Combined activities of Hhip and Patched are required for muscle pioneer and slow muscle cell development. (A–M) Overexpression of hhip mRNA inhibits muscle pioneer and slow muscle development. Control embryo (A–C), hhip mRNA-injected embryo (D–F), ptc1 mRNA-injected embryo (G–I), hhip and ptc1 mRNA-injected embryo (J–L). (A, B, D, E, G, H, J, K) Transverse sections of a 24 hpf embryo labeled with slow muscle marker S58 (green), fast muscle maker zm4 (red) (A, D, G, J) and Hoechst to mark nuclei (B, E, H, K). (C, F, I, L) Embryos labeled for nuclear slow muscle cell marker Prox1 (green) and the muscle pioneer marker 4D9 (red, Eng). Both hhip and ptc1 inhibit slow muscle (D, G) and muscle pioneer development (arrows in F, I), but fast muscle cells are relatively unaffected. Simultaneous injection of hhip and ptc1 mRNAs significantly enhances the phenotypes observed in the single injection experiments (J, L). (N–D2) Loss of Hhip or Ptc increases formation of muscle pioneer and slow muscle cells. (N–Q) Control embryo. (R–U) hhip-MO-injected embryo. (V–Y) ptc-MO-injected embryo. (Z–C2) hhip-MO and ptc-MO-injected embryo. (N, O, R, S, V, W, Z, A2) Transverse sections of 24 hpf embryo labeled with slow muscle marker S58 (N, R, S, V, Z) and stained with Hoechst to label nuclei (O, S, W, A2). (P, T, X, B2) Expression of eng1a in 24 hpf embryos. (Q, U, Y, C2) Embryos labeled with Prox1 and 4D9. An expansion of the slow muscle domain was observed in hhip-MO (arrow, R), ptc-MO (V, arrow) and hhip-MO + ptc-MO (Z, arrow)-injected embryos. In situ hybridization of eng1a and double labeling with Prox1 and 4D9 demonstrates that the number of muscle pioneer cells increases in hhip-MO-injected (T, U, D2) and ptc-MO-injected (X, Y, D2) embryos. Simultaneous injection of hhip-MO and ptc-MO enhances these effects (B2, C2 D2). This enhanced phenotype is similar to that observed in shh mRNA-injected embryos (D2). (M, D′) Numbers of slow muscle and muscle pioneer cells. Averages were calculated from the total number of cells labeled by the Prox1 antibody (slow muscle) and the total number of cells labeled by both the Prox1 and 4D9 antibodies (muscle pioneer cell) counted in four somites over the extended yolk per embryo at 24 hpf in 5–10 embryos. Data represent the average ± SEM. Significance: *P < 0.01, **P < 0.05, ***P < 0.075, # no significant difference, Student′s t test. Lateral views, anterior are towards the left (C, F, I, L, P, Q, T, U, X, Y, B′, C′). Scale bar (A, B, D, E, G, H, J, K, N, O, R, S, V, W, Z, A′): 150 μm (C, F, I, L, P, Q, T, U, X, Y, B′, C′), 100 μm.
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