The dorsalising effect of VAb@2cell is suppressed in MZvrtn mutants. (A) Targeted mutation of the vrtn gene. The Cas9/gRNA targeting sequence and the PAM region (red) are shown, and the mutated allele with the predicted peptide is indicated. (B) Representative images of indicated embryos at 27 hpf, showing smaller eyes and reduced head size in vrtn mutants. (C) Decreased rx3 and six3b expression domains in vrtn mutants at the bud stage. (D) Expression patterns of six3b and myod1 in flat-mounted embryos with equal somite numbers. (E,F) Analysis by scatter plots of head length and six3b expression area at the six-somite stage. The red broken lines in the insets define the parameters of measurement. Horizontal lines represent the mean±s.d. from three batches of embryos with the total number indicated. (G-J) Analyses by in situ hybridisation and qRT-PCR of the expression of gsc, dha/boz, bmp2b and otx2 in MZvrtn mutants. The expression of gsc and dha/boz remains unchanged at sphere stage (animal pole views), the expression of bmp2b is enhanced at 50% epiboly, and the expression of otx2 is reduced at 80% epiboly (lateral views with dorsal on the right). The relative expression level in wild-type embryos is normalised as 1; data are mean±s.d. from three independent experiments. (K) MZvrtn embryos are ventralised following VAb@2cell. (L) Statistical analysis of the ventralising effect. Numbers at the top represent total embryos from three independent experiments. (M,N) Analyses by in situ hybridisation and qRT-PCR of bmp2b expression at the shield stage in wild-type and MZvrtn embryos after VAb@2cell. The embryos are shown as lateral views. The relative bmp2b level in VAb@2cell wild-type embryos is set as 1; data are mean±s.d. from six independent experiments. Scale bars: 250 µm.
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