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ephb4b genetic mutants exhibit defects in DFC aggregation and laterality. (A) Illustration of the ephb4b mutant allele (Mt) generated by the Cas9 knockout approach. (B) ephb4b expression pattern in different types of embryos at 75% ES. The siblings were genotyped after being photographed and the proportion of embryos exhibiting the representative pattern is indicated. (C) Morphology of live WT and mutant siblings at 28hpf. (D-F) Heart jogging was detected by probing cmlc2 expression at 28hpf (D) and quantified (E). ns, not significant; **P<0.01. After in situ hybridization, each embryo was genotyped by PCR as shown in F. (G) DFC aggregation was examined by probing sox17 expression at 75% ES. Shown are representative DFC patterns in mutants only. The DFC region is enlarged in the lower panels. (H) Representative genotyping results for DFCs-III embryos. (I) Embryos at the bud stage were immunostained for GFP expression and observed by confocal microscopy. The AP axis is specified.
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