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Itga7 is required for NAD+-mediated reduction of fiber degeneration in dag1 morphants. (A-D, G–H) Anterior left, dorsal top, side-mounted embryos stained with phalloidin (red or white) or pY397 FAK (green). (A-B) Muscle morphogenesis proceeds normally in itga7 morphants. Phosphorylated FAK (green) outlines fibers and concentrates at the MTJ, and actin distribution in slow- and fast-twitch muscle fibers (red) appears normal in 26 hpf itga7 morphants compared to wild-types. (C) 3 dpf dag1;itga7 double morphant. (D) 3 dpf NAD+-supplemented dag1;itga7 double morphant. MTJ morphogenesis is disrupted in dag1;itga7 double morphants as displayed by wider MTJ angles (C). MTJ morphogenetic defects were rescued by NAD+ in dag1;itga7 double morphants (D), suggesting that another laminin receptor is sufficient for NAD+-mediated MTJ improvements. (E) Quantification of MTJ angles shows that dag1;itga7 double morphants have significantly wider MTJ angles than wild-types, and NAD+ significantly reduces this defect; **p<0.01, *p<0.05. (F) Quantification of incidence of dystrophy per embryo shows no significant difference in dag1;itga7 double morphants upon addition of exogenous NAD+, suggesting that Itga7 is required for NAD+-mediated reduction of dystrophy in dag1 morphants; N.S., not significant. However, fewer fibers appeared to detach in dag1;itga7 double morphants supplemented with NAD+ (D), again suggesting the involvement of another receptor for laminin in NAD+ action. (G–H) Mild fiber detachment is readily observed in 4 dpf itga7 morphants (G) and reduced in NAD+-treated itga7 morphants (H). (I) NAD+ treatment significantly decreases fiber degeneration in itga7 morphants; ***p<0.001. (J) Model of cell adhesion at the MTJ. The transition from a somite boundary to a MTJ involves the downregulation of Fibronectin and the upregulation of laminin and laminin receptors. Our results suggest that laminin receptors, Itga7 and Dag1, play a role in this transition, but the primary receptor involved is an unknown integrin. In maintenance of fiber adhesion at the MTJ, our data show that either Itga7 or Dag1 is required, but also suggest the involvement of an additional laminin receptor. Scale bar is 50 micrometers.
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