Figure 4

Figure 4

Dhi2059 long-finned phenotype was only able to be rescued by a kcnj13 loss-of-function mutation in an allelic-specific manner. (A) Illustration for principle of the genetic rescue experiment. Purple dots represent WT kcnj13 gene expression in both endogenous domains and ectopic expression in somites. The blue dots represent the loss-of-function kcnj13 endogenous expression by the jaguar (Kcnj13^G157E/) allele, which is driven by an intact promoter. The orange dots represent the overlapped expression. The green lines indicate pronephric ducts. (B) WT control fish. (C) Morphology of heterozygous jaguar mutant. (D) Morphology of homozygous jaguar mutant. (E) Morphology of kcnj13^Dhi2059/+ mutant. (F and G) Morphology of double heterozygous jaguar and Dhi2059 mutants, kcnj13^{Dhi2059/G157E}. (F) Male. (G) Female. Note, the caudal fin broke due to its large size before imaging. Although pigmentation patterns were altered, the long-finned phenotype still remained in kcnj13^{Dhi2059/G157E} mutants. (H) Quantitative comparison between kcnj13^Dhi2059/+ mutant and kcnj13^{Dhi2059/G157E} mutants. WT (n = 20); G157E, jaguar (G157E, n = 15); Dhi2059 (n = 19); Dhi2059; G157E (n = 21). * P < 0.0001 by unpaired Student’s t-test. (I) Location of CRISPRs against kcnj13 coding region. CRISPR and important functional domains are annotated with colored arrows. (J–L) illustration and morphology of CRISPR-induced insertion/deletion mutation in Dhi2019 mutants. All fish shown are injected F₀ adults. (J) CRISPR mutation is located on the WT allele (orange bar) of kcnj13^Dhi2059/+. (K) CRISPR mutation is located on the virial inserted allele (orange bar) of kcnj13^Dhi2059/+. (L) CRISPR mutations are located on both virial inserted alleles (orange bars) of kcnj13^{Dhi2059/Dhi2059}. (M–N) F3 generation adult fish with CRISPR mutant that linked with Dhi2059 inserted kcnj13 allele. (M) Morphology of heterozygous kcnj13^hi2059-CRmutant. (N) Morphology of homozygous kcnj13^{hi2059-CR/hi2059-CR} mutant. Long fins are completely rescued in both conditions. Bars, 5 mm. CR, CRISPR mutant; CRISPR, clustered regularly interspaced short palindromic repeats; gRNA, guide RNA; WT, wild-type.