Single cell transcriptomes of zebrafish germline reveal progenitor types and feminization by Foxl2l [scRNA-seq]

Sex determination is distinct from development of other organs for its diversity among species. In zebrafish, the sex determination in juvenile stage could be hermaphroditic or gonochoristic. However, the detail mechanism is still unclear. Here in this study, we found that the transcription factor that belongs to forkhead box family, Foxl2l, is required for determination of female fate in zebrafish. Foxl2l was expressed from larva stage and became female bias in juvenile stage. The expression was restricted in pre-meiotic germ cells. Disruption of Foxl2l function by CRISPR/Cas9 system resulted in all-male phenotype in the mutant. Germ cells in foxl2l mutant were arrested in pre-meiotic stage at 21 dpf, the critical period for female meiotic entry. To further verify the downstream mechanism of Foxl2l on female sex determination, wildtype or foxl2l mutant germ cells labeled by foxl2l:egfp at 21 days post fertilization (dpf) and germ cells labeled by ziwi:egfp at 26 dpf were analyzed by RNA-seq and single-cell RNA-seq respectively. Trunks of ziwi:egfp;foxl2l-/- or ziwi:foxl2l+/+ containing gonad were dissociated by 0.4% collagenase and 0.5% trypsin at 26 dpf. Germ cells were then sorted by fluorescence activated cell sorter. Around 3000 EGFP+ cell were collected in each genotype. cDNA libraries were constructed by Chromium Next GEM Single Cell 3' Library Construction Kit v3.1 kit (10x Genomics) and was sequenced performed by Illumina NextSeq 500.