MYH3‐associated distal arthrogryposis zebrafish model is normalized with para‐aminoblebbistatin

Figures

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  Figure 1. Generation of smyhc1 mutant lines and embryonic muscle development ASchematic to scale of human MYH3 gene on chromosome 17. Noncoding regions are displayed in pink. Coiled coil domain (840–1,933 bp) displayed in purple. Motor domain (86–779 bp) displayed in yellow. Actin binding site (656–678/758–772 bp) shown in cyan. Location of R672H mutation is enlarged and labeled.BSchematic 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.CAligned 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.DWestern 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…

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  Figure EV1. Transient expression of smyhc1 Smyhc1 immunohistochemistry stain of 3 dpf 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 do not display Smyhc1 in the muscle fibers at 3dpf. Data Information: Scale bar represents a length of 50 μm.

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  Figure 2. Embryonic phenotypes of smyhc1 mutants AMorphologies of smyhc1 +/+, smyhc1 −/+, and smyhc1 −/− embryos are grossly normal at 2 days postfertilization (dpf), despite complete paralysis of smyhc1−/− embryos. In contrast, the smyhc1 R673H/+ embryos have notochord kinks in larval stages (arrow head), while smyhc1R673H/R673H embryos have much more severe notochord kinks that compress and distort the body axis (arrow heads). The smyhc1R673H/− embryos completely phenocopy smyhc1 R673H/R673H embryos.BQuantification 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.

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  Figure 3. The smyhc1 R673H allele reduces survival in a dose‐dependent manner ASchema of in‐crosses used to generate clutches for survival assay.BThe 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.

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  Figure 4. smyhc1 mutants exhibit skeletal abnormalities in adulthood AGross morphology of smyhc1 mutant adults. Most smyhc1 −/− adult fish display dorsal tail curvature, while smyhc1 R673H/+ adults have a shortened body axis and variable spinal curves.BAlizarin 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.CQuantification 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.

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  Figure EV2. Examples of adult phenotypes resulting from various smyhc1 mutant genotypes

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  Figure EV3. Representative examples of each embryonic and adult graded phenotype AGrading scale for 2 dpf embryos. Normal is classified as 0 visible abnormalities, mild is classified as 1 notochord curve or kink, moderate is classified as multiple notochord curves or kinks, and severe is classified as multiple notochord kinks that severely distort the body. Arrowheads indicate notochord abnormalities.BGrading 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.

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  Figure 5. Motor deficits in smyhc1 mutants are variable during development ATime course of light‐triggered larval movements was manually counted at indicated times between 24 and 48 hpf. The smyhc1 −/− larvae (n = 36) were paralyzed up to 48 hpf. smyhc1 R673H/+ mutants (n = 15) twitched slightly less than their smyhc1 +/+ siblings (n = 21) at 34 hpf. 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.BDistance 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…

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  Figure 6. Slow skeletal muscle disorganization of smyhc1 mutants AConfocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin‐rhodamine to detect actin. Puncta of filamentous actin are indicated with white arrowheads, and misshapen myofibers are indicated with white arrows and outlined.BConfocal 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.CConfocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin‐rhodamine (red), anti‐α‐actinin antibodies (green), and DAPI (blue).DConfocal 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.EMyoseptal 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…

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  Figure EV4. Activated Caspase‐3 presence in smyhc1 mutant tissue AConfocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin‐rhodamine (red), Caspase‐3 antibodies (green), and DAPI (blue).BConfocal fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin‐rhodamine (red), Caspase‐3 antibodies (green), and DAPI (blue).Data Information: Scale bars represent a length of 50 μm.

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  Figure EV5. Tricaine fails to ameliorate the smyhc1 R673H larval phenotype, and blebbistatin improves the smyhc1 R673H larval phenotype AZebrafish embryos (2 dpf) treated with 40 mg/ml tricaine methanylsulfonate or control egg medium for 24 h starting at 1 dpf. Tricaine suppressed movement but failed to normalize notochord kinks and bends. Tricaine appears to have no major effect on anatomy.BZebrafish 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.

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  Figure 7. Para‐aminoblebbistatin ameliorates the smyhc1 R673H larval phenotype ARepresentative morphologies of smyhc1 +/+, smyhc1 R673H/+, and smyhc1 R673H/R673H larvae at 2 dpf after 24‐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. All 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.BRepresentative 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.