The Hhip membrane anchoring domain is required for synergistic interaction with Patched but not for negative regulation of Hedgehog. (A) Schematic of Hhip deletion mutants. Blue indicates hydrophobic region, red EGF-like domain and speckled blue the membrane anchoring domain. Asterisks indicate potential N-linked glycosylation sites. (B–H) Hhip lacking the membrane anchoring domain (D) and the EGF-like domain (E, arrow) still inhibits myod expression, whereas Hhip lacking amino acids 415–693 fails to inhibit myod expression. We scored the percentage of embryos with decreased myod at segmentation stages (G, n, total number of embryos) and the total number of cells labeled by Prox1 or both Prox1 and 4D9 at 24 hpf (H, Supplementary Fig. 3). The data represent the average ± SEM. Significance: *P < 0.01, **P < 0.05, # no significant difference, Student′s t test. (I, J) Hhip lacking the membrane anchoring domain fails to rescue ptc-MO-injected embryos. (I) ptc-MO induces ectopic eng1a expression in the myotome, and full-length hhip restores this expression (Supplementary Fig. 3). In contrast, hhipΔC22 does not suppress ectopic expression eng1a in ptc-MO-injected embryos. (J) Full-length hhip but not hhipΔC22 suppresses the number of muscle pioneer cells. The data represent the average ± SEM. Significance: *P < 0.05, **P < 0.1, Student′s t test. (B–F) Dorsal views, anterior towards the top, scale bar, 50 μm.
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