(A) Genomic region around the start codon (green box) of gjd2b gene was selected for TALEN design, where TALEN-1 recognition sequence (blue line) spans the 5′UTR, the start codon sequence, and a few nucleotides after the start codon. The TALEN-2 recognition sequence (purple line) begins after the 18 nucleotide spacer region. (B) Chromatograms of sequence read from wild-type (WT) and homozygous gjd2bncb215 fish generated in this study indicate an insertion of nucleotide G (red arrowhead) within the Xho1 restriction site (blue line). (C) Representative gel image after Xho1 restriction digestion analysis of an 800 bp amplicon (includes ~300 bp upstream and 500 bp downstream sequences from the point of insertion) shows undigested and partially digested bands in the homozygous and heterozygous mutants, respectively, compared to complete digestion in WT siblings. (D) Predicted amino acid sequences of various gjd2b mutant alleles generated in this study aligned with the WT Gjd2b sequence. gjd2bncb215 is predicted to code for the first six amino acid residues of Gjd2b followed by a nonsense sequence up to the 54th amino acid position. Presence of a premature stop codon terminates translation at this position. (E) Representative images of Gj2b-like immunoreactivity from WT and mutant fish. Staining was not completely abolished as the antibody also recognizes Gjd2a. (F) Gjd2b-like immunoreactivity is reduced in PNs of gjd2bncb215 compared to WT larvae (Mann–Whitney U test; ***p<0.001). Number of images analyzed is indicated in parentheses. Data used for quantitative analyses are available in Figure 2—source data 1.
