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Fig. 4 - Supplemental 1 Generation of the zebrafish raldh2 mutants by CRISPR–Cas9. (A) The nucleotide sequence surrounding the mutations in raldh2 is shown on the left. The target sequence of the crRNA is emphasized with an underline, while the inserted nucleotide sequence is indicated in orange. Below the DNA sequence, the predicted amino acid is displayed, with red letters highlighting the abnormal amino acid resulting from the frameshift mutation. The asterisk represents the termination codon. (B) A schematic representation of the predicted protein structure is shown, with the red box indicating the abnormal amino acid sequences resulting from the frameshift mutations. The total number of predicted amino acids is displayed to the right of the schematic. (C) The absence of pectoral fins (arrowhead) in raldh2 homozygous mutants is observed dorsally at 3 dpf. (D) The expression of tbx5a in the pectoral fin buds (arrowhead) is significantly reduced in raldh2 mutants. The phenotype of our raldh2sud118 mutants closely resembles the phenotype observed in previously isolated raldh2 mutants.