Fig. 6

fgfr4^-/- phenotype identified by inbreeding injected founder fish. Two single-guide RNAs (sgRNAs) targeting two different exons of the fgfr4 gene were co-injected with Cas9mRNA into wild-type embryos and raised to adults to generate founder fish (F₀). Six pairs of founder fish (F₀) were inbred and scored for mutant phenotypes. One of the six pairs showed multiple embryos with the same phenotype observable at 48 h post-fertilization. (A) Wild-type embryo at 48 h post-fertilization. White arrow indicates a normally sized hindbrain, white arrowhead indicates a normal heart chamber, and black arrow represents a normal tail length. (B) An fgfr4 compound heterozygous mutation displaying multiple phenotypes. White arrow indicates a reduced hindbrain region, white arrowhead points to a heart edema, and black arrow shows the shortened body axis. All phenotypes were only found in the compound heterozygous mutant embryos.