Fig. 4

Epistatic analysis of scube function in the Hedgehog pathway. Hedgehog pathway overexpression induces ectopic slow MyHC expression in zebrafish embryos. (A–F). Slow MyHC expression in treated and untreated embryos. (A) Slow MyHC localization in wildtype embryos. (B) scube triple MO embryos show severe reductions in slow MyHC expression. Representative embryo (B) has 15 slow MyHC positive fibers (arrowheads). (C) Overexpression of dnPKA RNA turns all muscle in the embryo into slow muscle. (D) Overexpression of dnPKA in scube triple MO embryos rescued the loss of slow muscle seen in scube triple MO embryos. (E) shh RNA overexpression induced ectopic slow MyHC expression in wildtype embryos. (F) shh RNA overexpression in scube triple MO embryos rescued the loss of slow muscle in scube triple MO embryos. Lateral views of<30 hpf embryos. F59 monoclonal antibody labels slow MyHC positive muscle fibers, Alexa 488 conjugated-donkey anti-mouse secondary antibody, 0.65 ·g of dnPKA RNA and shh RNA was injected at 100 μg/mL. Scale Bars: 100 μm. (G–O) Titration of shh RNA in scube triple MO embryos (G, I, K, M) and uninjected 10 somite embryos (H, J, L, N). Expansion of myod expression is seen at 0.16 ng in embryos not injected with Scube morpholinos (J) compared to 0.65 ng in scube triple MO embryos (K). (O) Graph showing percentage of embryos with expanded myod expression. (P–V3) Mosaic analysis of cells expressing SHH fused to GFP under the control of the pax3a promoter. Mosaic expression of pax3a:Shh-GFP generated ectopic slow muscle both cell autonomously (arrows) and non-cell autonomously (arrowheads) in both wildtype (Q–S′′) and scube triple MO embryos (T–V′′). (Q, R, S, T, U, V) F59 monoclonal antibody, (Q′, R′, S′, T′, U′, V′) anti-GFP antibody, (Q′′, R′′, S′′, T′′, U′′, V′′) merged images.