Generated myoglobin (mb) knockout lines, embryonic body length and genotype distribution. a Schematic representation of the zebrafish mb gene (top) and the three variants generated in this study. Exons are shown as grey bars, introns as black lines, and stop codon and UTR are white bars. The positions of guide RNAs and His-89 are indicated by numbered arrows and text, respectively. For generation of the mb^Auzf13.2 and the mb^Auzf13.6 lines, gRNAs 4, 7, 12, and 21 were used. For generation of the mb^Auzf13.2 gRNA 13 was used. Insertions are shown as a black bar in mb^Auzf13.2. Deletions are shown as dotted lines in mb^Auzf13.3 and mb^Auzf13.6. b Genotype frequency at 6 dpf from F2 or F3 mb^(±) in-crosses. Using an X²-test, no statistically significant deviation from the expected Mendelian distribution in mb^Auzf13.2 (X² = 3.372, df = 2, p = 0.1853), mb^Auzf13.3 (X² = 0.84921, df = 2, p = 0.654) or mb^Auzf13.6 (X² = 0.55882, df = 2, p = 0.7562) was found. c Length measurements of the three KO lines at 6 dpf from F2 or F3 mb^(/-) in-crosses. Using a one-way ANOVA test, no statistically significant difference in average length according to genotype for mb^Auzf13.2 (F(2) = 1.012, p = 0.366), mb^Auzf13.3 (F(2) = 1.932, p = 0.147) or mb^Auzf13.6 (F(2) = 0.128, p = 0.879) was found. d Frequency of enlarged pericardium at 6 dpf from F2 or F3 mb^(/-) in-crosses. Using a Fisher’s exact test, no statistically significant difference in occurrence of enlarged pericardium in mb^Auzf13.2 (p = 0.4747), mb^Auzf13.3 (p = 0.7556), or mb^Auzf13.6 (p = 0.5524) was found. e Frequency of deviation from normal body curvature. Using a Fisher’s exact test, no statistically significant deviation from normal body curvature in mb^Auzf13.2 (p = 0.6126), mb^Auzf13.3 (p = 0.6126), or mb^Auzf13.6 (p = 0.5794) was found

Characterization of adult (3–6 months post fertilization) mb^Auzf13.2 zebrafish resulting from in-crossed mb^Auzf13.2(±). a Genotype frequency distribution. Using an X²-test, no statistically significant deviation from the expected Mendelian distribution (X² = 0.18367, df = 2, p = 0.9123) was found. b Zebrafish length relative to mean length of mb^{Auzf13.2(+/+)} siblings. Using a one-way ANOVA test, no statistically significant difference in length according to genotype (F(2) = .474, p = 0.624) was found. c Adult hearts from wild-type and knockout (mb^{Auzf13.2(−/−)}) fish. d Ventricle size measured by its cross section. Using a one-way ANOVA test, no statistically significant difference in ventricle cross section area normalized to standard length when comparing genotypes (F(2) = 1.12, p = 0.341) was found. e Mb specific western blot of heart homogenate from the mb^Auzf13.2 with the wild-type (+ / +) and knockout (−/ −) genotype. Three different protein amounts (left to right: 2.28, 1.53, and 0.765 µg) of total protein were loaded. Principal component (PC) analysis of gene counts from RNA sequencing comparing knockout vs. wild-type zebrafish heart ventricles. f Scree plot for all PCs. g Plot of PC1 and PC2. h Volcano plot depicting log2 fold change (FC) in expression and the − log10(P_adj) values. Horizontal grey line indicated a P_adj value of 0.05. The vertical line depict log2FC of − 2 and 2