4-OHT–induced hwa mutations in GCs of young females. (A) Zebrafish hwa locus and hwa-gRNAs transgene structures. E1–E3, exons; gray bars, UTR. (B) Procedure for 4-OHT treatments of the double transgenic young fish and subsequent mating and embryonic analysis. Moon, night; Sun, daytime. (C) Immunostaining of Cas9 (green) and nuclei stained by DAPI (blue) in ovaries within the 4-OHT– or DMSO-treated females at 45 dpf. (D) Box plots showing the ratio of Cas9 (green) signal intensity in the nucleus to the cytoplasm of oocytes (left, mean ± SD), and the signal intensity ratio of Cas9 (green) to DAPI (blue) in the nucleus of oocytes (right, mean ± SD). Nu, nucleus. Cyto, cytoplasm. No. oocytes, the number of oocytes. P values of the t test (unpaired, two-sided) were also labeled. (E) The ratio of ventralized embryo types (left bar graph) and morphology of representative embryos at 24 hpf (right). Females: the serial number of treated transgenic females. No. embryos, the number of embryos produced by the corresponding females. (F) Left, bar chart showing the proportion of different mutated hwa genotypes. No. embryos, the number of embryos. Right, schematic showing different mutated genotypes of the hwa allele in the ventralized embryos. The light green boxes indicated the UTR of hwa, and the dark green boxes indicated the exons of hwa. Red lines and blue rectangles represented deletions and insertions, respectively. F1 and R1 are the forward and reverse genotyping primers of the hwa target site. Source data are available for this figure: SourceData F4.