Fig. S2

Fig. S2 Overexpression of hhip produces U-type somites. (A–D) Muscle pioneer cells are increased in hhip mutant embryos. hhip mutant embryos show an expanded midbrain–hindbrain boundary (A, B, arrow). The number of muscle pioneer cells is increased (C, D; hhip^+/+ 1.2 ± 0.6; hhip^-/- 2.3 ± 0.2). Prox1 positive slow muscle nuclei assume a sharp chevron shape in wild-type embryos (C). In contrast, slow muscle nuclei are disorganized in hhip mutant embryos (D, arrows). The number of slow muscle cells: hhip^+/+ 17.4 ± 1.3; hhip^-/- 15.4 ± 1.1. The data represent the average number of cells ± SEM counted in 4 somites over the yolk extension. (E, F) hhip mRNA-injected embryos develop U-type somites. Mutations that disrupt Hh signaling (van Eeden et al., 1996) and injection of ptc1 mRNA are known to cause U-type somites (Lewis et al., 1999b). Consistent with this, hhip mRNA-injected embryos develop U-type somites (50%, n = 48) and some have more closely spaced eyes (data not shown), another characteristic of embryos with disrupted Hh signaling. Somites of live embryos at 24 hpf. (G, H) Reduced myod expression is observed at 24 hpf in hhip mRNA-injected embryos (D, arrow, 14/61 injected embryos). (I–L) Hhip suppresses slow muscle cell development. Slow muscle cells labeled by F59 (I, J), nuclei stained with Hoechst (K, L). Consistent with S58 labeling (Fig. 2), the number of F59 labeled cells is reduced in hhip mRNA-injected embryos (F, arrowheads). (C–H) Lateral views, anterior towards the left, dorsal toward the top. (I–L) Cross-sections. Scale bar: E–L, 50 μm.