Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary
- University of California, Davis, United States
- Mount Desert Island Biological Laboratory, United States
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 dpf). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA sequencing studies which identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in 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.
Data availability
The raw and processed data reported in this paper are archived at NCBI GEO https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE191137) and in an interactively browsable forms at the Broad Institute Single-Cell Portal (https://singlecell.broadinstitute.org/single_cell/study/SCP928/40dpf-ovary-all-cells). Analysis code and objects are archived at github (https://github.com/yulongliu68/zeb_ov_ssRNAseq). Gene expression tables for the cell clusters identified are archived at Dryad: (https://datadryad.org/stash/share/CEd0Zs4oZKdinTWeJPKbWYjBq6hYq4QhVacQcFjf37E).
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Single-cell transcriptome reveals insights into the development and function of the zebrafish ovaryNCBI Gene Expression Omnibus, GSE191137.
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Single-cell transcriptome reveals insights into the development and function of the zebrafish ovaryDryad Digital Repository, doi:10.25338/B8FH12.
Article and author information
Author details
Funding
National Institutes of Health (R01 HD-081551)
- Yulong Liu
- Matthew E McFaul
- Lana N Christensen
- Bruce W Draper
National Science Foundation (IOS-1456737)
- Michelle E Kassack
- Lana N Christensen
- Bruce W Draper
National Institutes of Health (T32 training grant ES-0070599)
- Michelle E Kassack
National Institutes of Health (T32 training grant GM-007377)
- Matthew E McFaul
- Sydney R Wyatt
National Science Foundation (GRFP 2036201)
- Sydney R Wyatt
National Institutes of Health (R35 GM133689)
- Stefan Siebert
- Celina E Juliano
National Institutes of Health (R01 DK126021)
- Iain A Drummond
National Institutes of Health (R01)
- Caramai N Kamei
- Iain A Drummond
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#20200 and #20201) of the University of California, Davis.
Copyright
© 2022, Liu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary
eLife 11:e76014.
https://doi.org/10.7554/eLife.76014
Further reading
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The rete ovarii (RO) is an appendage of the ovary that has been given little attention. Although the RO appears in drawings of the ovary in early versions of Gray’s Anatomy, it disappeared from recent textbooks, and is often dismissed as a functionless vestige in the adult ovary. Using PAX8 immunostaining and confocal microscopy, we characterized the fetal development of the RO in the context of the mouse ovary. The RO consists of three distinct regions that persist in adult life, the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). While the cells of the IOR appear to form solid cords within the ovary, the EOR rapidly develops into a convoluted tubular epithelium ending in a distal dilated tip. Cells of the EOR are ciliated and exhibit cellular trafficking capabilities. The CR, connecting the EOR to the IOR, gradually acquires tubular epithelial characteristics by birth. Using microinjections into the distal dilated tip of the EOR, we found that luminal contents flow toward the ovary. Mass spectrometry revealed that the EOR lumen contains secreted proteins potentially important for ovarian function. We show that the cells of the EOR are closely associated with vasculature and macrophages, and are contacted by neuronal projections, consistent with a role as a sensory appendage of the ovary. The direct proximity of the RO to the ovary and its integration with the extraovarian landscape suggest that it plays an important role in ovary development and homeostasis.
