Schematic to scale of zebrafish smyhc1 gene on chromosome 24. Noncoding regions are displayed in pink. Coiled coil domain (842–1,929 bp) displayed in purple. Motor domain (85‐778 bp) displayed in yellow. Actin binding site (655–677 bp) displayed in cyan. Location of R672H mutation is enlarged and labeled.

Aligned DNA and amino acid sequences of MYH3 and smyhc1 alleles surrounding the smyhc1^R673H and MYH3 R672H substitutions. The smyhc1⁻ allele has a 7 base pair deletion that results in a frameshift in which one errant amino acid precedes a premature stop codon. The smyhc1^R673H allele results from a G>A transition single point mutation.

Western blot of whole zebrafish larvae, Smyhc1 stained with the F59 antibody. smyhc1^+/+ larvae express Smyhc1 at 24 hpf, but not at 48 hpf. smyhc1^−/− larvae do not express Smyhc1.

Smyhc1 immunohistochemistry stain of 24 hpf (hour postfertilization) zebrafish larvae. Filamentous actin is stained with phalloidin (red). Nuclei are stained with DAPI (blue). Smyhc1 is stained with the F59 antibody (green) (Elworthy et al, 2008). smyhc1^+/+, smyhc1^R673H/+, and smyhc1^R673H/R673H larvae display Smyhc1 in the muscle fibers at 24 hpf. smyhc1^−/− larvae at 24 do not stain positively for Smyhc1. Scale bar represents 50 μm.

Quantification of smyhc1^R673H embryonic phenotypes at 2 dpf. Representative examples of fish in each of the four phenotypic groups (normal, mild, moderate, and severe) are shown in Fig EV3.

The predicted mendelian (1:2:1) and experimental ratio of genotypes of progeny from smyhc1^R673H/+ in‐crosses demonstrates survival at the indicated ages. This ratio quickly skewed toward an overrepresentation of smyhc1^+/+ fish with no representation of smyhc1^R673H/R673H after 5 dpf. There is also dropout of smyhc1^R673H/+ fish into adulthood. Number of fish counted per group is indicated.

Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1^−/− adults, in contrast to the compression and fusion of vertebrae seen in smyhc1^R673H/+ adults. Distal tail regions are highlighted and enlarged.

Quantification of skeletal phenotype severity in adults. Representative examples of fish in each of the four phenotypic groups (normal, mild, moderate, and severe) are shown in Fig EV3. Scale bars represent a length of 5 mm.

Distance traveled during 5 min of spontaneous swimming at 6 dpf quantified by motion tracking software (Noldus Ethovision). At 6 dpf, both smyhc1^−/− (n = 24) and smyhc1^R673H/+ (n = 11) larvae traveled significantly less than smyhc1^+/+ larvae (n = 13). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.

Diagram of swim tunnel used to quantify adult swimming behavior. Water flow was gradually increased until the fish fatigued and were collected at the grate.

Time spent swimming in swim tunnel before fatigue in adults at 6 months postfertilization (mpf). smyhc1^+/+ (n = 6), smyhc1^−/− (n = 5), smyhc1^R673H/+ (n = 5).

Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin‐rhodamine. Puncta of filamentous actin are indicated with white arrowheads, distorted myofibers are indicated with arrows and outlined, and frayed myofibers are indicated with yellow arrowhead.

Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin‐rhodamine (red), anti‐α‐actinin antibodies (green), and DAPI (blue).

Confocal fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin‐rhodamine (red), anti‐α‐actinin antibodies (green), and DAPI (blue). Bundle of actin ringed with α‐actinin indicated with white‐bordered black arrowhead.

Myoseptal intervals of slow skeletal muscle at 1 dpf in smyhc1^+/+ (n = 54), smyhc1^−/− (n = 31), smyhc1^R673H/+ (n = 45), and smyhc1^R673H/R673H (n = 49). Distance between myosepta was measured perpendicular to rostral‐caudal body axis at defined mid‐body regions in phalloidin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.

Myoseptal intervals of slow skeletal muscle at 3 dpf in smyhc1^+/+ (n = 51), smyhc1^−/− (n = 32), smyhc1^R673H/+ (n = 64), and smyhc1^R673H/R673H (n = 38). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.

Z‐disk intervals (sarcomere length) of slow skeletal muscle at 1 dpf in smyhc1^+/+ (n = 380), smyhc1^R673H/+ (n = 293), and smyhc1^R673H/R673H (n = 268). Distance between z‐disks was measured in anti‐α‐actinin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.

Z‐disk intervals (sarcomere length) of slow skeletal muscle at 3 dpf in smyhc1^+/+ (n = 249), smyhc1^−/− (n = 278), smyhc1^R673H/+ (n = 246), and smyhc1^R673H/R673H (n = 252). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.

Representative images of smyhc1^+/+, smyhc1^R673H/+, and smyhc1^R673H/R673H larvae at 3 dpf after 48‐h treatment with 0.25% DMSO (control) or 25 μM para‐aminoblebbistatin. All larvae treated with para‐aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. Para‐aminoblebbistatin‐treated smyhc1^R673H/+ (n = 9) and smyhc1^R673H/R673H (n = 3) larvae are indistinguishable from smyhc1^+/+ unlike the untreated larvae which have markedly curved or distorted body axis.

Grading scale for adult skeletal abnormalities. Normal is classified as 0 visible abnormalities, mild is classified as 1 spinal curve or kink, moderate is classified as multiple spinal curves or kinks, or apparent vertebral fusion, and severe is classified as multiple spinal kinks or vertebral fusions that severely distort the body. Arrowheads indicate spinal abnormalities.

Confocal fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin‐rhodamine (red), Caspase‐3 antibodies (green), and DAPI (blue).

Zebrafish embryos (2 dpf) were treated with 25 μM blebbistatin or DMSO control for 24 h starting at 1 dpf. Blebbistatin appears to partially rescue smyhc1^R673H phenotype, but causes known off target effects. Note the severe dorsal tail curve and pericardial edema present in all larvae treated with blebbistatin regardless of genotype. Representative images of embryos at 48 hpf.