Fig. 1

Formation of Shh-Dependent Muscle Requires RyR Function

(A) Schematic illustration of the nuclei of Shh-dependent muscle cells in the somites of 26 hpf zebrafish embryos, presented relative to the notochord (ntc) in transverse section and in a superficial lateral view (anterior to left). Nuclei of different muscle types are color-coded: slow muscle pioneer cells ([MPs], gray), medial fast fibers ([MFFs], green), and superficial slow fibers ([SSFs], magenta).

(B, C, E, F, H, I, K, and L) Shh-dependent muscle fiber nuclei, marked by Prox1 (magenta) and En (green) expression, were visualized by immunostaining 26 hpf embryos: (B) wild-type (WT), (C) ryr1a and ryr3 MO-injected (ryr1a,3 MO), (E) vehicle-treated (0.5% DMSO) WT, (F) MZryr1a^(−/−);ryr1b^(−/−);MZryr3^(−/−) mutant, (H and I) azumolene-treated WT, (K) azumolene-treated MZryr1a^(−/−);ryr1b^(−/−);MZryr3^(−/−) mutant, and (L) 4-CmC-treated WT (supernumerary Prox1⁺ SSF nuclei are indicated with arrowheads). All nuclei co-expressing En and Prox1 were considered MPs. Scale bar, 25 μm.

(D and J) Quantification of distinct muscle cell types per somite. Data are represented as means ± SEM. As numbers of nuclei in WT and DMSO-treated embryos did not differ significantly, comparisons were made with control vehicle-treated embryos unless otherwise indicated. ^∗p < 0.01; ^∗∗∗p < 0.0001; n.s., not significant.

(G) Traces of GCaMP fluorescence in electrically stimulated muscle of 24 hpf Tg(act2b:GCaMP6f) embryos incubated from 6 to 24 hpf in indicated solutions. Data are represented as means ± SEM. The maximum change in fluorescence from baseline, arrow in (G) is shown for each recorded embryo in (G′) with horizontal lines representing means. ^∗p < 0.01, ^∗∗p < 0.001, ^∗∗∗p < 0.0001. See also Figure S2.