natter/fn1a^tl43c mutants display a variable cardiac phenotype.

(A)In situ hybridisation with the myocardial marker myl7 was performed on 24 hpf natter/fn1a^tl43c mutant embryos and WT siblings from four single pair natter/fn1a^tl43c heterozygous intercrosses. The proportion of embryos matching the image shown is indicated in the top right corner of each image. The remaining 6/41 mutant embryos displayed a linear heart tube phenotype as noted in (B). Dorsal views are shown with anterior to the top. (B) Quantification of the 24 hpf myocardial migration phenotype in natter/fn1a^tl43c mutants and WT siblings shown in (A). (C) Representative images of a 100 hpf homozygous WT sibling as well as a WT-like, a mild, and a severe natter/fn1a^tl43c mutant larva from a single pair natter/fn1a^tl43c heterozygous intercross. Lateral views are shown with anterior to the left. The mild mutant phenotype is distinguishable by mild pericardial oedema (white arrowhead). The severe mutant phenotype is distinguishable by a small head, pronounced pericardial oedema, and disorganised trunk muscle fibres (white arrows). (D) Confocal images of three hearts representative of each phenotype were selected from imaging 12 WT-like, 6 mild, and 6 severe Tg(myl7:EGFP) mutants from a single pair natter/fn1a^tl43c mutant incross at 100 hpf. For the fn1a^+/+ and fn1a^+/tl43c siblings, referred to collectively as fn1a^+/?, three representative images were selected from imaging 12 WT fn1a^+/? siblings from a single pair natter/fn1a^tl43c heterozygous intercross at 100 hpf. (E) Quantification of the 100 hpf cardiac phenotype in natter/fn1a^tl43c mutants and WT siblings from three single pair natter/fn1a^tl43c heterozygous intercrosses. The numbers on each bar in (B) and (E) indicate the total number of larvae assessed. All p-values were calculated using chi-squared tests. Scale bars, 100 µm in (A) and (D), and 1 mm in (C).

fn1a^bns692 mutants also display a variable cardiac phenotype.

(A) Schematic showing the WT, natter/tl43c, and bns692 fn1a alleles. Exons (n = 46) are indicated by black boxes. The start codons are represented by a green triangle and the stop codons by a red octagon. (B)In situ hybridisation with the myocardial marker myl7 was performed on 24 hpf fn1a^bns692 mutant embryos and WT siblings from four single pair fn1a^bns692 heterozygous intercrosses. The proportion of embryos matching the image shown is indicated in the top right corner of each image. The remaining 5/49 mutant embryos displayed a linear heart tube phenotype as noted in (C). Dorsal views are shown with anterior to the top. (C) Quantification of the 24 hpf myocardial migration phenotype in fn1a^bns692 mutants and WT siblings shown in (B). (D) Quantification of the 100 hpf cardiac phenotype in fn1a^bns692 mutants and WT siblings from three single pair fn1a^bns692 heterozygous intercrosses. The numbers on each bar in (C) and (D) indicate the total number of larvae assessed. All p-values were calculated using chi-squared tests. Scale bars, 100 µm.

fn1b expression levels correlate with the severity of the natter/fn1a^tl43c and fn1a^bns692 mutant cardiac phenotype.

(A)fn1b 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. (B, C)In situ hybridisation for fn1b was performed on 72 hpf larvae from two single pair fn1a^bns692 heterozygous intercrosses. The proportion of larvae matching the image shown is indicated in the top right corner of each image. (B) Lateral views with anterior to the left. (C) Ventral views with anterior to the top. (D) Quantification of the 100 hpf cardiac phenotype in mutant larvae injected with 350 pg fn1b mRNA and uninjected controls from five single pair fn1a^bns692 mutant incrosses. (E) Quantification of the 100 hpf cardiac phenotype in fn1a^bns692; fn1b^ya14Tg mutants and siblings from three single pair fn1a^bns692; fn1b^ya14Tg double heterozygous intercrosses. The numbers on each bar in (D) and (E) indicate the total number of larvae assessed. For (A), p-values were calculated by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons; for (D), p-values were calculated with chi-squared tests; and for (E), p-values were calculated by Fisher’s exact test with Bonferroni correction for multiple comparisons. Scale bars, 1 mm in (B) and 100 µm in (C).

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.