Maternal wnt8a mutants show no defects in dorsal-axis formation. (A) Maternal wnt8a mutant embryos (Mwnt8a-/-) at 30 dpf. Embryos were obtained by crossing WT male fish and female fish with wnt8aΔO1ΔO2/ΔO1ΔO2 germ cells. Lateral view with anterior to the left. The Mwnt8a-/- mutant embryos showed no apparent abnormality in dorsal axis formation (n=190). (B, C) Genotyping and RT-PCR of Mwnt8 mutant embryos. The TALEN target regions of ORF1 (upper panel) and ORF2 (lower panel) were amplified by PCR from the genome of a WT and eight Mwnt8a mutant embryos (B), and by RT-PCR from the RNA of a one-cell-stage WT and four Mwnt8a mutant embryos (C). The PCR products were separated on acrylamide gels. The positions of the PCR bands corresponding to the mutant DNAs are marked by asterisks. “+” and “-” indicate the presence and absence of reverse transcriptase (RT) in the cDNA synthesis. Note that all of the Mwnt8a mutant embryos had both WT and mutant alleles (n=8/8), and all four Mwnt8a mutants had only mutant ORF1 and ORF2 transcripts. (D) Expression of noto (shield stage, a, b), tbx6l (80% epiboly stage, c, d), otx2 (100% epiboly stage, e, f), and gsc (shield stage, g, h) in WT and Mwnt8a mutants. Mwnt8a mutants were obtained by crossing female fish with wnt8aΔO1ΔO2/ΔO1ΔO2 germ cells and WT (b, d, f) or wnt8aΔO1ΔO2/+ male (h) fish. Animal pole views with dorsal to the right (a, b, g, h). Lateral views with dorsal to the right (c-f). The expression of noto, tbx6l, otx2, and gsc was unaffected in the Mwnt8a mutants compared to WT embryos (noto, n=20; tbx6l, n=20; otx2, n=20; gsc, n=16). Scale bars: 500 µm in A; 200 µm in D.