Jaw Development: Chinless Wonders Dispatch

Anthony Graham

It has been suggested that the regionally restrictedexpression of Dlx genes acts to pattern the proxi-modistal axis of the pharyngeal arches during verte-brate development. Recently, clear evidence of thishas emerged from Dlx-5; Dlx-6 double mutants, inwhich the lower jaw is transformed to an upper jaw.

The earliest vertebrates were jawless animals withmouths that were only suitable for suspension feeding.Some 400 million years ago, however, there appeareda new group of vertebrates, the gnathostomes, whichpossessed biting jaws. This was a key event as itallowed these animals to become active carnivores,which in turn facilitated their success. The mostpopular model for the origin of jaws suggests that theupper and lower jaws evolved as modifications of themost anterior pharyngeal arch: the upper jaw frommodification of the more proximal skeletal elements ofthe first arch, and the lower jaw from rearrangement ofthe more distal elements. If this scenario is to hold,one would predict there must be genes that differenti-ate between the proximal and distal regions of thearches. Furthermore, one might also expect that muta-tions in such genes would cause proximal regions todevelop as distal or vice-versa. Excitingly, a recentpaper [1] has shown this to be the case. The home-obox genes Dlx-5 and Dlx-6 are both expressed in thedistal portion of the first pharyngeal arch, and whenboth are inactivated the lower jaw is transformed intoan upper jaw .

In mammals, there are six members of the Dlx familyof homeobox genes existing as three sets of linkedgene pairs: Dlx-2 and Dlx-1; Dlx-5 and Dlx-6; and Dlx-3 and Dlx-7. Each of these pairs is convergentlytranscribed and, by and large, the members of eachpair have coincident expression patterns [2] (Figure 1).One shared site of expression of all of the Dlx genes isin the neural-crest-derived mesenchyme of thepharyngeal arches, the source of the skeletal elementsof the mouth and pharynx [3]. Within the pharyngealarches, however, the Dlx gene pairs exhibit nestedpatterns of expression. Dlx-1 and Dlx-2 are expressedthroughout much of the proximodistal axis, while Dlx-5 and Dlx-6 are expressed in the more distalmesenchyme and Dlx-3 and Dlx-7 are expressed onlyat the distal end of the arches (Figure 1) [4,5]. Such anexpression pattern could be consistent with the Dlxgenes acting to establish a code, whereby differentdomains along the proximodistal axis of the archesare defined by combinatorial expression of Dlx genes.Thus, the maxilla and most proximal mandible wouldform from crest cells that express only Dlx-1 and

Dlx-2, while the other elements of the mandible wouldbe generated by crest additionally expressing Dlx-5and Dlx-6 and distally also Dlx-3 and Dlx-7 (Figure 1).

To date, however, mutational studies of the Dlx geneshave not clearly demonstrated that these transcriptionfactors function to provide a combinatorial code thatspecifies proximodistal identity. For example, micelacking the Dlx-1/Dlx-2 gene pair display defects ofproximal arch elements [5], but the distal skeletal struc-tures are unaffected in these animals. Therefore, to testthe role of Dlx genes in the patterning of the distal regionof the arches, Depew et al. [1] analysed the elements ofthe jaw in mice mutant for both Dlx-5 and Dlx-6.

Interestingly, Depew et al. [1] found that the distalregion of the first arch assumed a proximal identity inthese double-mutant animals. This was first assessedin the embryo using markers that distinguish betweendistal and proximal domains within the arches. Duringnormal development, the distal territory of the archesis marked through its expression of a number ofgenes, including Alx-4, Dlx-3, dHAND, Bmp-7 andPitx-1, while the proximal region displays elevatedexpression of wnt-5a, Meis-2 and Prx-2. In the Dlx-5;Dlx-6 double mutants, however, the expression of thedistal markers was lost, and instead the distal archterritory now expressed proximal markers.

More dramatically still, the morphology of the firstarch skeletal elements in these mutants was radicallyaltered. The Dlx-5; Dlx-6 mutants lack a number oflower jaw elements, and in their place they display anadditional, mirror-imaged group of upper jaw ele-ments. This transformation of the lower jaw to upperis also mirrored in the soft tissues. In the Dlx-5; Dlx-6mutants the ‘lower jaw’ displays, externally, a secondset of vibrissae, whiskers and, internally, ectopicruggae, the ridges of the palate.

With regard to the role of Dlx genes in patterning theproximo-distal axis of the arches, it is informative tocompare the results from the Dlx-1; Dlx-2 doublemutants with those from the Dlx-5; Dlx-6 doublemutants. In the Dlx-1; Dlx-2 mutants, there was afailure in the development of proximal structures [5],while in the Dlx-5; Dlx-6 mutants there was a cleartransformation of arch structures from distal, mandibu-lar, to proximal, maxillary [1]. Although these two setsof results seem somewhat at odds, both phenotypessuggest that the roles of the Dlx genes are to direct theresponse of the cells of the arches to the patterningcues. Thus, in Dlx-1; Dlx-2 mutants, the proximal cellslack expression of all Dlx genes, so they cannotrespond to these cues, the development of proximalstructures fails and some ectopic elements form. Incontrast, in the Dlx-5; Dlx-6 double mutants most ofthe arch mesenchyme expresses only Dlx-1 and Dlx-2,and so these cells behave as proximal cells and inresponse to patterning cues form the upper jaw.

These results, however, still do not reveal whetherthe nested expression of the Dlx gene pairs function to provide a combinatorial code that specifies proximodistal identity. A test of that would require a

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comparison of the effects of expressing Dlx-5 and Dlx-6 throughout the mesenchyme of the first arch inanimals that were either wild-type or mutant for bothDlx-1 and Dlx-2. If, in both cases an ectopic lower jawwas formed proximally, then one could conclude thatDlx gene pairs do not function combinatorially and thatthe specification of the lower jaw merely requires theexpression of Dlx-5 and Dlx-6. But if an ectopic lowerjaw was formed only if Dlx-5 and Dlx-6 were expressedproximally in wild-type animals — that is, in a Dlx-1/Dlx-2-expressing context — then one could con-clude that these gene pairs do function combinatorially.

Recent phylogenetic analyses have also providedinsights into how the vertebrate Dlx gene family likelyevolved [6]. The vertebrates are a subphyla of thephylum chordata, so to understand how the Dlx genefamily has evolved it is important to start with ananalysis of other chordates, specifically the cephalo-chordate Amphioxus — nearest extant relative of

the vertebrates — which has a single Dlx gene [7](Figure 2). The vertebrates, however, have more Dlxgenes and they often exist as linked gene pairs, sug-gesting that vertebrate evolution was accompanied bya tandem duplication event which generated a linkedgene pair (Figure 2). Subsequent to this there were anumber of duplication and gene-loss events, whichestablished the vertebrate Dlx family.

In the lineage leading to lamprey — extant jawlessvertebrates — this resulted in there being four Dlxgenes, one linked gene pair and two single genes [6](Figure 2). Importantly, although these Dlx genes areexpressed in the neural crest of the lamprey pharyn-geal arches, they do not exhibit nested expression[6,8]. Rather, they are all expressed throughout theproximodistal axis.

In the lineage leading to the gnathostomes, however,there were likely two full rounds of gene duplication,followed by the loss of one gene pair, generating the

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Figure 1. Genomic organisation of the Dlxgenes and their nested expression withinthe pharyngeal arches.

(A) The Dlx genes exist as three linkedgene pairs — Dlx2/Dlx-1, Dlx-5/Dlx-6 andDlx-3/Dlx-7 — which are convergentlytranscribed. (B) All the Dlx genes areexpressed in the neural crest derivedmesenchyme of the pharyngeal arches.Dlx2/Dlx-1 are expressed along the prox-imodistal extent of the arches, while Dlx-5/Dlx-6 are expressed more distally, andDlx-3/Dlx-7 are only expressed at themost distal region of the arches. Theupper jaw, maxillary apparatus (mx),develops from the proximal part of thefirst arch, while the distal regions givesrise to the lower jaw, the mandibularapparatus (md). The second arch con-tributes to the hyoid (hy).

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A B

Dlx2

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Dlx1/2/5/6

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Figure 2. A model for the evolution of theDlx gene family (based on [6]).

It is proposed that ancestrally chordateshad a single Dlx gene (orange), and thatthis condition is evident in Amphioxus.With the evolution of the vertebrates therewas a tandem duplication which gener-ated a linked Dlx gene pair. This genethen underwent rounds of duplication fol-lowed by gene loss. In the lineage leadingto lamprey, a jawless vertebrate, thisresulted in the presence of a single genepair and two solo Dlx genes. In thegnathostomes, there were two rounds ofduplication, followed by the loss of onelinked gene pair. Thus the mammals havethree linked gene pairs.

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Amphioxus Lamprey Mammals

Chordate ancestor

Gnathostomes

Dlx-6 Dlx-5

Dlx-7 Dlx-3

Dlx-1 Dlx-2X X

X

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Dlx-D Dlx-A

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Dlx content observed in, for example, mammals(Figure 2). Also importantly, all gnathostomes displaynested expression of the Dlx gene pairs. Thus, the evo-lution of jaws was associated with an increase in thenumber of Dlx gene pairs, and along with this was thenested proximodistal deployment of the gene pairs inneural crest of the pharyngeal arches. This would likelyhave facilitated the proximodistal regionalisation of thefirst pharyngeal arch and consequently the evolution ofthe upper and lower jaws.

References1. Depew, M.J., Lufkin, T. and Rubenstein, J.L.R. (2002). Specification

of jaw subdivisions by Dlx genes. Science 298:381-385.2. Panganiban, G. and Rubenstein, J.L.R. (2002). Developmental func-

tions of the Distal-less/Dlx homeobox genes. Development 129,4371–4386.

3. Graham, A. and Smith, A. (2001). Patterning the pharyngeal arches.Bioessays 23, 54–61.

4. Simeone, A., Acampora, D., Pannese, M., D’Esposito, M., Stor-naiuolo, A., Gulisano, M., Mallamaci, A., Kastury, K., Druck, T.,Huebner, K. et al. (1994). Cloning and characterization of themembers of the vertebrate Dlx gene family. Proc. Natl. Acad. Sci.USA 91, 2250–2254.

5. Qiu, M., Bulfone, A., Ghattas, I., Meneses, J.J., Christensen, L.,Sharpe, P.T., Presley, R., Pedersen, R.A. and Rubenstein, J.L.(1997). Role of the Dlx homeobox genes in proximodistal patterningof the branchial arches: mutations of Dlx-1, Dlx-2, and Dlx-1 and -2alter morphogenesis of proximal skeletal and soft tissue structuresderived from the first and second arches. Dev. Biol. 185, 165–184.

6. Neidert, A.H., Virupannavar, V., Hooker, G.W. and Langeland, J.A.(2001). Lamprey Dlx genes and early vertebrate evolution. Proc.Natl. Acad. Sci. USA 98, 1665–1670.

7. Holland, N., Panganiban, G., Henyey, E. and Holland, L. (1996).Sequence and developmental expression of AmphiDll, anamphioxus Distal-less gene transcribed in the ectoderm, epidermisand nervous system: insights into evolution of craniate forebrainand neural crest. Development 122, 2911–2920.

8. Shigetani, Y., Sugahara, F., Kawakami, Y., Murakami, Y., Hirano, S.and Kuratani, S. (2002). Heterotopic shift of epithelial-mesenchymalinteractions in vertebrate jaw evolution. Science 296, 1316–1319.