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Fig. 4
Later NAD+ supplementation improves MTJ structure, but NAD+ and EmergenC are required prior to initial muscle development to improve motility, fiber resilience, and fiber organization. (a–d) Anterior left, dorsal top, side-mounted embryos at 72 hpf stained for f-actin (phalloidin, gray). (a) Control embryo. (b) fkrp morphant. (c) fkrp morphant treated with NAD+ at 24 hpf. (d) fkrp morphant treated with EmergenC at 24 hpf. White arrowheads indicate single fiber detachments. (e) Fiber organization quantification. The anisotropy factor in embryos injected with fkrp MOs (n = 104 half-myotomes) is reduced compared to controls (n = 48 half-myotomes) and not improved with NAD+ supplementation at 24 hpf (n = 96 half-myotomes). (f) MTJ angle quantification. Fkrpmorphants treated with NAD+ (n = 381 MTJs) or EmergenC (n = 318 MTJs) at 24 hpf have significantly decreased MTJ angles compared to untreated morphants (n = 651 MTJs). (g) Fiber detachment quantification. Although there is a trend towards reduced muscle degeneration, fkrp morphants receiving NAD+ (n = 55 embryos) or EmergenC (n = 41 embryos) at 24 hpf do not have a significant reduction in the percent of myotomes with dystrophy compared to untreated morphants (n = 98 embryos). (h) Escape response quantification. The number of touches required to induce an escape response is elevated in embryos injected with fkrp MOs (n = 128 embryos) compared to controls (n = 114 embryos). Morphants treated with NAD+ at 24 hpf (n = 81 embryos) have a worsened escape response compared to untreated morphants. Morphants treated with EmergenC at 24 hpf (n = 52 embryos) do not exhibit a significant change in escape response compared to untreated morphants, but significantly differ from NAD+-treated morphants. Scalebars are 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ns non-significant