected complexities in the various cell types of the ovaries including follicle cells, theca cells, and interstitial stromal cells. For germ cells, they were able to provide molecular profiles of pre-meiotic, meiotic and early-stage oocytes. For the ovarian mesenchyme, they describe follicle cells and pre-follicle cell subtypes, theca cell subpopulations, and other stromal cell subpopulations, including a putative stromal progenitor. Many of these cell subtypes were validated by their expression of molecular markers and their histologies. The authors also produced reverse genetic evidence of the role of newly identified genes in the regulation of progenitor functions in germ cells stem cells and pre-follicle cells. Finally, they used steroid biosynthesis as a test case for the utility of this cell atlas in elucidating cellular and molecular functions.
Major strengths:
The hypothesis that Foxl2l is a marker of oocyte progenitors was well supported by histological evidence and the localization of nanos2 and rec8a. Furthermore, the sex ratio skewing observed in the homozygous knock-in allele recapitulates its role in medaka as a necessary transcription factor to commit to female germ cell differentiation.
Similarly, disruption of the wnt9b gene perturbs sex determination, and partially phenocopies previous findings of wnt4a, suggesting this pathway is important in early follicle cell specification.
Another important finding is the source of E2 biosynthesis in the zebrafish which appears to favor Hsd17b1 to catalyze the E2 synthesis, which is expressed primarily in follicle cells. This mirrors the 2 cell hypothesis of E2 production in mammalian follicles but does not rely on cyp19a1. This is supported both by molecular evidence and careful analyses of previously uncharacterized follicular structures.
Weaknesses:
The identification of a putative stromal progenitor is one of the more exciting findings of the study. Yet their molecular signature and spatial position within the ovary were not validated.
Likewise, the presence of distinct subpopulations of theca is not fully explored. It is unclear whether the subsets are associated with different maturation stages of the follicle, and thus might represent immature versus differentiated theca.
Likely impact:
This dataset identified novel cell types and molecular pathways which will likely be the subject of fertile investigation. Furthermore, many of the genes identified herein are likely to be developmentally relevant and should be the subject of further gene knockout analysis.
The data provides strong evidence of conservation of function of key gonadal sex-determining genes, including those of the Wnt and Foxl2 family, which have orthologs in mammals and fish. Therefore, this new understanding of ovarian cells provides a framework for using zebrafish as a model for ovarian development and sexual differentiation in humans, and perhaps for the study of developmental disorders and diseases.
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Reviewer #3 (Public Review):
This scRNAseq study provides definitive markers for various ovarian cell types in the zebrafish and new cell sub-types, as well as specific ovarian germ cell stages and early oocyte developmental stages, which will be valuable for future functional studies. Importantly, this study makes clear the strongly conserved nature of oogenesis between zebrafish and mammals, demonstrating conserved expression signatures for various cell types. Beautiful FISH analysis of definitive marker genes of particular subpopulations was used to determine where the subpopulation resides in the ovary. The gene motif enrichment analysis to identify putative transcriptional regulators of gene modules is quite compelling and valuable in providing testable frameworks of regulatory pathways. A further strength is the functional …
Reviewer #3 (Public Review):
This scRNAseq study provides definitive markers for various ovarian cell types in the zebrafish and new cell sub-types, as well as specific ovarian germ cell stages and early oocyte developmental stages, which will be valuable for future functional studies. Importantly, this study makes clear the strongly conserved nature of oogenesis between zebrafish and mammals, demonstrating conserved expression signatures for various cell types. Beautiful FISH analysis of definitive marker genes of particular subpopulations was used to determine where the subpopulation resides in the ovary. The gene motif enrichment analysis to identify putative transcriptional regulators of gene modules is quite compelling and valuable in providing testable frameworks of regulatory pathways. A further strength is the functional analysis of two genes that were mutated to test for predicted functions.
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