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Fig. 2 (A to E) GFP-negative F1 sibling embryos developed normally. (F to J) Typical Mnanog defective phenotypes observed among GFP-positive embryos. All embryos are lateral view with the dorsal region on the right (A to D and F to I) or upward (E and J). (K) Stacked columns show the ratio of Mnanog-like defective embryos among GFP-positive offspring in Tg(zpc:zcas9) background. Higher efficiency in generating maternal mutants was observed by expressing three sgRNAs. Numbers on the top of each column represent the total GFP-positive embryos scored. WT, wild-type fish; Tg1 to Tg4, different mutation-carrying F0 founders. (L) Single sgRNA expression in Tg(zpc:zcas9) leads to indel in nanog maternal transcripts, resulting in frameshift and premature termination of translation. The four exons of nanog are represented by black boxes, with gray indicating untranslated regions (UTRs). Mutant transcripts were identified in two independent embryos at 3 hpf. The dashed line corresponds to deleted nucleotides; red bases indicate insertions. Red letters in protein sequences indicates amino acid residues translated after frameshift mutation. The arrowhead represents the position of amino acid in wild-type protein. (M to P) Nuclear accumulation of Nanog protein in 3-hpf wild-type embryos revealed by immunofluorescence. (Q to T) Absence of Nanog protein expression in 3-hpf Mnanog embryos. (P and T) Magnified images of framed regions in (O) and (S). Hoechst 33342 (blue) was used to stain nuclei. (U to X) In situ hybridization (ISH) analysis shows the expression of sox17 at 7.5 hpf and mxtx2 at the sphere stage. (Y) Procedure to rescue Mnanog phenotype by mRNA injection. (Z) Stacked columns show a nearly complete rescue of Mnanog mutant defects after injection of the wild-type nanog-myc mRNA. Unij, Uninjected. Numbers represent total embryos scored from two experiments. Scale bars, 50 μm (P and T) and 250 μm (others).