Fig. 4. The Shh-Gli1 pathway mediates the interaction between EZ-I and CNCCs. (A) Explant assay. Fragments were dissected from EZ-I and anterior apical neural crest ridges (see also Fig. S1 in the supplementary material) and were either cultured alone or co-cultured. (Right panel) RT-PCR for Gapdh, Shh and Gli1 in untreated explants (-), after treatment with 10 µM cyclopamine (Cyc) or 10 µM Shh-N (Shh). The forehead of a 13-somite embryo was used as a control (whole head). Note the strong, cyclopamine-sensitive induction of Gli1 expression occurring only in the co-cultures. (B) CNCC-endoderm interaction. Frontal section of a 13-somite chicken head grafted with 5-somite quail CNCCs and processed for QCPN immunodetection (dark nuclei). (Inset) Post-migratory donor CNCCs (green) contact the ventral aspect of the host endoderm (red) triggering a mutual exchange of inductive signals (arrows). (C) Endodermal fate map indicating the induction by EZ-I of the mesethmoid cartilage (blue). The 150 µm EZ-I domain is adjacent to the EZ-II domain, which drives Meckel's cartilage morphogenesis (Couly et al., 2002). I and II, endoderm zones I and II; AIP, anterior intestinal portal; en, endoderm; Mc, Meckel's cartilage; mes, mesethmoid; n, neural tube; nc, neural crest; r1-r8, rhombomeres 1-8.