Hhip and Patched act synergistically in muscle development. (A–J) hhip acts downstream of Hh and upstream of Smo. (A, B) Injection of hhip-MO rescues eng1a expression in shh mutants. Expression of eng1a is absent in myotomes of 25% of the siblings derived from crosses between shh+/- embryos (A, n = 243). In contrast, 90% of shh mutant embryos injected with hhip-MO exhibit increased eng1a expression in the myotome (B, n = 84). (C–F) The effect of hhip-MO is suppressed in smo and gli2 mutant embryos. smo mutant embryos show no eng1a expression in the myotome (C, n = 70) and injection with hhip-MO fails to rescue eng1a expression (D, n = 69). gli2 mutant embryos show no eng1a expression (E, n = 216) and hhip-MO injection fails to rescue eng1a expression (F, n = 181). (G–J) The effect of hhip mRNA injection is suppressed by dnPKA. (G) eng1a expression in control embryo. (H) Injection of dominant negative PKA induces ectopic expression of eng1a in the myotome (arrow, 23/34 injected embryos). (I) In contrast, eng1a expression is suppressed by injection of hhip mRNA (arrow). (J) No apparent difference can be detected between dnPKA-injected embryos and embryos coinjected with hhip mRNA + dnPKA (arrow, 26/36 injected embryos). (K–X) Hhip and Ptc can replace each other. (K–M, W) Ptc1 rescues the phenotype of hhip-MO-injected embryos. eng1a expression in control (K), 3.0 μg/μl hhip-MO-injected (L) and 3.0 μg/μl hhip-MO and 100 ng/μl ptc1 mRNA-injected embryos (M). (N, Q, T) Control embryo, (O, R, U) 1.0 μg/μl ptc-MO-injected embryo, (P, S, V) 1.0 μg/μl ptc-MO and 100 ng/μl hhip mRNA-injected embryo. (N, O, W) 90% of ptc-MO-injected embryos show ectopic expression of eng1a. (P, W) The effects of ptc-MO injection are suppressed by overexpression of hhip. (Q–S) 59% of ptc-MO-injected embryos exhibit U-type somites (S). In contrast, only 36% of ptc-MO + hhip mRNA-injected embryos show U-type somites. (T–V, X) Double labeling of Prox1 and 4D9. The numbers of slow muscle and muscle pioneer cells increase in ptc-MO-injected embryos (U, X) compared to control embryos (T, X). The numbers of slow muscle and muscle pioneer cells are rescued by coinjection of hhip mRNA (V, X). (W) Percentage of embryos in which eng1a is ectopically induced in myotomes; n, number of embryos examined. Ramps indicate increasing concentrations of ptc1 mRNA (left) or hhip mRNA (right), 10, 50, 100, 200 ng/μl ptc mRNA and hhip mRNA, respectively. (X) Average numbers of slow muscle and muscle pioneer cells per somite counted in 4 somites over the yolk extension in 5–10 embryos. The data represent the average ± SEM. Significance: *P < 0.075, **P < 0.01, # no significant difference, Student′s t test. (A–V) Lateral views, anterior toward the left; scale bar: 100 μm.
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