Figure 3.

Figure 3.

Obscna and obscnb knockout zebrafish (A) Schematic representation of generation of knockouts using CRISPR/Cas9 gene editing technology in zebrafish. (B, C) Amino acid sequence illustrating Obscna and Obscnb protein. Blue letters indicate the frameshift mutation resulting in premature stop codon indicated by asterisk. The resulting genotyped PCR products after cutting with restriction enzymes (obscna; BbVCI; New England BioLabs, Ipswich, MA, USA) and obscnb; SacI (Thermo Scientific, Vilnius, Lithuania) are shown using gel electrophoresis. DNA sequences of obscna and obscnb knockouts are displayed with chromatography, and mutation sites are shown in the box. (D) Obscurin immunolabeling of obscna^+/−;obscnb^+/− and obscna^−/−;obscnb^−/− zebrafish larvae at 5dpf. Longitudinal sections of 10-month-old zebrafish EOMs immunolabeled with (E) α-bungarotoxin labeling multiple nerve endings (arrowheads) in obscna^+/−;obscnb^+/− and obscna^−/−;obscnb^−/− adult zebrafish. (F) F-actin (green) and Tenascin (red) immunolabeling in whole EOMs of 10-month-old zebrafish showing MTJs of myofibers of obscna^+/−;obscnb^+/− and obscna^−/−;obscnb^−/− adult zebrafish. (G) myomesin (green) labeling M-band, desmin (red) labeling Z-discs (desmin-positive myofiber on the left column, and desmin-negative myofiber on the right column). (H) Cross-sections of 10-month-old zebrafish EOMs: subsarcolemmal localization of desmin in obscna^−/−;obscnb^−/− and distributed throughout the myofiber in obscna^+/−;obscnb^+/− (arrowheads indicate sarcolemma, open arrowheads indicate cytoplasm). (I) Percentage of myofibers showing desmin distribution either subsarcolemmaly or throughout the myofiber relative to the total number of myofibers. ****P < 0.0001.