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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.

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism., eLife digest Before a cell divides, it creates a copy of its genetic material (DNA) and evenly distributes it between the new ‘daughter’ cells with the help of a complex called the mitotic spindle. This complex is made of long cable-like protein chains called microtubules. To ensure that each daughter cell receives an equal amount of DNA, structures known as centrosomes organize the microtubules during the division process. Centrosomes have two rigid cores, called centrioles, which are surrounded by a matrix of proteins called the pericentriolar material. It is from this material that the microtubules are organized. The pericentriolar material is a dynamic structure and changes its size by assembling and disassembling its protein components. The larger the pericentriolar material, the more microtubules can form. Before a cell divides, it rapidly expands in a process called centrosome maturation. A protein called pericentrin initiates the maturation by helping to recruit other proteins to the centrosome. Pericentrin molecules are large, and it takes the cell between 10 and 20 minutes to make each one. Nevertheless, the cell can produce and deliver large quantities of pericentrin to the centrosome in a matter of minutes. We do not yet know how this happens. To investigate this further, Sepulveda, Antkowiak, Brust-Mascher et al. used advanced microscopy to study zebrafish embryos and human cells grown in the laboratory. The results showed that cells build and transport pericentrin at the same time. Cells use messenger RNA molecules as templates to build proteins. These feed into protein factories called ribosomes, which assemble the building blocks in the correct order. Rather than waiting for the pericentrin production to finish, the cell moves the active factories to the centrosome with the help of a molecular motor called dynein. By the time the pericentrin molecules are completely made by ribosomes, they are already at the centrosome, ready to help with the recruitment of other proteins during centrosome maturation. These findings improve our understanding of centrosome maturation. The next step is to find out how the cell coordinates this process with the recruitment of other proteins to the centrosome. It is also possible that the cell uses similar processes to deliver other proteins to different parts of the cell.

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment ofPCNTmRNA, its translation near the centrosome, and requirement of intact polysomes forPCNTmRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.