Fig 4

Fig 4 A genetic modifier links itgα5 to the phenotypic variation in fn1a mutants.

(A) Outline of the breeding scheme to select fn1a mutants that display milder phenotypes. Multi-pair mutant incrosses were performed and the embryos raised at 28.5°C. Larvae were phenotyped at 100 hpf and the WT looking larvae raised to establish the next generation. Quantification of the 100 hpf cardiac phenotype in natter/fn1a^tl43c(A’) and fn1a^bns692(A”) mutants from single pair mutant incrosses over 3 generations. natter/fn1a^tl43c F3 [28.5°C] and F3 [32°C] zebrafish were raised during development at 28.5°C and 32°C, respectively. (B) Outline of the breeding strategy used to map the genetic modifier in natter/fn1a^tl43c mutants, see materials and methods for a detailed description. A single dominant linkage peak was identified on chromosome 23 (B’) mapping to the itgα5 locus (B”). 4 additional peaks with a SNP index ≥0.5 were identified on chromosomes 1, 3, and 23 (B’). (C) Quantification of the 100 hpf cardiac phenotype in uninjected and 1 ng itgα5 MO injected mutant larvae from five single pair fn1a^bns692 mutant incrosses. (D) Quantification of the 100 hpf cardiac phenotype in uninjected and 250 pg itgα5 mRNA injected mutant larvae from five single pair fn1a^bns692 mutant incrosses. The numbers on each bar in (C) and (D) indicate the total number of larvae assessed. (E)itgα5 mRNA levels were analysed by RT-qPCR in individual 72 hpf protruding mouth stage larvae collected from single pair natter/fn1a^tl43c and fn1a^bns692 heterozygous intercrosses. Each data point represents an individual larva. Expression is relative to homozygous WT siblings in each clutch. Eight clutches were examined for each allele from experiments described in S2 and S4 Figs. (E’) The data from (E) are displayed with a shortened Y-axis to show the increased variance in itgα5 mRNA levels in severe mutants. For (C) and (D), p-values were calculated using a chi-squared test; and for (A) p-values were calculated by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons.