Fig. S3

hhipΔC22 fails to suppress the phenotype of ptc-MO-injected embryos. (A–J) eng1a and Eng protein expressions are reduced in hhipΔC22-injected embryos. (A–E) Expression of eng1a. (F–J) Embryos double labeled with anti-Prox1 and 4D9. Embryos injected with hhip, hhipΔC22 and hhipΔ614–693 exhibit decreased eng1a, Prox1 and 4D9 double labeled muscle pioneer cells (B, C, D, G, H, I) and U-type somites (B, C, D, dotted line). There is no obvious difference in eng1a expression between control and hhipΔ415–693-injected embryos. (K–T) Hhip lacking the membrane anchoring domain fails to restore the phenotypes of ptc-MO-injected embryos. (K–O) Expression of eng1a. (P–T) Embryos double labeled with anti-Prox1 and 4D9. hhip suppresses ectopically induced eng1a in ptc-MO-injected embryos (L, Q). In contrast, hhipΔC22 fails to suppress ectopically induced eng1a expression (M, R). Side views, anterior to the left, dorsal to the top, 24 hpf, scale bar: A–T, 100 μm. (U–W) HhipΔC22 fails to localize to the cell surface. COS7 cells transfected with myc-tagged Hhip and then labeled with anti-myc antibody. Hhip protein is detected at the cell surface (U, arrows), whereas HhipΔC22 and HhipΔ614–693 proteins are not located on the cell surface (V, W, arrows). (X) Western blot analysis of Hhip protein. COS7 cells transfected with myc-tagged Hhip and then labeled with anti-myc antibody.