FIGURE 1

Isolation of germ cells from juvenile zebrafish with defined sex. (A). The flow chart for the generation of W- and Z-hybrids. Sex-reversed ZW^SR Nadia males were crossed with ZW females to obtain the WW females in the F2 generation. The WW and ZZ fish were each mated with the Tg (ziwi: GFP) in the TL genetic background to obtain W- or Z-hybrid offspring with fluorescent germ cells. (B). Gel electrophoresis showing the detection of the W-fragment in the genome of 10 offsprings from the WW mother after PCR amplification (lanes 1–10). Lanes 11 and 12 are genomic DNAs from the fin of a male and a female of the Nadia strain as controls, respectively. The W fragment is 86 bps in length. The star marks nonspecific PCR bands. (C). The gonad of a Tg (ziwi: GFP) fish with fluorescent germ cells. (D). The flow chart for transcriptome analysis. Larvae were first staged to ensure proper development before their gonads were dissected. Their germ cells were then dissociated and sorted by FACS before RNA-seq analysis. (E). A representative FACS plot shows the selection of GFP-positive germ cells (P1). (F). A typical FACS data set showing the procurement of 232 GFP⁺ germ cells (pink group, 0.3% of all cells). Blue-group (P3) and pink-group (P4) cells were obtained after cell size quantity control. The green group (P2) selects for live cells that do not stain with propidium iodide. Black dots represent other cells and cell debris.