An endothelin-1 switch specifies maxillomandibular identity

Abstract

Articulated jaws are highly conserved structures characteristic of gnathostome evolution. Epithelial-mesenchymal interactions within the first pharyngeal arch (PA1) instruct cephalic neural crest cells (CNCCs) to form the different skeletal elements of the jaws. The endothelin-1 (Edn1)/endothelin receptor type-A (Ednra)-->Dlx5/6-->Hand2 signaling pathway is necessary for lower jaw formation. Here, we show that the Edn1 signaling is sufficient for the conversion of the maxillary arch to mandibular identity. Constitutive activation of Ednra induced the transformation of upper jaw, maxillary, structures into lower jaw, mandibular, structures with duplicated Meckel's cartilage and dermatocranial jaws constituted by 4 dentary bones. Misexpression of Hand2 in the Ednra domain caused a similar transformation. Skeletal transformations are accompanied by neuromuscular remodeling. Ednra is expressed by most CNCCs, but its constitutive activation affects predominantly PA1. We conclude that after migration CNCCs are not all equivalent, suggesting that their specification occurs in sequential steps. Also, we show that, within PA1, CNCCs are competent to form both mandibular and maxillary structures and that an Edn1 switch is responsible for the choice of either morphogenetic program.

Fig. 1.

Transformation of maxillary components in Ednra^Edn1/+ mice. (A and B) Facial appearance of E18.5 wild-type (A) and Ednra^Edn1/+ (B) mice. Vibrissae are absent in mutant mice that have open eyelids (arrows) and an anterior shift of the ear position (arrowheads). (C–L) Bone and cartilage elements of E18.5 wild-type (C, E, G, I, and K) and Ednra^Edn1/+ (D, F, H, J, and L) skulls. (C and D) Lateral views of wild-type and Ednra^Edn1/+ craniofacial skeletons. (E and F) The Ednra^Edn1/+ mutant shows mirror-image duplication of the mandibular elements (dentary, MC, and malleus) at the expense of the maxillary elements (maxilla, jugal, squamosal, and incus). (G and H) Caudal views of wild-type and Ednra^Edn1/+ craniofacial skeletons. The dentaries are removed to show the normal and transformed maxillae. Large parts of the palatine, pterygoid, lamina obturans, and ala temporalis are missing or severely deformed in the Ednra^Edn1/+ mutant. (I and J) Ectotympanic and gonial bones are also duplicated in association with the ectopic MC in Ednra^Edn1/+ mutants. (K and L) Caudal views of the premaxilla-maxilla junction. The tip of the transformed maxilla-dentary in the Ednra^Edn1/+ mutant contains an ectopic incisor and fuses to the premaxilla. (M and N) Parasagittal sections of E18.5 wild-type (M) and Ednra^Edn1/+ (N) mice stained by Mallory trichromic. The Ednra^Edn1/+ mutant shows an ectopic incisor in addition to the orphotopic 1. Fusion between the premaxilla and transformed maxilla-dentary is observed in the mutant. at, ala temporalis; bs, basisphenoid; dnt, dentary; etm, ectotympanic; fmx, frontal process of maxilla; gn, gonial; hy, hyoid; in, incus; jg, jugal; lo, lamina obturans; ma, malleus; mx, maxilla; pl, palatine; pmx, premaxilla; ps, presphenoid; ptg, pterygoid; sq, squamosal; tg, tongue; UI, upper incisor; vbf, vibrissae follicle; vm, vomer; *, ectopic structure.

Fig. 2.

Comparison of craniofacial skeletons among E15.5 Ednra-null (Ednra^lacZ/lacZ) (A), wild-type (B) and Edn1-misexpressing (Ednra^Edn1/+) (C) mice. Activated Edn1/Ednra signaling (colored in pink) correlates to the formation of MC and associated mandibular structures. at, ala temporalis; dnt, dentary; hy, hyoid; in, incus; ma, malleus; mx, maxilla; pl, palatine; pmx, premaxilla; *, ectopic structure.

Fig. 3.

Histological and whole-mount neuromuscular analysis of Ednra^Edn1/+ mice. (A and B) Frontal sections of E18.5 wild-type (A) and Ednra^Edn1/+ (B) mice stained by Mallory trichromic. Constitutive activation of Edn1/Ednra signaling results not only in the transformation of maxillary skeletal elements, but also in the appearance of new muscular components associated to the ectopic dentary, which can be interpreted as a duplicated masseter muscle. (C and D) Cranial nerves of E10.5 wild-type (C) and Ednra^Edn1/+ (D) embryos stained by antineurofilament antibody. Ectopic branches of the mandibular (arrow) and facial (arrowheads) nerves are indicated. dnt, dentary; mm, masseter muscle; pl, palatine; tg, tongue; gV, trigeminal ganglion; V₁, ophthalmic nerve; V₂, maxillary nerve; V₁, mandibular nerve; VII, facial nerve; IX, glossopharyngeal nerve; *, ectopic structure.

Fig. 4.

Gene expression analysis of the pharyngeal arches in Ednra^Edn1/+ embryos. (A–T) Whole-mount in situ hybridization for Dlx2 (A and B), Fgf8 (C and D), Dlx5 (E and F), Dlx6 (G and H), Dlx3 (I and J), Pitx1 (K and L), Goosecoid (M–P), and Hand2 (Q–T) in E10.5 wild-type (A, C, E, G, I, K, M, O, Q, and S) and Ednra^Edn1/+ (B, D, F, H, J, L, N, P, R, and T) embryos. Arrows indicate the junction of maxillary and mandibular arches. Yellow arrowheads indicate ectopic gene expression in the maxillary arch. hy, hyoid arch; md, mandibular arch; mx, maxillary arch.

Fig. 5.

Morphological transformation in Ednra^Hand2/+ ES cell-derived chimeric mice. (A) Representative surface appearance of E18.5 Ednra^Hand2/+ chimeric mice. Mutant mice demonstrate loss of vibrissae follicles, open eyelids (arrows), and polydactyly (arrowheads). (B–E) Craniofacial bone and cartilage elements of E18.5 Ednra^Neo/+ ES cell-derived (B) and Ednra^Hand2/+ ES cell-derived (C–E) chimeric mice. Whereas Ednra^Neo/+-chimeric mice show normal skeletal morphology (B), Ednra^Hand2/+-chimeric mice demonstrate transformation of the maxilla into dentary-like bone (C), which fuses to the premaxilla and contains an ectopic incisor at the tip (D). The condylar and angular processes of the dentary, gonial, ectotympanic, and squamosal bones were often malformed or absent (E). (F–I) Craniofacial skeletons of E15.5 wild-type (F and H) and Ednra^Hand2/+-chimeric (G and I) mice. Ectopic formation of MC-like, rod-shaped cartilage is present often with small and deformed ala temporalis in Ednra^Hand2/+-chimeric mice. The malleus, incus, stapes, and styloid process are deformed. agp, angular process; at, ala temporalis; cdp, condylar process; crp, coronoid process; dnt, dentary; etm, ectotympanic; fmx, frontal process of maxilla; gn, gonial; hy, hyoid; in, incus; jg, jugal; ma, malleus; mx, maxilla; pmx, premaxilla; sp, styloid process; sq, squamosal; st, stapes; UI, upper incisor; *, ectopic structure.