Research Article

Specification of Jaw Subdivisions by Dlx Genes

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Science  11 Oct 2002:
Vol. 298, Issue 5592, pp. 381-385
DOI: 10.1126/science.1075703


The success of vertebrates was due in part to the acquisition and modification of jaws. Jaws are principally derived from the branchial arches, embryonic structures that exhibit proximodistal polarity. To investigate the mechanisms that specify the identity of skeletal elements within the arches, we examined mice lacking expression ofDlx5 and Dlx6, linked homeobox genes expressed distally but not proximally within the arches.Dlx5/6–/– mutants exhibit a homeotic transformation of lower jaws to upper jaws. We suggest that nestedDlx expression in the arches patterns their proximodistal axes. Evolutionary acquisition and subsequent refinement of jaws may have been dependent on modification of Dlx expression.

The diversification and radiation of vertebrates was impelled by developmental innovations. Those particularly affecting the head include the elaboration of the brain, neural crest cells (NCCs), ectodermal placodes, an endoskeleton, and jaws (1–4). Large-scale gene duplications, including that of the Dlx gene family, have been tied to these innovations (5, 6), the nexus of which is manifest in the gnathostome (jawed vertebrate) skull. The six known murine Dlx genes are variously expressed in, and regulate the development of, the branchial arches (BAs), brain, placodes, and skeleton including the BA-derived jaws (7–12). Here, we show that deletion ofDlx5 and Dlx6 results in a repatterning of the skull, including a homeotic transformation of the lower jaw into an upper jaw. This transformation supports a model of patterning within the BAs that relies on a nested pattern of expression of Dlxgenes. Expansion and expression of the Dlx gene family correlates with elaboration of the gnathostome jaw.

The BAs are segmentally repeated structures in the embryonic vertebrate head arising from the ventrolateral surfaces. The most rostral arch (BA1) gives rise to most, though not all, of the jaw apparatus and associated soft tissues. BA1 has two principal proximodistal subdivisions, the maxillary (mxBA1, proximal) and mandibular (mdBA1, distal) arches, which contribute to the upper and lower jaws, respectively. It has been thought that a prototypical gnathostome BA likely contained a proximodistal series of five chondrocranial elements (2, 13) (Fig. 1A). Subsequent evolution has modified this pattern. The mammalian BA1 chondrocranium has only two major components, the derivatives of the palatoquadrate (PQ, mxBA1 derivative) and Meckel's cartilage (MC, mdBA1derivative) (14, 15). These elements are further associated with an ordered series of dermatocranial bones.

Figure 1

Hypothesized role of Dlx genes in BA patterning. (A) Diagram of a proto-gnathostome neurocranium (Nc) and associated BA (1 to 7) skeletal derivatives. Gnathostome BA are metameric structures within which develop a proximodistal series of skeletal elements. Inter-BA identity is regulated by Hox,Pbx, and Otx genes. It is hypothesized that the nested expression of Dlx genes regulates intra-BA identity. (B) In situ hybridization of Dlx2 andDlx5 (E10.5) and diagram highlighting the nestedDlx expression within BA mesenchyme. AP, anteroposterior; BA, branchial arch; BA1, first branchial arch; BA2, second branchial arch; Bb, basibranchial; Cb, ceratobranchial; Eb, epibranchial; Hb, hypobranchial; hy, hyoid arch; md, mdBA1; mx, mxBA1; ; Pb, pharyngeobranchial; PD, proximodistal.

Patterning of the BA requires the establishment of both inter-BA and intra-BA identities (Fig. 1A) (16). Evidence implicates Hox, Pbx, and Otx homeobox gene regulation in the former task (17–20); less attention has been paid to the latter task. Intra-BA identity may be controlled by the Dlx genes (7,9–11, 16). The six mammalianDlx genes are genomically linked, convergently transcribed gene pairs (Dlx2/1, Dlx5/6, andDlx3/7) that share regulatory elements and similar expression patterns (5, 6, 21) (Fig. 1and fig. S1). In the BA mesenchyme, the Dlx gene pairs are expressed in nested patterns: Dlx1/2 throughout most of the proximodistal axis, with Dlx5/6 and Dlx3/7progressively restricted distally (9, 11) (Fig. 1, fig. S1).

The correlation of this nested expression pattern with a proximodistal BA skeletal series suggests the hypothesis that a Dlx code establishes identity within this series (Fig. 1A, fig. S1). This has been addressed in Dlx1–/– ,Dlx2–/– , Dlx1/2–/– , and Dlx5–/– mice (7,9–11). Dlx1–/– ,Dlx2–/– , andDlx1/2–/– mice evince progressively more severe alterations of the elements derived from proximal BAs. AlthoughDlx1 and Dlx2 are expressed in distal BAs (Fig. 1), derivative structures appeared normal inDlx1/2–/– mutants (9). Thus, perhaps Dlx3, 5, 6, and 7 compensate forDlx1/2 function distally, an idea tested with theDlx5–/– mice. Reflective of Dlx5expression (Fig. 1B), distal defects, particularly in the proximal mdBA1, were seen (10, 11). Generation ofDlx5/6–/– mutants (22) allowed us to examine the prediction that the loss of these distally expressed genes would result in distal BAs having proximal properties (fig. S1).

Because the deleted Dlx5/6 allele has LacZinserted under the control of the Dlx6 promoter and BA enhancer (Dlx6LacZ ) (22), we examined wild-type and mutant embryos at embryonic day 10.5 (E10.5) forDlx6LacZ expression. Mutant mdBA1 and BA2 form and contain NCCs expressing Dlx6LacZ (Fig. 2A and fig. S2) (23). Moreover, the mutant mdBA1 and BA2 maintain their mesenchymal expression of Dlx1 and Dlx2 at E10.5 (Fig. 2B, fig. S2) (23). The loss of Dlx5/6 and maintenance of Dlx1/2 suggested the possibility that the distal BAs may be respecified to proximal BA fates.

Figure 2

Characterization of distal BA molecular identity at E10.5. (A) β-Galactosidase expression demonstrating the maintenance of theDlx6LacZ+ cell population inDlx5/6+/– (phenotypically wild-type) andDlx5/6–/– embryos. (B toJ) In situ hybridization of wild-type andDlx5/6–/– embryos. (B) Maintenance ofDlx2 expression. [(C) to (G)] Loss of distal identity. Mesenchymal expression of dHAND, Dlx3,Alx4, and Pitx1 is lost inDlx5/6–/– mutants, as is mandibular midline ectodermal expression of Bmp7 (arrowheads) [(H) to (J)] Acquisition of proximal, maxillary-like identity. Expression of Wnt5a, Meis2, and Prx2is increased and expanded in mdBA1 ofDlx5/6–/– mutants. Mutant structures noted in red. hrt, heart; olf, olfactory pit and frontonasal primordia; and otc, otic vesicle.

We examined whether Dlx5/6–/– mutants evinced changes at E10.5 in the expression of genes that have been implicated in mdBA1 development (16, 24–32). Mutant BA expression of dHAND [a downstream target ofDlx6 (33)], Dlx3, and Alx4was lost (Fig. 2, C to E). Alhough proximal mdBA1 Bmp7expression was maintained, expression at the distal midline of mdBA1 was lacking (Fig. 2F). Mesenchymal Pitx1 expression was lost, although ectodermal expression slightly extended further ventrocaudad (Fig. 2G, fig. S2). Expression of Msx1 andMsx2 in mdBA1 was reduced, although Prx1 was expanded (fig. S2). Barx1 was expanded distad in mdBA1 (fig. S2); BA2 and BA3 expression, however, was lost. Therefore,Dlx5/6–/– mutants lack expression domains of several genes implicated in mdBA1 development (Alx4,dHAND, Dlx3, Dlx5/6, Bmp7, and Pitx1), while maintaining expression of genes also known to participate in mxBA1 development (Dlx1, Dlx2,Msx1, Msx2, and Prx1) (9,16, 26–28). Of note,Dlx5/6 regulation of Alx4, Barx1, andDlx3 provides evidence for conservation of invertebrate genetic circuitry, because Drosophila Distal-less regulates homologs of these genes (aristaless, BarH1, andDistal-less itself, respectively) (34–36).

Although few, if any, genes solely expressed within proximal BA have been characterized, we assayed the possible acquisition of mxBA1 molecular identity in Dlx5/6–/– mutants by examining the expression of genes (Wnt5a,Meis2, and Prx2) that are normally expressed in a graded manner within BA1 (higher in proximal BA1 than in distal BA1) (37–39). In theDlx5/6–/– mutants, the expression ofWnt5a, Meis2, and Prx2 was more intense and expanded laterad and caudad within mdBA1 at E10.5 (Fig. 2, H to J). Thus, the levels of these three genes in mutant mdBA1 more closely resemble normal mxBA1 than mdBA1.

We examined the morphologic consequences of the loss of mdBA1 and gain of mxBA1 molecular properties in Dlx5/6–/– mutant BA1. We operationally defined as “proximal” both chondrocranial (e.g., ala temporalis of the alisphenoid and incus) and dermatocranial (maxilla, palatine, pterygoid, lamina obturans of the alisphenoid, jugal, and squamosal) mxBA1 skeletal elements affected by the loss of Dlx1/2, and as “distal” the remainder of the BA1-derived elements (from mdBA1: the malleus, body and rostral process of MC, dentary, ectotympanic, and gonial) (9,16).

Dlx5/6–/– mutants die at postnatal day 0 (P0), and often, although not exclusively, exhibit exencephaly (Figs. 3 and 4; and fig. S3). Sensory capsular defects seen inDlx5–/– single mutants (10,11) are exacerbated with the loss of Dlx5/6; the nasal capsules are nearly absent, and the trabecular basal plate is severely truncated, as are the pars canalicularis and tegmen tympani of the otic capsule (Fig. 3, C and D; Fig. 4, A and F; fig. S3). Exencephalic and nonexencephalic mutants showed the same BA phenotypes (fig. S3). The distomedial tissues of BA1 often failed to become fully opposed and were cleft (Fig. 3B; Fig. 4, E and F).

Figure 3

Morphologic transformation of the mandibular arch at E16. (A and B) Mandibular morphology of wild-type (A) and Dlx5/6–/– mutants (B). In both fused (arrowhead, left) and cleft (center, right) states, the mutant lower jaw (UJ*) is transformed, appearing as a mirror image (arrows) of the upper jaw (UJ). (C and D) Skeletal staining of E16 wild-type andDlx5/6–/– mutant (exencephalic) littermates, with expanded views, demonstrates the transformation of the body of MC into a second ala temporalis (at*), attached, with the maxillary-derived ala temporalis (at), to the neurocranial basal plate (bp). Note the truncated styloid (black arrowhead), the ectopic projection from the hyoid toward the styloid (purple arrowhead), and an adjacent stapes. These data appear in an expanded form in fig. S3.

Figure 4

Morphologic transformation of the mandibular arch at P0. (A to G) Mandibular to maxillary homeotic transformation revealed by bone (red) and cartilage (blue) staining at P0. (A) Staining highlights the loss of nasal structures (arrowhead), severely hypoplastic upper incisors (UI) lacking premaxillary bone, the transformation of the dentary (dnt) into a maxilla (mx*), and rudimentary lower incisors (LI) without alveolar bone of attachment. [(B) and (C)] Wild-type (B) endogenous andDlx5/6–/– mutant (C) endogenous and ectopic ala temporali [in (B) ectopic outlined in yellow, endogenous in black] in situ and dissected (C). [(D) and (E)] Staining reveals the transformation of the dentary in cleft (upper left) and not cleft (lower left) mandibular states. In the noncleft state, the ectopic maxillary (mxp*) and palatine (pl*) palatal shelves reach the midline. (F) Basal and lateral schemas (incorporating the range of defects seen in several cases) of wild-type andDlx5/6–/– mutant elements demonstrating the nature of the homeotic transformation. A mandibular-cleft P0Dlx5/6–/– neonate is included for reference. mxBA1 elements are in yellow, mnBA1 in lavender, BA2 in turquoise, neurocranium in steel blue, premaxillary-derived UI in orange, and all other ossified elements in black. Transformed elements are labeled in red with an asterisk. (G) Wild-type and Dlx5/6–/– mutant neonates, minus superficial ectoderm (upper) or sectioned (lower), reveal concomitant soft tissue [vibrissae (compare arrowheads) and rugae] transformations. at, ala temporalis; fp, frontal process; gn, gonial; iof, infraorbital foramen; in, incus; jg, jugal; Lmo, lower molar; lo, lamina obturans; ma, malleus; mmx, maxillary molar alveolus; nsc, nasal capsule; pl, palatine; pm, premaxillary; pt, pterygoid; rp, rostral process; rtp, retrotympanic process; rug, rugae; sq, squamosal; tbp, trabecular basal plate; ty, ectotympanic; V2, maxillary branch, trigeminal; vbf, vibrissae follicle; Vg, trigeminal ganglion; wt, wild type.

Assays of E14.5 to P0 skeletal preparations revealed the presence of proximal BA1 skeletal elements, whereas distal BA elements were missing, having been replaced by a second set of “proximal” elements (Figs. 3 and 4, fig. S3). Although affected by aberrant olfactory placodal development (below), the mxBA1-derived maxilla and palatine bones were apparent, as were pterygoid, squamosal and, usually, a diminutive jugal. A clearly identifiable ala temporalis and associated lamina obturans were present (Figs. 3 and 4, fig. S3). The body of MC was transformed into a second ala temporalis, attached to the neurocranial base adjacent to the mxBA1-derived ala temporalis (Fig. 3, C and D; Fig. 4, B and C; fig. S3). mdBA1 contained dematocranial derivatives nearly identical in shape and size to the mxBA1-derived maxillae (Fig. 3D; Fig. 4, A and D to F; fig. S3). These ectopic maxillae (mx*) had frontal processes with infraorbital foramina, molar alveolae, and palatal shelves, which in mutant mandibles that were not cleft extensively abutted at the midline (Fig. 4, E and F). Ectopic laminar intramembranous bones developed, juxtaposed to the ectopic lamina obturans, which appear to be duplicated squamosals (Fig. 3F). Ectotympanic and gonial bones failed to form; instead, a second set of palatine and pterygoid bones developed in conjunction with the ectopic maxillae (Fig. 4, E and F). The malleus, which normally constitutes the proximal end of MC, appeared to have been transformed into a cartilaginous structure often fused to the incus; we have taken this as an ectopic incal structure. In some cases, the duplicated maxillae were associated with incisors, which usually lacked their associated alveolar bone (Fig. 4, A and F). These incisors were not in close association with each other, although occasionally they were accompanied by a cartilaginous nodule, the remnant of the midline rostral process of MC. Thus, within BA1 two sets of proximal skeletal elements developed.

Although the nature of their transformation was more ambiguous, skeletal derivatives of BA2 were also affected (Figs. 3D and 4F, fig. S3). The styloid process was truncated, and the hyoid extended an ectopic process toward it. The lesser horns projected to the cranial base. Stapes were present (often lacking foramena), as were associated ectopic cartilages.

The proximalization of the skeletal structures in BA1 is mirrored by a duplicated set of soft tissue structures normally restricted to the maxillary arch. A second set of mystacial vibrissae (40), induced by signals from the underlying mesenchyme (41), developed in the soft tissue of mdBA1 (Fig. 4G). Moreover, a second set of rugae developed in conjunction with the ectopic palatal shelves in the mutant mdBA1 (42) (Fig. 4G).

The fact that the mdBA1-to-mxBA1 transformation produces a mirror-image duplication (Fig. 3B, red arrows) suggests the presence of a source of positional information centered midway along the maxillary-mandibular axis (Fig. 5). Fgf8, whose expression in the BA1 ectoderm of the Dlx5/6–/– mutants is maintained (fig. S2), is a candidate for such a patterning signal (16, 29, 43, 44). Interpretation of this patterning signal would then depend on the combination of Dlx genes expressed in the mesenchyme (Fig. 5, A and B).

Figure 5

Schemae of BA1 patterning. (A andB) Lateral and frontal views of E10.5 BA1 and FNPs depicting the relationship of the nested, mesenchymal BA1 expression ofDlx1/2/5/6 (Dlx1/2 + in yellow; Dlx1/2/5/6 + in lavender) and hypothesized ectodermal sources of patterning information (e.g.,Fgf8 expression) from the BA1 ectoderm centered at the junction of mxBA1 and mdBA1 (dark blue zone and arrows). This is interpreted by the subjacent Dlx +mesenchyme: Where solely Dlx1/2 +, proximal structures develop; where Dlx1/2/5/6 +, distal structures develop. Additional information is hypothesized to be supplied by factors (e.g., BMPs) expressed in the distal mdBA1 ectodermal midline and the olfactory ectoderm at the junction of the FNPs and mxBA1 (red patches and arrows). The jaws are kept in register by juxtaposing a common central domain (blue) with two caps (red) of positional information. (C) Schema of BA1 patterning and molecular identity in the absence ofDlx5/6. (D) Schema of regulatory control exerted by Dlx5/6 on distal BA1 (green arrowheads indicating positive control and red line indicating inhibition). dml, distal midline; lFNP, lateral FNP; mFNP, medial FNP.

For proper functioning, jaw articulation and dentition must be appropriately aligned. Functional registration within the upper jaw requires the correct integration of the frontonasal prominences (FNPs; sources of the premaxillae and upper incisors) with mxBA1. This integration was lost in Dlx5/6 –/– mutants, in which nasal capsular and premaxillary structures were minimal, upper incisors rudimentary, and the adjacent parts of the maxillae subsequently misshapen (Figs. 3 and 4, fig. S3). Thus, without theDlx5/6+ olfactory placodal induction of the underlying FNP mesenchyme (Figs. 1B and 2A) (11,16), the mutant upper jaw lacked integration of the molar-bearing maxillae with incisor-bearing premaxillae. In some respects, the endogenous Dlx5/6 –/– mutant maxillae resemble their ectopic counterparts more than wild-type maxillae, which suggests that they are sensitive to patterning information from the olfactory placodes or tissue at the junction of the FNPs and mxBA1 (Fig. 5, A and B).

These tissues are the sources of secreted signaling molecules, including bone morphogenetic proteins (BMPs), and numerous transcription factors are expressed in the NCCs of both the mxBA1 and the adjacent FNPs (e.g., Msx1and Msx2). These same signaling molecules and transcription factors are expressed at, and adjacent to, the distal midline of mdBA1, where they regulate lower incisor development (16, 26, 29). Lack of mesenchymal Dlx5/6 in mdBA1 results in a loss of proper distal mdBA1 midline development, as suggested by the loss of ectodermal Bmp7 expression, mandibular clefting, and a deficiency in integrating the distal midline (lower incisor) with more proximal parts of mdBA1. Therefore, formation of the jaw apparatus appears to involve juxtaposing three ectodermal patterning centers: the olfactory placode/FNPs, the core of mxBA1 and mdBA1, and distal midline mdBA1 (Fig. 5) (16, 29,43–45).

We conclude that loss of Dlx5 and Dlx6 results in a homeotic transformation of the lower jaw into an upper jaw and that cellular identity within an arch relies on a nested pattern ofDlx expression. Although lampreys (a type of agnathan, or jawless vertebrate) express Dlx genes in their BAs, theirDlx expression is not nested, and their BA cartilage is uniform and unsegmented (6, 44, 46). Thus, perhaps the advent of nested Dlx BA expression, by providing specification of intra-BA identity, contributed to the transition from jawless to jawed vertebrates. This transition may have occurred in conjunction with a change in Hox gene expression (47). Delineating the underlying mechanism(s) responsible for the nesting of Dlx genes expression will likely yield greater insight into gnathostome evolution.

Supporting Online Material

Figs. S1 to S3

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