Jaw muscularization requires Dlx expression by cranial neural crest cells

Abstract

The origin of active predation in vertebrates is associated with the rise of three major, uniquely derived developmental characteristics of the head: (i) migratory cranial neural crest cells (CNCCs) giving rise to most skeletal skull elements; (ii) expression of Dlx genes by CNCCs in the Hox-free first pharyngeal arch (PA1); and (iii) muscularization of PA1 derivatives. Here we show that these three innovations are tightly linked. Expression of Dlx genes by CNCCs is not only necessary for head skeletogenesis, but also for the determination, differentiation, and patterning of cephalic myogenic mesoderm leading to masticatory muscle formation. In particular, inactivation of Dlx5 and Dlx6 in the mouse results in loss of jaw muscles. As Dlx5/6 are not expressed by the myogenic mesoderm, our findings imply an instructive role for Dlx5/6-positive CNCCs in muscle formation. The defect in muscularization does not result from the loss of mandibular identity observed in Dlx5/6(-/-) mice because masticatory muscles are still present in EdnRA(-/-) mutants, which display a similar jaw transformation. The genesis of jaws and their muscularization should therefore be seen as an integrated Dlx-dependent developmental process at the origin of the vertebrate head. The role of Dlx genes in defining gnathostome jaw identity could, therefore, be secondary to a more primitive function in the genesis of the oral skeletomuscular system.

Fig. 1.

Head muscle phenotypes observed in EdnRA^−/− and Dlx5/6^−/− mutant mice. Mallory trichromic staining on frontal (A–C) and parasaggital (D–F) sections of E18.5 wild-type, EdnRA^−/−, and Dlx5/6^−/− mice. The EdnRA^−/− and Dlx5/6^−/− mutants show a duplicated maxillary bone (mx*) associated with head muscle defects. In the EdnRA^−/− mutant, the masseter muscle (mm) is present but has an abnormal morphology and orientation (B–B′ and E); all masseter components pass through the infraorbital foramen of the transformed lower jaw (black arrowhead in B). The pterygoid and temporal muscles are indistinguishable and form a single muscular mass (E). In the Dlx5/6^−/− mutant, the masseter muscle is replaced by connective tissue [see the masseter region (mr) in C and C′]. In the two mutants, the tongue is considerably reduced, the muscles fibers are totally disorganized (B, B′′, C, C′′, E′, and F′), and sublingual muscles are either absent or replaced by a few muscle fibers (B′′′ and C′′′). dg, digastric muscle; dnt, dentary bone; e, eye; jg, jugal bone; lpt, lateral pterygoid muscle; mh, mylohoid muscle; mm, masseter muscle; mr, masseter region; mx, maxillary bone; mx*, duplicated maxillary bone; pt, pterygoid muscle; sbr, sublingual region; tg, tongue; tp, temporal muscle; um, upper molar. (Scale bar: 500 μm in A–C; 700 μm in D–F and D′–F′; 100 μm for other panels.)

Fig. 2.

Expression of early myogenic markers in Dlx5/6^−/− embryos. Whole-mount in situ hybridization for Capsulin (A and B), Tbx1 (C and D), and Pax3 (E and F) on E9.5–E10.5 wild-type and Dlx5/6^−/− embryos. In the mandibular and hyoid arches at E9.5, the expression of the early markers of myogenic specification, Capsulin and Tbx1 (A–D), is not affected by the Dlx5/6 inactivation. The persistence of Pax3 expression in the hypoglossal cord of E10.5 Dlx5/6^−/− embryos (E and F) indicates that somitic myoblast migration during tongue development is not altered. e, eye; h, heart; hc, hypoglossal cord; hy, hyoid arch; md, mandibular arch. (Scale bar: 300 μm in A–D; 450 μm in E and F.)

Fig. 3.

β-Galactosidase staining on E10.5, E11.5, and E12.5 Myf5^nLacZ/+ and Dlx5/6^−/−;Myf5^nLacZ/+ mutant embryos. In E10.5 Myf5^nLacZ/+ embryos, Myf5-LacZ is expressed in the extraocular premuscle mass and in the mandibular and hyoid arches (A). At this stage, the expression of Myf5-LacZ is not perturbed by Dlx5/6 inactivation in the hyoid and extraocular region, but is reduced in the mandibular arch (B). At E11.5 and E12.5 in Myf5^nLacZ/+ embryos, Myf5-LacZ is expressed in the extraocular and masticatory premuscle masses (C and E). This expression is dramatically reduced in the ocular and mandibular region of Dlx5/6^−/−;Myf5^nLacZ/+ mutant embryos (D and F). e, eye; hy, hyoid arch; md, mandibular arch; mx, maxillary bud; mx*, duplicated maxillary bud; peom, extraocular premuscle mass; pmm, masticatory premuscle mass. (Scale bar: 500 μm in A and B; 650 μm in C and D; 750 μm in E and F.)

Fig. 4.

Expression of MyoD in EdnRA^−/− and Dlx5/6^−/− mutant embryos. (A–C) MyoD whole-mount in situ hybridization on E11.5 wild-type (A), EdnRA^−/− (B), and Dlx5/6^−/− (C) embryos. MyoD expression in the masticatory premuscle mass (pmm) (A) is reduced in EdnRA^−/− and Dlx5/6^−/− mutants (B and C). (D–O) MyoD immunostaining on frontal sections in the masseter (D–I) and tongue (J–O) region of wild-type (D, G, J, and M), EdnRA^−/− (E, H, K, and N), and Dlx5/6^−/− embryos (F, I, L, and O) at E12.5 (D–F and J–L) and E14.5 (G–I and M–O). In EdnRA^−/− mutants, the masticatory and masseter muscle mass (E and H) are MyoD positive, but are reduced and show an abnormal morphology. In the Dlx5/6^−/− mutant, only a few cells express MyoD in the masseter region (F and I), resulting in an absence of masticatory muscle masses. In EdnRA^−/− and Dlx5/6^−/− mutants at E12.5, only a few cells express MyoD in the tongue and sublingual region (K and L). At E14.5, the myogenic marker is expressed in the vestigial tongue of the two mutants (N and O), the genioglossus and geniohyoid muscles are never present, and sublingual muscles are barely differentiated in EdnRA^−/− (N) and are absent in Dlx5/6^−/− mutants (O). dg, digastric muscle; e, eye; gg, genioglossus; gh, geniohyoid; md, mandibular bud; mh, mylohyoid; mm, masseter muscle; mr, masseter region; mx, maxillary bud; mx*, duplicated maxillary bud; oc, otic capsule; peom, extraocular premuscle mass; pmm, masticatory premuscle mass; pmr, masticatory premuscle region; psbl, sublingual premuscle mass; ptg, tongue premuscle mass; sbl, sublingual muscle; tg, tongue. (Scale bar: 400 μm in A–C; 70 μm in D–F and J–L; 150 μm in G–I and M–O.)

Fig. 5.

Whole-mount in situ hybridization on E11.5 wild-type (A and C) and EdnRA^−/− (B and D) embryos using Dlx5 (A and B) and Dlx6 (C and D) probes. In wild-type embryos (A and C), Dlx5 and Dlx6 are expressed in the mandibular and the hyoid arches and in the proximal part of the maxillary bud. In EdnRA^−/− mutants, expression of Dlx5 and Dlx6 is lost in distal but not in proximal pharyngeal arches, including the duplicated maxillary bud (mx*), the hyoid arch (hy), and the maxillary bud (mx) (B and D). In these mutants, the pattern of Dlx5 and Dlx6 expression in the maxillary bud is a mirror image of that found in the duplicated maxillary bud. e, eye; hy, hyoid arch; md, mandibular arch, mx, maxillary bud; mx*, duplicated maxillary bud; oc, otic capsule. (Scale bar: 650 μm.)

Fig. 6.

Summary diagram. (Left) The territory of Dlx5/6 expression in E11.5 PA1 of the three mouse genotypes analyzed in this study. (Right) The skeleto-muscular defects observed in these mice are summarized. Muscles are shown as red, bones as green, and the tongue, which is a somite-derived muscle, as yellow. In both EdnRA^−/− and Dlx5/6^−/− mutants, lower jaw skeletal elements undergo a mirror image transformation into upper-jaw-like skeletal structures. Although in EdnRA^−/− mutants the masseter muscle persists, no PA1-derived muscles are differentiated in Dlx5/6^−/− mutants. dnt, dentary bone; md, mandibular bud; mm, masseter muscle; mx, maxillary bone or maxillary bud; mx*, duplicated maxillary bone or duplicated maxillary bud; pmx, premaxillary bone; sbl, sublingual muscles; tg, tongue.

Fig. 7.

Roles of Dlx genes during chordate evolution. The “new head” characterized by the simultaneous appearance of migratory skeletogenic CNCCs, Hox-negative Dlx-positive PA1 and PA1 muscularization may or may not include Myxynoidea (39). In this study, we show that CNCC expression of Dlx genes not only determines jaw identity in Gnathostomata, but also plays a role in organizing mouth muscularization in vertebrates. The expression of Dlx genes in PA1 CNCCs might have had a determining role in the transition between filter feeding and active predation. CNCCs, cranial neural crest cells; PA1, first pharyngeal arch.