UC Davis

Zebrafish are an established research organism that has been used to make 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). As such, forward genetic screens have identified only a few genes required for gonad development. In addition, bulk RNA sequencing studies used to 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 used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. In total, we profiled 10,658 germ cells and 14,431 somatic cells. In Chapter 1, I introduced germline stem cells in zebrafish and germline stem cell niches identified in other organisms. In Chapter 2, we used our single-cell data to identify the major cell types of the ovary. We then focused on the germ cell cluster, which contains the developmental stages of the germ cells from germline stem cells to early meiotic oocytes. From these data, we discovered a novel progenitor-stage germ cell marker in zebrafish, foxl2l, and revealed that it is required for female sex development. Furthermore, we identified multiple transcription factors that potentially have a role in germline stem cell regulation. As described in Chapter 3, we explored our somatic cell data, which represents all known somatic cell types, including follicle cells, theca cells, and interstitial stromal cells. Further analysis of these data revealed an unexpected number of somatic 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. Our results reveal novel insights into ovarian development and function and our Atlas will provide a valuable resource for future studies. In Chapter 4, I discuss future directions that should be pursued as a result of this dissertation.