General NMII protein structure and homology. a) Schematic of the hexameric structure of an NMII protein. b) Zebrafish to human gene and protein homology. Zebrafish contain two myh9 genes, myh9a, and myh9b, compared to human MYH9.

Myh expression in WT larvae by whole-mount in situ hybridization for myh9a, myh9b, and myh10 at 48, 72, and 96 hours post fertilization. Antisense probe expression pattern and sense controls shown for each time point. Scale bars = 1 mm and apply to all images for the same time point.

myh^−/− mutant phenotypes between 24 hpf and 5 dpf. a–e) Representative bright-field images of myh9a^−/−, myh9b^−/−, and myh10^−/− homozygous mutants compared to WT at 24 hpf, 48 hpf, 72 hpf, 96 hpf, and 5 dpf. Arrowheads indicate location of pericardial edema in myh9b^−/− mutants. f) Quantification of myh9b pericardial edema phenotype development and reversal from 48 hpf to 6 dpf in offspring generated from an myh9b^+/− parent incross. The χ² analyses determined that at 72 hpf the edema phenotype development is Mendelian, and at 96 hpf and beyond it is no longer Mendelian. Scale bars for all images = 0.5 mm. n = 610 for phenotype development and reversal quantification in f.

Kaplan–Meier survival curve for the myh^−/− mutants through 40 dpf. Only myh9b^−/− mutants demonstrated decreased survival rates compared to WT, myh9a^−/−, and myh10^−/− mutant populations (log-rank tests P < 0.001). WT, n = 100. myh9a^−/−, n = 100. myh10^−/−, n = 100. myh9b^−/−, n = 38.

myh9a, myh9b, and myh10 gene expression in zebrafish myh mutants at 96 hpf. Whole larvae qRT-PCR. a) Quantification of myh9a gene expression for each of the myh mutants. b) Quantification of myh9b gene expression for each of the myh mutants. c) Quantification of myh10 gene expression for each of the myh mutants. The eef1al1 gene was used for data normalization to determine fold change. +/− SEM is shown for all graphs. N = 3 independent experiments for each gene analyzed. *P < 0.05, **P < 0.01, and ***P < 0.005.

NMIIA and NMIIB protein levels in the zebrafish myh mutants. a) Representative western blot of whole larvae extracts at 96 hpf for NMIIA protein with alpha-tubulin control for each of the myh mutants. b) Quantification of NMIIA protein in the myh mutants. c) Representative western blot of whole larvae extracts at 96 hpf for NMIIB protein with alpha-tubulin control for each of the myh mutants. d) Quantification of NMIIB protein in the myh mutants. +/− SEM bars shown for all graphs. N = 3 independent experiments for each protein analyzed. *P < 0.05, **P < 0.01, and ***P < 0.005.

Double mutant phenotype quantification between 24 and 72 hpf and representative images at 72 hpf. a) Phenotype quantification for myh9a^−/−; myh9b^+/−double mutant parent incrosses and representative image of a 72 hpf double homozygous mutant. The χ² calculations determine at 24, 48, and 72 hpf myh9a;myh9b double mutant phenotype development is non-Mendelian. b) Phenotype quantification for myh9b^+/−; myh10^−/− double mutant parent incrosses and representative image of a 72 hpf double homozygous mutant. The χ² calculations determine at 24, 48, and 72 hpf myh9b;myh10 double mutant phenotype development is non-Mendelian. c) Phenotype quantification for myh9a^+/−; myh10^+/− double mutant parent incrosses and representative image of a 72 hpf double heterozygous mutant. The decrease in numbers throughout each evaluation period in a–c) was due to death among the population of fish studied.

Summary of genetic interactions found through the analysis of the zebrafish myh mutant lines. myh9b is required for the expression of myh9a and myh10. myh10 is required for the expression of myh9b.