Fig. S5

Inhibition of endocytosis induces slow muscle cell development. (A–F) Internalization of Hhip is suppressed by MDC in COS7 cells. COS7 cells were transfected with myc-tagged Hhip and then incubated with MDC for 30 min. Cells were labeled with anti-myc antibody and anti-β-catenin antibody. Hhip protein is distributed both intracellularly (A) and at the cell surface (B, C arrow). In contrast, Hhip protein is primarily detected at the cell surface after MDC treatment (D–F). (G–N) MDC mimics the phenotype of hhip-MO-injected embryos. Embryos were treated with 200 μM MDC from 40% epiboly to 24 hpf then labeled with Prox1 (green) and 4D9 (red). (H) The number of muscle pioneers increases in embryos treated with endocytosis inhibitor (H, arrow). Numbers of muscle pioneer cells: DMSO-treated embryos, 2.6 ± 0.16; MDC-treated embryos, 3.7 ± 0.38 (G). The data represent the average number of cells ± SEM counted in 4 somites over the yolk extension. Significance: P < 0.05, Student′s t test. (I–J) Transverse sections of 24 hpf embryos labeled with S58 (I, J) and stained with Hoechst for nuclei (K, L). Dorsal to the top. (M, N) Inhibition of endocytosis increases expression of nkx2.2. Embryos were treated with 200 μM MDC from 40% epiboly stage to 24 hpf. The nkx2.2 expression domain is expanded in MDC-treated embryos (N; 83%, n = 12) compared to controls (M, n = 8). Scale bar: (G–L) 50 μm, (M, N) 100 μm.