Genetic compensation triggered by actin mutation prevents the muscle damage caused by loss of actin protein

Figures

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  Fig 1. Quantitative RT-PCR analysis for zebrafish α-actin genes. mRNA expression of acta1a, acta1b, actc1a, and actc1b genes in the A) head (comprising the heart tissue) and B) tail (comprising predominantly skeletal muscle tissue) during zebrafish development. Error bars for A) and B) represent SEM for three independent biological replicates each consisting of a pooled sample of 20–30 embryos.

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  Fig 2. Characterization of muscle phenotypes in actc1b-/- mutants and Actc1b morphants. A) Antibody labelling against Actinin2 and Phalloidin of actc1b-/- mutants and wildtype siblings with Actinin2 (green) and F-actin (red) at 2 dpf and 6 dpf showing normal muscle morphology. Scale bar represents 50μm. B) Locomotion assays show a significant reduction in distance travelled by actc1b-/- mutants compared to siblings (actc1b+/- and actc1b+/+) zebrafish. Error bars represent SEM for three independent experiment (n = 6,11,16 for actc1b+/+; n = 24,23,18 for actc1b+/-; and n = 13,9,10 for actc1b-/- per experiment), *p<0.05 using a one-way ANOVA. C) Locomotion assays showing a significant reduction in distance travelled by Actc1b ex2 and UTR morphants compared to both Standard Control MO injected and uninjected zebrafish. No significant difference in locomotion is observed for Standard Control MO injected and uninjected zebrafish. Error bars represent median values and interquartile range (pooled samples from 3 independent experiments n = 45,48,46 for Actc1b ex2 MO; n = 45,48,33 for Actc1b UTR MO; n = 45,48,47 for Standard Control MO; and n = 48,47,45 for uninjected zebrafish), #p<0.0001 using…

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  Fig 3. Characterization of phenotypic severity following Actc1b morpholino knockdown. A) actc1b-/- and wildtype siblings (actc1b+/- and actc1b+/+) injected with either an Actc1b ex2, Actc1b UTR or Standard Control morpholino were stained with Actinin2 (green) and F-actin (red) and phenotypes were scored as either wildtype, mild (small outgrowth of aggregates at the myosepta (arrowheads)) or severe (large outgrowth of aggregates at the myosepta (arrowheads) and Actinin2 positive aggregates throughout the muscle fibers (arrows)). Scale bar represents 50μm. B) Quantification of phenotypic severity for actc1b-/- and wildtype siblings injected with Actc1b ex2 and Actc1b UTR MOs compared to Standard Control MO injected zebrafish. Error bars represent SEM for three independent experiments (for Actc1b ex2 MO: n = 26,14,16 actc1b+/+, n = 31,35,29 actc1b+/- and n = 12,11,23 actc1b-/-, for Actc1b UTR MO: n = 23,21,17 actc1b+/+, n = 28,30,23 actc1b+/- and n = 8,11,8 actc1b-/- and for Standard Control MO: n = 11,8,10 actc1b+/+, n = 11,13,11 actc1b+/- and n = 9,12,4 actc1b-/-), #p<0.0001 using a Chi-square test. C) Locomotion assays show a significant reduction in distance travelled by actc1b+/+…

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  Fig 4. Quantification of RNA and protein levels following Actc1b morpholino knockdown. A) Representative data from western blot analysis for α-actin protein expression in actc1b-/- and their wildtype siblings (actc1b+/- and actc1b+/+) at 2 dpf injected with either an Actc1b UTR, Actc1b ex2, or Standard Control MO. β-tubulin was used as a loading control. B) Quantification of western blot analysis from three independent replicate experiments of A) (S5 Fig) consisting of a pooled sample of 20 tails whereby α-actin protein levels were normalized against the -tubulin loading control. Error bars represent SEM for three independent experiments, *p<0.05, **p<0.01, and #p<0.0001 using a two-way ANOVA. C) Quantitative RT-PCR analysis for zebrafish acta1a, acta1b, actc1a, and actc1b genes in tail samples from actc1b+/+, actc1b+/-, and actc1b-/- at 2 dpf. Error bars represent SEM for three independent experiments each consisting of a pooled sample of 30 tail samples, *p<0.05 indicates difference from actc1b+/+ using a one-way ANOVA. D) Quantitative RT-PCR analysis for zebrafish acta1a, acta1b, actc1a, and actc1b genes in whole embryos from Actc1b ex2 and Actc1b UTR morphants compared to Standard…

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  Fig 5. Characterization of phenotypic severity following Actc1a morpholino knockdown. A) actc1b-/- and wildtype siblings (actc1b+/- and actc1b+/+) injected with either an Actc1a splice or Standard Control MO were stained with Actinin2 and phenotypes were scored as either wildtype, mild (small outgrowth of aggregates at the myosepta (arrowheads)) or severe (large outgrowth of aggregates at the myosepta (arrowheads) and Actinin2 positive aggregates throughout the muscle fibers (arrows)). Scale bar represents 50μm. B) Quantification of the phenotypic severity for actc1b-/- and wildtype siblings (actc1b+/+ and actc1b+/-) injected with Actc1a splice compared to Standard Control MO injected zebrafish. Error bars represent SEM for three independent experiments (for Actc1a MO n = 8,13,7 actc1b+/+, n = 27,16,16 actc1b+/- and n = 7,15,7 actc1b+/+ and for Standard Control MO n = 7,3,7 actc1b+/+, n = 16,21,20 actc1b+/- and n = 4,8,6 actc1b-/-), *p<0.05 indicates a significant difference in phenotype proportions using a Chi-square test.