Fig. 2

Defective PGC development in znf706 maternal and maternal-zygotic cKO mutants. A: Representative pictures of WT and Tg(piwil1:zCas9;ca15b:mCherry;U6:znf706 gRNAs) embryos at 28 hpf showing PGC positions labeled by mCherry. B: Bar plot showing the ratio of WT and maternal cKO embryos at 28 hpf containing ectopic PGCs (ectopic, orange) or without ectopic PGCs (normal, blue). C: Bar plot showing qRT-PCR analysis of ddx4 and dnd1 mRNAs in WT and znf706 MZ cKO mutant embryos at 1-cell stage. D: ISH of ddx4 (upper) and dnd1 (lower) showing germ plasm distribution in WT and znf706 MZ cKO mutant embryos at 4-cell stage. The dashed boxed areas were enlarged for better viewing. E: ISH of ddx4 revealing PGCs in WT and znf706 MZ cKO mutant embryos at shield, 18-somite, and 24 hpf stages. The dashed boxed areas were enlarged for better viewing. F: Histogram showing the proportion of embryos in (D) and (E) with normal (blue), impaired ddx4 signal (green), and ectopically located PGCs (orange) at indicated stages. Blue square represented embryos with normal PGC number and proper location (D–F); green square for embryos with reduced ddx4 signal (D–F); orange square for embryos with at least one ectopic PGC location (E–F). G: Immunostaining of Ddx4 showing PGCs in WT and znf706 MZ cKO embryos (left) at 18-somite and 24 hpf stages. Scatter plots (right) illustrating the discrepancy in PGC counts. Scales bars, 100 μm (A); 50 μm (D, shield and 18-somite stages in E); 250 μm (24 hpf in E); 20 μm (G). n, number of embryos. ∗∗∗∗, P < 0.0001; ∗∗, P < 0.01; ∗, P < 0.05. cKO, conditional knockout; MZ, maternal-zygotic; PGCs, primordial germ cells; WT, wild-type; ISH, in situ hybridization; IF, immunofluorescence staining; hpf, hours post fertilization.