Fig. 3

a Schematic of the mating strategy used to generate smad1-cKO (or smad9-cKO) embryos (“Methods” section). PGCs were labeled with kop:Eos (green) or piwil1:mCherry (red), and cKO embryos identified by gsc:tdTomato or cryaa:CFP. b Genome editing efficiency of three smad1 gRNA target sites assessed by targeted amplicon sequencing in somatic cells and PGCs sorted from sibling and smad1-cKO embryos at 15-somite stage. NGS, next-generation sequencing. N. reads, number of aligned reads per target site. Representative images of sibling and smad1-cKO embryos at the 13-somite stage (c) and 1 dpf (d). Embryos were dorsally viewed (c), or laterally viewed with anterior to the left (d). PGCs were kop:Eos-positive (green). The gsc:tdTomato-positive (red) notochord (NT) was indicated by arrow (c). e ISH-detected ddx4 expression in sibling and smad1-cKO embryos at 2 dpf. The gonad region was enlarged for better visibility. f Quantification of PGC numbers per embryo in sibling and smad1-cKO groups at the indicated stages. g The proportion of mislocated PGCs in sibling and smad1-cKO embryos at 1 dpf. h Sex ratios of sibling and smad1-cKO adult fish at 4.5 months post-fertilization (mpf). i Rescue of PGC numbers in smad1-cKO embryos by smad1-nanos3 3′UTR mRNA injection (250 pg/embryo). Left, representative images of 2 dpf embryos with a lateral view and anterior to the left. PGCs were kop:Eos-positive (green); right, box plot showing PGC numbers per embryo. In f and i, the bottom, top edges, and whiskers represent the min and max values and 1.5 times the IQR, respectively. The median is indicated by the center line. Each dot represented one embryo. Statistical analysis was performed using two-sided Student’s t-test (f, i) or two-sided Fisher’s exact test (g, h). Ne, number of embryos analyzed; Nc, number of PGCs quantified; Nf, number of fish tested. Source data are provided as a Source Data file.