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Fig. 5 Cu2+-stressed zebrafish embryonic muscle genes exhibited changes in the promoter methylation level, and the ectopic expression of smyd5 could phenocopy the defective myofibrillogenesis observed in Cu2+-stressed embryos. A, Heat map for the methylation levels of genes rap3ip, fgfbp2b, arpc1a, and smyd5 in the control and the Cu2+-stressed larvae based on bisulfite sequencing data. B, Graphical representation of methylation patterns in the promoter domain of gene smyd5 in the Cu2+-stressed and the control larvae at 96 hpf (B1); (B2) Dynamic changes of DNA methylation for a representative locus (in the red box in B1) in the smyd5 promoter. Open circles represent un-methylated CpG base, and filled circles represent methylated CpG base. C, Expression of muscle cell methylated genes rap3ip, fgfbp2b, arpc1a, and smyd5 in the Cu2+-stressed embryos and larvae at 24 hpf (C1) and 96 hpf (C2), respectively. D, Representative control embryos and larvae (D1, D3, D5 and D7) and the embryos and larvae with the ectopic expression of smyd5 mRNA (D2, D4, D6, D8 and D9). Black embryos in panels D1 and D2 indicated dead embryos, and red arrows in panel D4 indicated abnormal embryos with shorter bodies. D5-D9, lateral view, anterior to the left. E, Muscle fiber specification in embryos with the ectopic expression of smyd5 mRNA. E1-E3, fast muscle fiber marker mylpfa; E4-E6, slow muscle fiber marker smyhc1l; E7-E9, muscle cell marker smyd1b; E10, E11, E12, calculation of mylpfa, smyhc1l, and smyd1b expression level in each embryo from different groups, respectively. Each dot represents the signal level in a representative individual embryo. Each experiment was repeated two or three times with similar results, and a representative result is shown. E1-E9, lateral view, and anterior to the left. D5-D9, scale bar, 60 μm; E1-E9, scale bar, 250 μm. *P < .05, **P < .01, ***P < .001. NS, not significant