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. 2022 May 19;11:e76014. doi: 10.7554/eLife.76014

Figure 1. Single-cell RNA sequencing of 25,089 single cells isolated from 40-day-old zebrafish ovaries.

(A) Single-cell uniform manifold approximation and projection (UMAP) plot of the 40-day-old zebrafish ovary. Cells are color-coded by computationally determined cell clusters. (B) Gene expression plots of known cell-specific marker genes identify the major cell type (labeled in A) that each cluster corresponds to. Cells expressing the indicated gene are colored purple, and the relative intensity indicates relative expression levels (intensity scale for each plot is on the right).

Figure 1.

Figure 1—figure supplement 1. Workflow for single-cell RNA sequencing library preparation, data cleaning, analysis, and validation.

Figure 1—figure supplement 1.

(A) Experimental pipeline for the production of the single-cell RNA-seq library. Briefly, 40-day post-fertilization (dpf) ovaries were isolated from Tg(piwil1:egfp) zebrafish and dissociated to single cells using two dissociation methods. The whole ovary dissociation method favored dissociation of somatic cells but led to loss of germ cells. A less stringent method was used to dissociate germ cells from somatic cells, followed by purification of germ cells by fluorescent-activated cell sorting (FACS). (B–D) FACS pseudocolor scatter plots with gating overlays. (B) The R2 gate selected for GFP+ single cells based on GFP fluorescence intensity (X-axis) and side scatter to measure cell size (Y-axis). (C) The R3 gate selected for GFP signal (X-axis) relative to cell autofluorescence (Y-axis). The R4 and R5 gates selected for cell size (Y-axis) and GFP signal (X-axis). The R5 gate contained smaller cells that were likely premeiotic, meiotic and early-stage oocytes, while R4 gate selected for larger cells that were likely more advanced oocytes. (E–H) Representative images of DAPI-stained nuclei from cells obtained from either the R5 (E–G) or R4 gates. (E) The prominent single nucleolus (n) contained within this nucleus is indicative of a premeiotic germ cell. (F, G) The diffuse chromatin in these nuclei is characteristic of premeiotic oocyte progenitor cells. (H) The presence of condensed and synapse chromosomes is characteristic of cells that have entered meiosis. Note that the nucleus in (H) is 30 µm in diameter while those in (E, F) are between 7 and 10 µm in diameter. Scale bars 10 µm. (I, I’). Fluorescence (I) or bright-field (I’) micrographs of GFP+ cells obtained from gate 5. Scale bars, (E–H) 10 µm; (I, I’) 250 µm.
Figure 1—figure supplement 2. Major cell-type statistics.

Figure 1—figure supplement 2.

(A) Single-cell uniform manifold approximation and projection (UMAP) plot of 40-day-old zebrafish ovary with combined clusters based on major cell types. (B–D) Violin plot of percent of mitochondria genes (B), number of genes per cell (C), and number of transcripts per cell (D), in each major cell type in the final dataset.
Figure 1—figure supplement 3. Differential expression heatmap and top markers of major cell types.

Figure 1—figure supplement 3.

(A) Gene expression heatmap of differentially expressed genes between major cell types. Yellow represents highly expressed genes, purple represents lowly expressed genes, and black represents no expression. (B) Top 5 marker genes computationally identified in each major cell type based on statistical significance.