Fig. 2

Foxl2l is required for female differentiation. (A) In situ hybridization detects foxl2l transcripts in indifferent gonads at 11 days post fertilization (dpf) (a, a’). Expression of foxl2l becomes female biased when female differentiation initiates at 22 dpf (b, b’), and becomes female-specific at the initiation of male differentiation at 30 dpf (c, c’). In adults, foxl2l is expressed in cystic cells in ovary but not in testis (d, d’). mpf: months post fertilization. All samples were collected from Nadia strain. Scale bars are 50 µm in (a, a’), 100 µm in (b, b’, c, c’), 20 µm in (d, d’). (B) Domain structure of wild-type (WT) and the predicted mutant of Foxl2l with 10 base pairs insertion (10i) or 22 base pairs insertion (22i) generated by CRISPR/Cas9 system. (C) Sex ratios of WT (+/+), foxl2l+/22i heterozygous (+/-), and foxl2l22i/22i homozygous (-/-) mutants at 4 months of age reveal that foxl2l mutant fish are all males. The total numbers of fish in each genotype and gender are labeled in white. Dot: sex ratio of each batch. (D) All foxl2l10i/10i mutant males (-/-) are fertile. Dot: individual fish used in the test, n=3 in WT and heterozygous mutants, n=6 in homozygous mutants. (E) The foxl222i/22i homozygous (-/-) mutants lack meiotic oocytes at 20 dpf shown by histological staining. Black dashed circle: germ cell cysts with one to three nucleoli in each cell. Yellow dashed circle: meiotic oocyte. Scale bars represent 20 µm. (F) The foxl2l22i/22i mutants (-/-) express male markers (tekt1 or odf3b) detected by RNA fluorescence in situ hybridization (FISH) at 28 dpf. Scale bars are 10 µm.