Fig. 3

Fig. 3 Defective PGC development in MZznf706 mutants by CRISPR/Cas9. A: Schematic diagram showing WT and predicted mutant Znf706 protein structures. Red asterisk, the mutated site; brown square, the mutated amino acid sequence. B: Morphology of MZznf706 mutants and WT embryos. C: Relative expression levels of znf706 in WT and MZznf706 embryos at 1-cell stage detected by qRT-PCR. D: Left, ISH of ddx4 showing germ plasm and PGCs in WT and MZznf706 embryos at 4-cell, shield, and 26 hpf stages. The dashed boxed areas were enlarged for better viewing. Blue square represented embryos with normal PGC number and proper location; green square represented embryos with reduced ddx4 signal; orange square represented embryos with ectopically located PGCs (>1). Right, histogram showing the proportion of embryos as exemplified in ddx4 ISH (left). E: Representative images of PGCs in WT and MZznf706 embryos, labeled by GFP in the Tg(kop:egfp-CAAX-nanos3 3′UTR) transgenic background. The dashed boxed areas were enlarged for better viewing. F: Scatter plots illustrating the discrepancy in PGC counts (E). Fifteen larvae were observed for each group. G: Fluorescence images (left) showing PGCs in WT embryos, MZznf706 embryos (-), and MZznf706 embryos injected with WT znf706 mRNA (Rescued). Bar plot (right) showing the ratio of embryos with ectopic PGCs (ectopic, orange) or without ectopic PGCs (normal, blue). WT OE, overexpression of WT znf706 mRNA; Mut OE, overexpression of mutant form znf706 mRNA. H: Scatter plots illustrating the PGC number per embryo (G). Fifteen larvae were observed for each group. I: Bar plot showing the sex ratio of MZznf706 and WT adults. n, number of fish per batch. J: Morphology of MZznf706 and WT adults. K: Morphology of WT embryos and offspring derived from MZznf706 incrossing. Scale bar, 100 μm (B, D, E, G, K). n, number of embryos (D and G). ns, not significant; ∗∗∗∗, P < 0.0001; ∗∗∗, P < 0.001; ∗∗, P < 0.01; ∗, P < 0.05. PGC, primordial germ cell; WT, wild-type; Mut, mutant; MZ, maternal-zygotic; ISH, in situ hybridization; hpf, hours post fertilization; OE, overexpression; dpf, days post fertilization.