Fig. 3

RyR Function Is Required for Shh-Dependent Gli-Mediated Gene Expression

(A–H) Dorsal view images of live Tg(8xGli:mCherry-NLS-Odc1) embryos at 12 hpf (A, C, E, and G) or 24 hpf (B, D, F, and H) that had been treated with vehicle (0.5% DMSO), cyclopamine, azumolene, or ryanodine. At 12 hpf, the position of the notochord (ntc) just ventral to the FP is outlined by dashed lines. (A) In control 12 hpf embryos, mCherry is expressed in nuclei of cells responding to Shh, including adaxial cells (arrowhead) and FP cells (arrow). (B) At 24 hpf, mCherry is expressed in nuclei of slow muscle cells (arrowhead) and cells in the ventral neural tube (arrow). (C–H) mCherry expression is reduced in embryos treated with each drug.

(I) Tail-transected Tg(8xGli:mCherry-NLS-Odc1) embryos were soaked in vehicle or 4-CmC from 16 to 18 hpf, fixed, and imaged.

(J–L) Potentiation of RyR channel activity with 4-CmC treatment results in increased numbers of presumptive slow muscle nuclei that express the mCherry reporter (J and K are lateral views, arrowhead indicates nuclei of slow muscle cells and arrow indicates cells in the ventral neural tube). (L) Quantification of mCherry⁺ nuclei per somite in 4-CmC-treated embryos. Each point represents a single somite and the horizontal line represents the mean. ^∗∗p < 0.001.

(M–Q) RyR activity affects endogenous ptch2 expression as detected by whole-mount in situ hybridization in 24 hpf embryos. (M–Q) Lateral views reveal expression in somites and (M′–Q′) transverse sections reveal expression in slow muscle cells surrounding the notochord and in the ventral neural tube. Compared with WT embryos, ptch2 expression is diminished in azumolene-treated and MZryr1a^(−/−);MZryr2a^(−/−);MZryr3^(−/−) mutant embryos, and it is enhanced in 4-CmC-treated embryos.

Scale bars, 25 μm.