An early role for sonic hedgehog from foregut endoderm in jaw development: ensuring neural crest cell survival

Abstract

We have investigated the role of Sonic hedgehog (Shh) in the development of facial structures by depriving chicken embryos of the most anterior sources of this morphogen, including the prechordal plate and the anterior ventral endoderm of the foregut, before the onset of neural crest cell (NCC) migration to the first branchial arch (BA1). The entire forehead, including the foregut endoderm, was removed at 5- to 10-somite stage (ss), which led to the absence of the lower jaw when the operation was performed before 7-ss. If the embryos were deprived of their forehead at 8- to 10-ss, they were later on endowed with a lower beak. In embryos that were operated on early, the NCCs migrated normally to BA1 but were subjected to massive apoptosis a few hours later. Cell death did not occur when forehead excision was performed at a later stage. In this case, onward expression of Shh in the ventral foregut endoderm extended caudally over the excision limit, and we hypothesized that absence of Shh production by the endoderm in embryos that were operated on early could be responsible for the NCC apoptosis and the failure of BA1 development. We thus provided exogenous Shh to the embryos that were operated on before 7-ss. In this case, the development of the lower jaw was rescued. Therefore, Shh derived from the ventral foregut endoderm ensures the survival of NCCs at a critical stage of BA1 development.

Fig. 1.

Shh expression in pharyhgeal endoderm. (A and A′) Sagittal section (50 μm) of a 5-ss chicken embryo showing Shh expression in midline cells, PcP, anterior ventral foregut endoderm (black arrowhead), and, more posteriorly, in the AIP (asterisk). (B and B′) At 6-ss, Shh transcripts are still present in the anterior ventral endoderm (black arrowhead) but remain absent in the dorsal foregut endoderm (white arrow). The PcP, the most anterior source of Shh at this stage, is in close contact with the rostral ventral endoderm. (C and C′) At 8-ss, a caudal extension of Shh expression in the ventral foregut endoderm has occurred and overpasses the prosencephalic–mesencephalic boundary (dashed line). (D and E) Schematic representations of Shh expression and the level of forehead excision (arrows) on chicken embryos at 6- and 8-ss. End, endoderm; Tel, telencephalon; Di, diencephalon; FP, floor plate; No, notochord. (F) Sagittal section of a chicken embryo immediately after forehead excision at 6-ss. Remaining ventral foregut endoderm (black arrowhead) is devoid of Shh transcripts. (G) At 8-ss, Shh-expressing foregut endoderm (black arrowhead) is present. (H–S) Forehead quail/chicken chimeras. (H) Chimera immediately after the graft of a quail forehead on a 6-ss chicken embryo. (I) A sagittal section (7 μm) of an E3.5 chimera grafted at 6-ss. QCPN immunostaining (brown) shows quail cells forming the ventral Di and the most anterior mesencephalon, the facial ectoderm (FE), Rathke’s pouch (Rp), Sessel’s pouch (Sp), dorsal and ventral pharyngeal endoderm (Ph) of BA1, and ectoderm of BA1. (J) E4.5 quail/chicken chimera with indications (lines 1–3) of proximodistal (P→D) serial sections. (K–S) Proximodistal serial sections were treated with QCPN (K–M) or were in situ hybridized for Shh (N–P) or BMP4 (Q–S). At E4.5, quail cells (K–M) show the same distribution observed in E3.5 embryos (I), forming the ectoderm of the maxillary bud (Mx) of Rathke’s pouch, the epithelium ventral to the diencephalon, and the endoderm of Sessel’s pouch and the anterior part of the pharynx. (N–P) Shh mRNAs are present in Di, Sp, and Ph. (Q–S) Bmp4 mRNAs are present in the ectoderm of both the Mx and mandible (Mb).

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.

Fig. 3.

Whole-mount in situ hybridization in E3.5 to E4.5 chicken control and surgically altered embryos. (A–I) Fgf8, Bmp4, and Pitx1 mRNAs are present in the oral epithelium and ectoderm of BA1 in control (A–C) and forehead-excised embryos after 8-ss (G–I) but not in embryos whose foreheads were excised at 6-ss (D–F). (J–L) Embryos whose foreheads were excised at 6-ss and replaced by a Shh bead show patterns of expression of those genes (black arrows) and a well developed BA1 (‘1’), in contrast to nontreated embryos, which exhibit a reduced sized BA1 and misexpression of these genes (D–F). An asterisk marks the expression of Pitx1 in the suborbital zone in F. In the absence of eyes, the two foci of expression join in a single transversal spot.