Fig. 2.

Role of Shh from poregot endoderm on NCC survival and facial skeletogenesis. (A–D) Cell death analysis by Nile blue sulfate (NBS) on chicken embryos in toto. (A) E2.5 control chicken embryo. (B–E) NBS staining was strong in BA1 (‘1’) of embryos whose foreheads were excised at 6-ss (white arrowhead) (B) but not at 8-ss (C) or after forehead excision at 6-ss and replacement by heparin beads (white arrowheads) soaked with Shh (100 μg/ml) (D and E). However, cell death was abundant in the remaining of the anterior head region (B–D, white arrows). (F–I) Whole-mount in situ hybridization for Shh at E3.5 showing the presence of Shh transcripts at the level of the oral cavity (OC) in a control embryo (F), an embryo excised at 8-ss (H), and a 6-ss excised embryo grafted with an Shh bead (I) compared with a nongrafted embryo (G), which does not show Shh expression at the level of the oral cavity. (F′–I′) Cross sections (50 μm) at the level of BA1 (arrows in F–I). Shh mRNAs are present in the pharyngeal (Ph) endoderm of a control embryo (F′), an embryo excised at 8-ss (H′), and an Shh-grafted embryo (I′). In contrast, an embryo excised at 6-ss (G′) is deprived of Shh mRNAs in BA1 pharyngeal endoderm. (J–Q) Morphology (J–M) and skeletal (N–Q) analyses of E11 to E12 embryos by using alcian blue staining for cartilage and alizarin red for bone. (K and O) Absence of upper and lower beak in an embryo excised at 6-ss. The proximal part of Meckel’s cartilage (quadratoarticular cartilage) alone was preserved (O, black arrow). (L and P) After forehead excision at 8-ss, a normal lower beak similar to control (J and N) developed, also with the presence of hypoplasic bone elements of the maxilla (white arrow). (M and Q) An E12 embryo that was excised at 6-ss and treated with an Shh bead; cartilages and bones corresponding to mandible and maxilla are present.