Chai & Maxson [2006] Recent Advances in Craniofacial Morpholgenesis

SPECIAL ISSUE REVIEWS–A PEER REVIEWED FORUM

Recent Advances in CraniofacialMorphogenesisYang Chai1* and Robert E. Maxson, Jr2

Craniofacial malformations are involved in three fourths of all congenital birth defects in humans, affectingthe development of head, face, or neck. Tremendous progress in the study of craniofacial development hasbeen made that places this field at the forefront of biomedical research. A concerted effort amongevolutionary and developmental biologists, human geneticists, and tissue engineers has revealed importantinformation on the molecular mechanisms that are crucial for the patterning and formation of craniofacialstructures. Here, we highlight recent advances in our understanding of evo–devo as it relates tocraniofacial morphogenesis, fate determination of cranial neural crest cells, and specific signalingpathways in regulating tissue–tissue interactions during patterning of craniofacial apparatus and themorphogenesis of tooth, mandible, and palate. Together, these findings will be beneficial for theunderstanding, treatment, and prevention of human congenital malformations and establish the foundationfor craniofacial tissue regeneration. Developmental Dynamics 235:2353–2375, 2006. © 2006 Wiley-Liss, Inc.

Key words: cranial neural crest (CNC) cell; ectoderm; endoderm; evolution; mesoderm; mandible; palate; toothdevelopment

Accepted 30 March 2006

INTRODUCTION

Development of the craniofacial re-gion is a complex process with manyfeatures that reflect strong evolution-ary forces controlling morphology. Thevertebrate craniofacial region housesand protects the brain and providesthe scaffold on which the sensory andfeeding organs are located. The abilityto sense and devour prey is fundamen-tal to animal survival. Variations incraniofacial anatomy and functionprovide the major driving force in evo-lutionary adaptation.

One of the key features of craniofa-cial development is the formation of

cranial neural crest (CNC) cells. Thespecification, emigration and migra-tion, proliferation, survival, and ulti-mate fate determination of the CNCplay an important role in regulatingcraniofacial development. Unlike thetrunk neural crest, CNC cells give riseto an array of cell types during embry-onic development. For example, CNCcells form most of the hard tissues ofthe head such as bone, cartilage, andteeth, whereas hard tissues in the restof the body are formed from mesodermcells. Genetic disorders, environmen-tal insults, or the combination of bothcan alter the fate determination of

CNC cells and result in craniofacialmalformations. Significant progresshas been made in recent years towardthe understanding of how this impor-tant population of pluripotent cells isinitially established in the early em-bryo and of the molecular mechanismsthat mediate neural crest cell lineagesegregation, differentiation, and finalcontribution to a particular tissuetype (Shah et al., 1996; LaBonne andBronner-Fraser, 1999; Chai et al.,2003; Le Douarin et al., 2004).

The tissues of the head are com-posed of cells from all three germ layerorigins: ectodermal, endodermal, and

1Center for Craniofacial Molecular Biology School of Dentistry University of Southern California, Los Angeles, California2Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center and Hospital, Keck School of Medicine,University of Southern California, Los Angeles, CaliforniaGrant sponsor: National Institute of Dental and Craniofacial Research; Grant sponsor: NIH; Grant numbers: DE 014078; DE012711;DE017007; DE12941; DE12450; Grant sponsor: March of Dimes Birth Defects Foundation.*Correspondence to: Yang Chai, Center for Craniofacial Molecular Biology, University of Southern California, 2250 AlcazarStreet, CSA 103, Los Angeles, CA 90033. E-mail: [email protected]

DOI 10.1002/dvdy.20833Published online 5 May 2006 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 235:2353–2375, 2006

© 2006 Wiley-Liss, Inc.

mesenchymal. As seen in the develop-ment of many organs, craniofacialmorphogenesis depends upon continu-ous and reciprocal tissue–tissue inter-actions, with tooth, palate, and mandi-ble development as classic examples.Research on the development of thehead requires a thorough understand-ing of normal morphology, cell move-ment, cell signaling, gene/gene interac-tion, and transcriptional regulation intime and space.

Investigation of craniofacial devel-opment uses different animal speciesas models as with other areas of re-search in developmental biology.Studies in mice combine the power ofgenetics and genome manipulationtogether with in vitro organ culturetechniques, leading to great progressin recent years. Avian embryos(chicken and quail) are easily acces-sible and are used for grafting/transplantation experiments. Ze-brafish craniofacial developmentalstudies have emerged more recently,and mutant screens have led to theidentification of new cell signalinginteractions (Trainor and Krumlauf,2000; Yelick and Schilling, 2002).Central to these models and tech-niques lies morphology. The head isa very complex structure, and a de-tailed analysis and appreciation ofmorphology are essential to under-standing the mechanism of craniofacialmalformations. Ultimately, “molecularmorphology,” the combination of classicmorphology and molecular biology, pro-vides the basis for understanding theevolutionary changes in head structureand formation that in turn helps to clar-ify key developmental principals as wellas the mechanism of craniofacial mal-formations.

Mouse models are extremely valu-able in our effort to gain a betterunderstanding of human craniofa-cial birth defects. The remarkableprogress being made in the humangenome, in parallel with exquisitefunctional genomic investigations invarious mouse models, has resultedin the discovery of morphoregulatorygenes that determine craniofacialmorphogenesis. For example, wehave uncovered genes that are criti-cal for determining cranial– caudalaxis, dorsal–ventral patterning,left–right symmetry and segmenta-tion in early forming neurulation as

well as branchial arches. More re-cently, many animal models withspecific craniofacial malformationshave facilitated human genetic link-age analysis, in which a genetic de-fect has been identified as the causeof congenital malformation(s) (Thya-garajan et al., 2003; Murray andSchutte, 2004). Overall, geneticallymutated mouse models highlight theenormous challenges of uncoveringcomplex genetic mechanisms under-lying craniofacial development andmalformations.

Progress and understanding of thekey control processes of head develop-ment have advanced to an extentwhere developmental biologists areinteracting with tissue engineers todevise cell-based approaches to treatclinical malformations in humans.The prospect of harnessing develop-mental processes to repair or replacedamaged or diseased craniofacial tis-sues and organs is an exciting newarea that links basic animal researchwith human genetics and providesgreat promise in our effort to reducethe pain and suffering associated withcraniofacial malformations.

In this review, we highlight somerecent advances in our understandingof evo–devo (evolutionary–develop-ment) as it relates to craniofacial de-velopment, the fate determination ofcranial neural crest cells, craniofacialpatterning and organogenesis. Wealso review new discoveries on the de-velopment of craniofacial bones, sig-naling interactions, and specificity incraniofacial morphogenesis. Finally,we will discuss how research advance-ments will be beneficial for the under-standing, treatment, and preventionof human congenital malformationsand prospectus of tissue engineering.

EVOLUTION

Perhaps no other anatomical featuremore closely epitomizes vertebratesthan the head. Comprising paired sen-sory elements, a muscularized masti-catory apparatus, and a cartilaginousor bony braincase, the head developsfrom complex and massive movementsof mesenchymal cells derived frommesoderm and neural crest. Thesecells interact with each other and witha variety of craniofacial epithelia toproduce the intricate structures of the

face, skull, teeth, and jaw. How thistrajectory of inductive interactions oc-curs in molecular terms is a key ques-tion in developmental biology; how ithas been modified during evolution toproduce the stunning variety ofcraniofacial structures in vertebratesis of major interest in evolutionary bi-ology.

We begin our review with a briefconsideration of some current issuesconcerning the evolution of the head(for comprehensive treatments, seeSantagati and Rijli, 2003; Kuratani,2004, 2005; Northcutt, 2005; Morriss-Kay and Wilkie, 2005; Depew et al.,2005). We focus on recent findingsthat pertain to two broad questions:First, how did the head evolve in thechordate ancestor of vertebrates? Sec-ond, how, over shorter evolutionaryperiods, did modifications of the headdevelopmental program produce theevolutionary novelties that made pos-sible the huge array of craniofacialmorphologies evident in vertebrates?

Although the details of how thehead first evolved are as elusive asfossils of the early chordates that oncelived in pre-Cambrian seas, there isbroad agreement with a scenario putforward by Northcutt and Gans (1983)and modified recently by Northcutt(2005). Their New Head hypothesispostulates that the ancestral verte-brate was an animal similar to themodern day cephalochordate Am-phioxus. Like Amphioxus, this animalwas a filter feeder that lacked pharyn-geal arch muscles to move waterthrough the gills. Also lacking was abraincase and the structures charac-teristic of the rostral head of verte-brates, including olfactory bulbs andtelencephalic vesicles.

According to the New Head hypoth-esis, this stem organism underwentan evolutionary transition from filterfeeding to active predation (Fig. 1).Underlying this transition were sev-eral key innovations, including the de-velopment of muscularized jaws andgill arches, a nerve plexus that en-abled the animal to detect and captureprey, and cartilaginous and skeletalelements that provided a fixed spatialorganization for this plexus. In princi-ple, these new structures could be pro-duced in two different ways: the ante-rior portion of the trunk could berestructured, or a section could be

2354 CHAI AND MAXSON

added onto the trunk de novo. Thelatter possibility—that the head is aneomorph—is a central proposal ofthe New Head hypothesis. Also key tothis hypothesis is the idea that the celltype most critical for the evolution ofthe new head was the neural crest.

The neural crest is induced by an in-teraction between cells of the neuralplate and adjacent surface ectoderm(reviewed by Meulemans and Bronner-Fraser, 2004). Nascent neural crestcells delaminate from the developingneural tube, take on a mesenchymalcharacter and migrate to distant sitesin the developing embryo. In the cranio-facial region, they give rise to a widevariety of cell types and tissues, includ-ing intramembranous bone, cartilage,muscle, and nerves (Creuzet et al.,2005; Noden and Trainor, 2005). North-cutt and Gans (1983) proposed that theneural crest emerged in an early verte-brate ancestor and served as a keysource of evolutionary novelty thatmade the New Head possible. Theybased this view on the apparent unique-ness of neural crest to vertebrates andon the ability of neural crest both tocontribute to structures that form thenew head as well as to serve as a sourceof patterning information (Noden, 1978;Couly et al., 1993; Jiang et al., 2002;Schneider and Helms, 2003).

That the fate of the neural crestincludes elements of the rostral mostportion of the new head and that theneural crest has a key role in pat-terning these tissues are clear(Couly et al., 1993; Jiang et al., 2002;Gross and Hanken, 2005; Evans andNoden, 2006). What is less clear iswhether the neural crest is unique tovertebrates. Efforts to identify neu-ral crest-like cell populations in ex-tant nonvertebrate chordates haveproduced mixed results. Yu et al.(2002) found that amphioxus hascells that express an amphioxus ho-mologue of the crest marker FoxD3.AmphiFoxD is expressed in the an-terior neural plate but not in cellsbordering the neural plate, as is thecase for FoxD3 in vertebrates. Am-phiFoxD is also expressed in axialand paraxial mesoderm. Yu and co-workers speculate that an amphi-FoxD congener in a common ances-tor of vertebrates and amphioxushad a role in the development of themesoderm, and subsequently, during

early vertebrate evolution took on afunction in the early neural crest.

On the other hand, recent workfrom Jeffery’s group has shown that aurochordate (ascidian) possesses cellsthat exhibit neural crest-like behavior(Jeffery et al., 2004). These cells mi-grate from the neural tube and ex-press the neural crest markers hnkand Zic1. Ultimately they give rise topigment cells, leading Jeffery and co-workers to propose that the neuralcrest first arose as pigment cell pre-cursors in a common ancestor of ver-tebrates and ascidians, and later ac-quired additional fates.

Whether such neural crest-like cellsexist in other nonvertebrate chordategroups remains to be determined. Re-sults to date show that, although cellswith neural crest-like properties arepresent in some nonvertebrates, cellspossessing the full spectrum of neuralcrest behavior are unique to verte-brates. Thus, the idea that the neuralcrest is a major source of New Headstructures remains intact (Northcutt,2005).

Evolutionary Novelty in theCraniofacial Complex

The first vertebrates exhibiting a“New Head” were probably similar tomodern day jawless fishes (ag-nathans; Fig. 1). These organisms,which include lampreys and hagfish,are similar to higher vertebrates intheir embryology, but lack jaws, pos-sessing instead a filter-feeding appa-ratus (Kuratani et al., 2001). A majorquestion is what were the sources ofevolutionary novelty that made possi-ble changes that led to more complexcraniofacial morphologies of later ver-tebrate groups? One approach to thisproblem has been to carry out compar-ative embryological studies on thelamprey.

Kuratani and colleagues have ex-amined marker gene expression in theJapanese lamprey, comparing expres-sion patterns with those seen in gna-thostomes (Shigetani et al., 2002). Agenerally accepted view of jaw evolu-tion is that the mandibular arch, themost rostral of the pharyngeal arches,was modified to produce the mandibleand maxilla. In ancient vertebrates,the arches were morphologically sim-ilar. The lamprey lacks a jaw but has

upper and lower lips with distinctmorphologies. These authors exam-ined the expression of Dlx and Msxhomeobox genes, which, in amniotes,are expressed in neural crest-derivedmesenchymal cells along the futureproximal–distal axis of the mandibu-lar arch. Additional markers with re-gion-specific expression included Fgf8and Bmp4.

The expression patterns of thesegenes in lamprey embryos appearedat first to support the view that lam-prey larval lips and gnathostome jawsare homologous. However, DiI label-ing of neural crest populations sug-gested a more complex picture. Neuralcrest cells contributing to the upperand lower lips are derived from fore-brain and midbrain crest populations,unlike the situation in gnathostomesin which mandibular arch crest de-rives from more posterior populations.The lamprey Dlx1 homologue is ex-pressed in different regions of the neu-ral crest compared with gnathos-tomes. This shift in expression iscorrelated with a shift in expression ofFgf8, an upstream regulator of Dlx1 inboth lamprey and gnathostomes, inthe epidermis. This shift is seen as thecause of a corresponding shift in thefate of subpopulations of neural crest.

An evolutionary change such as thisone in a jawless gnathostome ancestorcould have led to the emergence of thejaw. If this scenario is correct, thenthe gnathostome jaw is an evolution-ary innovation resulting from achange in the topographic location ofan epithelial–mesenchymal interac-tion. This change resulted in a shift infate of neural crest cells and, thus, tothe innovation that became the verte-brate jaw.

Recent work from the Tabin andChuong labs has identified potentialsources of evolutionary novelty inthe epithelial–mesenchymal interac-tions that pattern the avian beak(Abzhanov et al., 2004; Wu et al.,2004). This work suggests that evo-lutionary changes in beak morphol-ogy in birds may be caused by simi-lar shifts in epithelial–mesenchymalsignaling. Darwin’s finches, in thecourse of their radiation into variousecological niches in the Galapagos,evolved diverse beak morphologies.By examining the expression of var-ious growth factors among Galapa-

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gos finches, Tabin’s group found thatthe expression of Bmp4 in the mes-enchyme of the upper beak wasclosely associated with a particularmorphology, a broad shape (Abzh-anov et al., 2004). By misexpressingbone morphogenetic protein-4

Fig. 1. The New Head and the evolution of the jaw. Schematics of the oral region of Amphioxus, alamprey (jawless vertebrate), and a gnathostome (jawed vertebrate) are shown with their phylogeneticrelationships (redrawn from Shigetani et al., 2002, and Holland et al., 2004). As proposed by Shigetaniet al. (2002), the neural crest-derived mesenchyme of lampreys and gnathostomes is subdivided intopostoptic (po) and mandibular arch (ma) subdomains. Growth factors are secreted by the epidermis (redline). In the lamprey, these induce homeobox genes (brown and blue) over the entire length of the neuralcrest-derived mesenchyme, whereas in the gnathostome, such signals are effective only in the man-dibular component. Thus, it is proposed that a shift in epithelial–mesenchymal interactions results in theevolutionary emergence of the jaw. Ulp, upper lip; llp, lower lip; prc, prechordal cranium.

Fig. 2. Neural crest boundary relationships inskull vaults of mouse, chicken, and frog. Skullsare shown in the dorsal aspect. In the mouse,the coronal suture (CS) marks a neural crest–mesoderm boundary (blue line; neural crest an-terior) (Jiang et al., 2002). Note also that thefrontal (F) and interparietal (IP) bones are neuralcrest-derived (blue). In the chicken, the neuralcrest–mesoderm boundary lies within the fron-tal bone (F) and does not coincide with a suture(Noden, 1975; Le Lievre, 1978; Noden andTrainor, 2005; Evans and Noden, 2006). Thesolid blue frontoparietal (FP) bone of the frog,Xenopus, indicates that neural crest contributesover its entire length (Gross and Hanken, 2005).The extent to which mesoderm contributes tothis bone is not known. See Figure 3 for adetailed depiction of neural crest- and meso-derm-derived bones of the skull vault. P, pari-etal bone.

Fig. 3. Contribution of ectoderm, mesoderm, and endoderm during craniofacial development.A: Neural crest cells are formed at the junction of neural and surface ectoderm. These cells undergoepithelial–mesenchymal transformation, become ectomesenchyme, and travel into multiple destina-tions. B: Side view of an E9.5 mouse embryo shows unsegmented paraxial mesoderm in the head andmesoderm-derived somites in the trunk. OP, optic vesicle; OV, otic vesicle. C: Transverse section of thedeveloping first branchial arch that is covered by surface ectoderm. The core of the first arch containscranial neural crest (CNC) -derived (blue) and paraxial mesoderm-derived (pink) cells. The pharyngealendoderm (yellow) lines the inner aspect of the branchial arch. D: Schematic drawing of an adult mouseskull shows both the CNC- and paraxial mesoderm-derived elements (modified from Noden andTrainor, 2005). Mesoderm-derived cells are in pink, and CNC-derived cells are in blue.


(Bmp4) in chick embryos, a similarbroad shape could be produced.

The Chuong group (Wu et al.,2004) addressed a related questionby examining the basis of morpho-logical differences between theshapes of chicken and duck beaks.Ducks have two growth zones in thefrontonasal mass, whereas chickenshave only one. This difference ac-counts for the difference in beakmorphology between these two birds.Bmp4 is expressed in the growthzones. By manipulating the locationof Bmp4 expression, Chuong’s groupwas able to modulate the shape ofthe beak. Together, these studies onbeak development demonstrate thatsubtle changes in the topography ofBmp signaling within the developingbeak can account for dramatic evolu-tionary shifts in beak morphology.

Schneider and Helms (2003) ad-dressed the role of the neural crest inbeak patterning and the evolution ofbeak shape. These authors exchangedneural crest cells fated to contributedto the beak between quail and chicks,which exhibit distinct beak morpholo-gies. They sought to test the tripartitehypothesis that neural crest cells con-tain patterning information for beakmorphology, that such cells possess anautonomous program by which theyexpress this information, and thatsuch cells influence the fates of non-neural crest cells.

Reciprocal transplantation experi-ments confirmed each of these points.Thus, for example, quail neural crestcells, when transplanted into duckembryos gave rise to beaks like thosefound in quail. That donor neuralcrest cells pattern host structures wasshown by an examination of egg toothmorphology. Duck and quail egg teeth,which are derived from epidermis, notneural crest, exhibit distinct morphol-ogies. The results showed that the mor-phology was characteristic of the trans-planted tissue. Transplanted neuralcrest cells express molecular markerscharacteristic of the donor species, con-firming the idea that such cells expressan autonomous molecular program. Itis not hard to envisage that evolution-ary shifts in neural crest populationscould produce profound morphologicalchanges.

Tissue Boundaries inCranial Evolution

The finding that the neural crest canprovide patterning information sug-gests that evolutionary shifts in neu-ral crest–non-neural crest boundariescould be an additional source of nov-elty (Fig. 2). Recent studies have ad-dressed the significance of boundariesin the development and evolution ofthe skull vault (Jiang et al., 2002;Merrill et al., 2006; Evans and Noden,2006).

As discussed above, the skull vaultdevelops from populations of mesen-chymal cells with distinct embryolog-ical origins, neural crest, and headmesoderm. The extent of the contribu-tion of neural crest to calvarial bonesof birds has been controversial. Sev-eral studies using quail–chick chime-ras or retroviral infection to producefate maps (Noden, 1975; Le Lievre,1978; Evans and Noden, 2006) sug-gest that the neural crest–non-neuralcrest boundary lies within the frontalbone (reviewed in Noden and Trainor,2005). The frontal bones develop afterthe fusion of two intramembranouscenters (Jollie, 1981; discussed in No-den and Trainor, 2005). The more ros-tral of these is of neural crest origin,the caudal of mesoderm origin. Thus,the boundary lies at the interface ofthese two ossification centers. Laterstudies, however, have suggested thatthe entire chick cranial vault, includ-ing the frontal and parietal bones, is ofneural crest origin (Couly et al., 1993).

Similarly, in Xenopus, dye-markingexperiments have shown that crestcontributes to the full length of thefrontoparietal bone, the major bone ofthe skull vault in anuran amphibians(Gross and Hanken, 2005). In mouse,in contrast, the frontal bone appearsto be derived entirely from neuralcrest, whereas the parietal bone is de-rived from mesoderm (Jiang et al.,2002; Ishii et al., 2003). Thus, in themouse, the interface between the fron-tal and parietal bones—the coronalsuture—is a boundary between neuralcrest and mesoderm (Merrill et al.,2006).

If we make the assumption that theboundary in the chick is within thefrontal bone (Noden, 1975; Le Lievre,1978; Evans and Noden, 2006), then,as pointed out by Noden and Trainor

(2005), the situation may not be sub-stantially different from the mouse.On the other hand, if, in the chicken,crest indeed contributes to the entireskull vault, as it apparently does inXenopus, then there may have been anevolutionary shift in the neural crest–mesoderm boundary in the ancestor ofbirds and mammals (Gross and Han-ken, 2005).

It is also possible that, as pointedout by Gross and Hanken (2005), am-phibians, birds, and mammals mayeach exhibit distinct contributions ofneural crest to the skull vault. Wenote as well that there remains someuncertainty about the homology rela-tionships between the frontal, pari-etal, and frontoparietal bones of thesethree taxa—an uncertainty that clearlycomplicates the interpretation of neuralcrest labeling studies.

In the mouse, the coronal suture islocated at the neural crest–mesoderminterface and is a major growth centerof the skull vault. Boundaries betweendissimilar cell populations often serveas important signaling centers thatfunction in growth control (reviewedin Dahmann and Basler, 1999). Thelocation of the tissue boundary at thecoronal suture in mammals might rep-resent an evolutionary innovationthat makes possible, for example, newmodes of mastication, locomotion, orcranial growth. Further analysis ofcontributions of neural crest to theskull vault in extant vertebrates willbe required to illuminate this issue.

FATE DETERMINATION OFCRANIAL NEURAL CRESTCELLS

The vertebrate neural crest is a pluri-potent cell population derived fromthe lateral ridges of the neural plateduring early stages of embryogenesis(Fig. 3). The functions of neural crestcells include coordination of variousvisceral activities, such as in the pe-ripheral nervous system; protection ofthe body from external conditions,such as by melanocytes; and partici-pation in craniofacial skeletal devel-opment (Le Douarin et al., 2004).

The formation of the neural crest isa classic example of embryonic induc-tion, in which tissue–tissue interac-tions and the concerted action of sig-naling pathways are critical for the


induction of neural crest precursorcells. After induction, neural crestcells disperse from the dorsal surfaceof the neural tube, undergo epithelial–mesenchymal transformation, and mi-grate extensively through the embryo,giving rise to a wide variety of differ-entiated cell types. The migration,proliferation, and differentiation ofneural crest cells along multiple dis-tinctive pathways have been studiedextensively in various animal models(Bronner-Fraser, 1993; LaBonne andBronner-Fraser, 1999; Le Douarin etal., 2004; Basch et al., 2004). Recentstudies suggest that fate determina-tion of the neural crest is strongly in-fluenced by environmental cues. CNCcells interact with and are consequentlyinstructed by pharyngeal endoderm, ec-toderm, and mesoderm before givingrise to various types of tissues in thecraniofacial region.

During craniofacial development,neural crest cells migrate ventrolater-ally as they populate the craniofacialregion. The proliferative activity ofthese crest cells produces the fronto-nasal process and the discrete swell-ings that demarcate each branchialarch. As these ectodermally derivedcells migrate, they contribute exten-sively to the formation of mesenchy-mal structures in the head and neck.Cell labeling studies have demon-strated that neural crest cells arisingfrom rhombomeres 1–3 (r1–3) of ante-rior hindbrain migrate into the firstbranchial arch and, thereafter, residewithin the maxillary and mandibularprominences (Osumi-Yamashita etal., 1990; Serbedzija et al., 1992; Bron-ner-Fraser, 1993; Selleck et al., 1993;Lumsden and Krumlauf, 1996). Themigration of these rhombencephaliccrest cells is regulated by growth fac-tor signaling pathways and theirdownstream transcription factors be-fore the CNC cells become committedto several different tissue types suchas bone, cartilage, tooth, and cranialnerve ganglia (Noden, 1983, 1991;Lumsden, 1988; Graham and Lums-den, 1993; Le Douarin et al., 1993;Echelard et al., 1994; Imai et al., 1996;Trainor and Krumlauf, 2000). Recentstudies provide strong supportive evi-dence that the CNC cells are develop-mentally “plastic,” i.e., their fate is notpredetermined before they reach theirfinal destination; rather, these pro-

genitor cells must be instructed by sig-nals from other tissues to generate skel-etal elements of appropriate shape andsize in the craniofacial region. Tissuesthat provide the instructive signalingfor CNC fate specification include, butare not limited to, the pharyngealendoderm, the branchial arch ectoderm,and the isthmic organizer at the mid-brain–hindbrain boundary (Baker andBronner-Fraser, 2001; Trainor et al.,2002; Couly et al., 2002; Le Douarin etal., 2004).

The Pharyngeal Endoderm

Most of our knowledge about thecritical function of pharyngealendoderm in regulating the fate ofCNC cells and the patterning of mid-dle and lower face derives from stud-ies using chick, quail, or zebrafishembryos as models. These embryosallow relatively easy manipulationcompared with the mouse model.Surgical removal of pharyngealendoderm during early stages ofchick embryogenesis resulted in de-fects in facial bone and cartilage devel-opment (Couly et al., 2002). During thedevelopment of the first branchial arch,pharyngeal endoderm is thought toprepattern the orofacial epithelium,which in turn will provide instructivesignals to pattern the CNC-derivedmesenchyme (Haworth et al., 2004).In zebrafish studies, fibroblast growthfactor (FGF) signaling has been shownto be critical for the development ofpharyngeal endoderm itself and forthe mediation of the endoderm to reg-ulate facial skeletal morphogenesis(Ruhin et al., 2003; Crump et al.,2004; Helms et al., 2005). On the otherhand, a recent study has shown thatpharyngeal endoderm is not criticalfor the normal development of the up-per and lower face. Instead, it is theectoderm that is critical for providingthe instructive information for facialmorphogenesis (Aoki et al., 2002).Furthermore, CNC cells also containintrinsic information that can affectfacial development, although this con-tribution may depend upon the collec-tive number of neural crest cellspresent at a given time and position(Schneider and Helms, 2003; Tuckerand Lumsden, 2004). Overall, there isoverwhelming evidence to support thenotion that the pharyngeal endoderm

has a critical role in regulating thefate of CNC. Discovery of the criticalsignaling pathway involved in thisregulatory process awaits the develop-ment of mouse pharyngeal endoderm-specific gene inactivation models.

Orofacial Ectoderm

Early patterning of the oral ectoderm isindependent of the neural crest (Veitchet al., 1999). In the mouse model, thebranchial arch epithelium is correctlypatterned, despite the CNC migrationdefect (Gavalas et al., 2001). After theinteraction with pharyngeal endoderm,the orofacial ectoderm is roughly di-vided into proximal and distal domains.The establishment of the ectodermaldomain greatly influences the fate de-termination of migrating CNC cells asthey populate the branchial arch. Atlater stages of embryonic develop-ment, CNC cells interact with andprovide instructive signals to the over-lying epithelium and have an effect onepithelial patterning (Miletich andSharpe, 2004). Tooth development is aclear example of the consistent shift ofinstructive signal between orofacialectoderm and the CNC-derived mes-enchyme (see below for detailed dis-cussion).

Concerted Action of Growthand Transcription Factors inDetermining the Fate,Expansion, and Survival ofCNC Cells

Recent studies have addressed the in-fluence of growth factors on the fate ofmultipotent progenitor cells, such asthe neural crest, during embryogene-sis. It turns out that signaling centers,such as the isthmic organizer, rely ongrowth factor signaling in regulatingthe fate of CNC cells. Specifically,FGF8 signaling from the isthmic orga-nizer can alter Hoxa2 expression andconsequently control branchial archpatterning, demonstrating that neu-ral crest cells are patterned by envi-ronmental signals (Trainor et al.,2002). Several growth factor signalingpathways have been shown to be crit-ical for CNC fate determination, ofwhich transforming growth factor-be-


ta/BMP (TGF-�/BMP) signaling is aclassic example. Members of theTGF-� superfamily of growth factorsare expressed at sites where neuralcrest cells commit to form particularcell types. TGF-� superfamily mem-bers promote alternative fates fortrunk neural crest cells; BMP signal-ing promotes neurogenesis by induc-ing MASH1 expression, whereasTGF-� signaling favors smooth mus-cle differentiation (Shah et al., 1996).In contrast, CNC cells react to TGF-�signaling differently. TGF-� controlsthe differentiation of CNC cells to glialcells, and TGF-�, BMP, and Wnt to-gether control chondrocyte differenti-ation. Apparently, one of the majordifferences between CNC and TNC isthe expression of Hox genes, which mayaccount for the differential responsive-ness between CNC and TNC to thesame environmental cues (Abzhanov etal., 2003). Furthermore, the function ofTGF-� in regulating neural crest celldifferentiation is sensitive to the TGF-�expression level, such that TGF-� maypromote alternative cell fates or induceapoptosis (Hagedorn et al., 2000). Dur-ing early mouse craniofacial develop-ment, TGF-� subtypes are present inthe CNC-derived mesenchyme duringcritical epithelial–mesenchymal inter-actions related to the formation of var-ious organs (Lumsden, 1984; Hall et al.,1992; Chai et al., 1994; Lumsden andKrumlauf, 1996; Chai et al., 2003). Tar-geted null mutation of Tgfb2 or haplo-insufficiency of Smad2 results in a widerange of developmental defects, includ-ing craniofacial malformations such assmall mandible, dysmorphic calvaria,and cleft palate (Sanford et al., 1997;Nomura and Li, 1998). Significantly,many affected tissues have neuralcrest-derived components and simulateneural crest deficiencies; thus, TGF-�signaling may provide significant in-structive information to specify the fateof CNC during early craniofacial devel-opment.

Members of the TGF-� superfamilyregulate the expression of transcriptionfactors to influence cell fate decisionsinstructively during embryogenesis(Shah et al., 1996; Dorsky et al., 2000).For example, the expression patterns ofTgfb2 and transcription factor Msx1have significant overlaps during earlytooth and palate development whenCNC-derived cells become specified to

form dental and palatal mesenchyme,respectively, suggesting an epistatic re-lationship between these two genes(Ferguson, 1994; Ito et al., 2003). In thepalatal mesenchyme, overexpression ofTGF-� suppresses transcriptional activ-ity of the Msx1 gene (Nugent andGreene, 1998). We have shown recentlythat compromised TGF-� signaling af-fects the expression of Msx1, which inturn controls the progression of theCNC cell cycle by regulating cyclin D1expression (Ito et al., 2003). In parallel,in vitro studies also suggest that Msx1gene expression maintains cyclin D1 ex-pression and holds cells in an undiffer-entiated state by promoting prolifera-tion (Hu et al., 2001). In general, TGF-�signaling specificity in regulating down-stream target gene expression is deter-mined by the interaction with other fac-tors (such as BMP, Wnt, FGF, and soon) in a temporal- and spatial-specificmanner (Massague, 2000). The close in-tertwining of TGF-� signaling withother pathways appears to be a criticalcomponent of specific cell fate determi-nation mediated by TGF-� family mem-bers.

In addition to regulating the fate ofCNC cells, the concerted action ofgrowth and transcription factors alsocontrols cell proliferation and death.For example, BMP signaling controlsMsx gene expression by directly regu-lating Msx2 promoter activity (Brug-ger et al., 2004). There is also an ap-parent feedback for BMP signaling bythe Msx genes, because loss of Msx1and Msx2 results in altered Bmp4 ex-pression in the CNC cells (Ishii et al.,2005). Msx1 and Msx2 function redun-dantly in regulating the survival andproliferation of CNC cells duringcraniofacial development. This regu-lation is most likely carried outthrough the control of cell cycle pro-gression (Hu et al., 2001; Han et al.,2003, Ishii et al., 2005). Recently,studies in Xenopus have shown that aMyc-mediated Id3 expression level iscritical for determining whether theneural crest cells will proliferate ordie, thus determining the size of theneural crest population (Kee andBronner-Fraser, 2005; Light et al.,2005). It is currently unknownwhether Id3 plays a similar role dur-ing mouse development.

Finally, it is important to addressthe issue of prepatterning of premi-

gratory neural crest cells. Noden’s ex-periment suggested that chick CNCcells are predetermined according totheir rostrocaudal origin in the neuraltube (Noden, 1983). However, recentwork has demonstrated that adjacenttissues, such as the isthmus, provideinstructive signals to regulate down-stream target genes to determine thefate of CNC cells (Trainor et al., 2002).Furthermore, by providing criticalfeedback to the surface ectoderm,CNC can provide species-specific pat-terning information during craniofa-cial development, highlighting the im-portance of tissue–tissue interactionin regulating organogenesis (Schnei-der and Helms, 2003; Helms et al.,2005). In the mouse model, it has beendemonstrated elegantly that CNCcells retain a remarkable degree ofplasticity, even after their migrationinto the branchial arch (Zhao et al.,2006). The second arch CNC cellshave a cell-autonomous requirementfor the Hoxa2 gene for their intrinsicpatterning program (Santagati et al.,2005). In vitro studies have also begunto address the plasticity of postmigra-tory CNC cells (Zhao et al., 2006).Taken together, it is clear that thefate of mouse postmigratory CNC cellsis determined through the reciprocalsignaling between neural crest mesen-chyme and the surrounding environ-ment, where timing is an essentialcomponent.

CRANIOFACIALPATTERNING AND TISSUE–TISSUE INTERACTION

The principal goal of developmental bi-ology is to understand how tissues areinduced and patterned to generate dif-ferent organs at the correct location andtime. Evidence suggests that signalsfrom the anterior visceral endoderm,anterior neural ridge, and the meso-derm are required for the developmentof head structures (Spemann, 1938;Shimamura and Rubenstein, 1997;Beddington and Robertson, 1999; De-pew et al., 2002a). Furthermore, the for-mation of vertebrate “New Head” de-pends upon the presence of CNC cellsand additional sensory placodes. Multi-ple molecules have been identified ascritical regulators at these signalingcenters. However, the challenge re-mains to determine how various signal-


ing centers coordinate with each otherand build complicated structures thatmake the head.

In the facial region, each branchialarch contains a central blood vessel,the aortic arch, which is surroundedby cells of paraxial mesoderm. Themesoderm core is enveloped by themore peripherally located CNC cells.The inner surface of the branchialarch contains cells derived from thepharyngeal endoderm, while the outersurface is covered by the ectoderm(Fig. 3). The spatial relationship of theboundary of cells with different em-bryonic origin is crucial for normal or-ganogenesis and may reflect the regu-latory interactions that control thedevelopment of complex structures inthe craniofacial region.

Ectoderm

Craniofacial ectoderm plays a criticalrole in regulating the fate of CNC cellsduring craniofacial morphogenesis,whereas the establishment of ecto-derm identity is independent of CNCcells. Later, the continuous and recip-rocal interaction between the ecto-derm and the CNC-derived ectomes-enchyme controls the position, size,and shape of craniofacial organs dur-ing embryogenesis. It is imperative toappreciate the inductive capability ofectoderm and CNC cells in the contextof time. This concept is best illus-trated by the seemingly contradictoryconclusions regarding the ability ofthe oral ectoderm or the CNC-derivedectomesenchyme to induce tooth mor-phogenesis.

Tissue recombination experimentsshow that the tooth inductive signalfirst resides in the oral ectoderm andthen shifts into the underlying CNC-derived ectomesenchyme at a later de-velopmental stage. In mouse, dentalepithelium before embryonic day (E)12 is capable of inducing tooth forma-tion when combined with nondentalmesenchyme, whereas dental mesen-chyme after E12 can induce tooth for-mation when combined with nonden-tal epithelium (such as the secondbranchial arch epithelium; Mina andKollar, 1987).

Growth and transcription factorsare responsible for establishing thepatterning of craniofacial develop-ment. In the first branchial arch oral

ectoderm, FGF8 and BMP4 are criti-cal for setting up the proximal–distalaxis during development. In the upperand middle face, where structures de-rive from the frontonasal process,Sonic hedgehog (Shh) and FGF signal-ing appear to be critical for setting upa boundary in the neural and surfaceectoderm (Helms et al., 2005). Incraniofacial patterning, definitive evi-dence only supports the critical func-tion of ectodermal FGF signaling, asconditional inactivation of the Fgf8signaling resulted in the disappear-ance of the proximal portion of themandible (Trumpp et al., 1999). Morerecently, studies have shown that sig-naling through FGFR1 is critical forthe neural crest independent pattern-ing of the pharyngeal ectoderm. Ecto-derm FGF signaling patterns the pha-ryngeal region to create a permissiveenvironment for the entry of CNCcells (Trokovic et al., 2003, 2005).

Craniofacial ectoderm regulates theexpression of transcription factors tospecify the fate of CNC cells. For ex-ample, FGF8 signaling controls theexpression of two Lim-homeobox do-main genes, Lhx6 and Lhx7, in theCNC-derived ectomesenchyme. Lhx6and Lhx7 are mainly expressed in theoral side of ectomesenchyme, whileGsc is expressed in the aboral region(Tucker et al., 1999). Endothelin-1 inthe mandibular epithelium controls theexpression of Gsc. Mutations in eitherEndothelin-1 or Gsc result in mandibu-lar development defects, demonstratingthe endothelin-mediated Gsc expres-sion is critical for controlling the pat-terning of mandible (Clouthier et al.,1998). In addition, BMP signaling hasbeen shown to be a critical regulator forMsx1 function, which is exclusively ex-pressed in the CNC-derived ectomesen-chyme (Chen et al., 1996; Tucker et al.,1998a). Conditional inactivation ofBmpr1a in the oral ectoderm results intooth agenesis (Andl et al., 2004). Inhi-bition of BMP signaling by noggincauses ectopic Barx-1 expression in thedistal, presumptive incisor ectomesen-chyme and a transformation of toothidentity from incisor to molar, thusdemonstrating the significance of BMPsignaling in patterning of craniofacialstructures (Tucker et al., 1998b). Takentogether, it is clear that ectoderm-mediated signaling plays an importantrole in determining the patterning of

craniofacial structures. At this point,one cannot help but ask the question ofwhat is responsible for setting up thesignaling domain in the ectoderm. Itturns out that pharyngeal endoderm iscritical for this process.

Pharyngeal Endoderm

The pharyngeal endoderm makes alimited contribution to craniofacialdevelopment, but it serves as an in-dispensable inducer during tissue–tissue interactions, controllingcraniofacial development (Fig. 3).The traditional view that the neuralcrest plays the key role in patterningthe branchial arches must be recon-sidered, because studies have foundthat the early ablation of CNC cellsdid not affect the development andfunction of endoderm cells. Thesecrestless branchial arches were pat-terned normally and had a sense ofindividual identity, strongly sug-gesting that the patterning ofendoderm is not dependent on CNCcells (Veitch et al., 1999). Hox genesare known to be critical for the iden-tity of neural crest cells. Hoxa1 andHoxb1 double mutant mice had pat-terning defects in the hindbrain;specifically, rhombomere 4 lost itsability to generate neural crest cells.Consequently, the second branchialarch crest population was not gener-ated. Despite the neural crest defect,the formation of the second branchialarch endoderm and epithelial region-alization was normal (Gavalas et al.,2001).

Limited information is available re-garding the patterning of pharyngealendoderm. Retinoic acid signalingclearly plays a crucial role in regulatingpharyngeal endoderm development asinactivation of retinaldehyde-specificdehydrogenase type 2 (Raldh2), the reti-noic acid synthetic enzyme, resulted inthe absence of all branchial arches cau-dal to the first (Niederreither et al.,1999). Inhibition of retinoid action bypan-retinoic acid receptor (pan-RAR)specifically perturbed the developmentof the third and fourth branchialarches, although the first and secondbranchial arches formed normally(Wendling et al., 2000). Recent studieshave shown that Raldh2 is expressed inthe lateral mesoderm flanking theendoderm during early embryonic de-


velopment and may pattern the pha-ryngeal endoderm development(Niederreither et al., 2003; Grahamet al., 2005). Tbx-1 is another impor-tant gene for pharyngeal endodermdevelopment. Tbx-1 mutant miceshow failure to form caudal pouches,whereas the first pharyngeal pouchdevelops normally (Jerome and Pa-paioannou, 2001; Lindsay et al.,2001). Of interest, Tbx-1 is also ex-pressed in the lateral mesodermflanking the endoderm before thepatterning of the pharyngealendoderm (Brown et al., 2004).

The pharyngeal endoderm exerts itsregulatory function through tissue–tis-sue interactions. For example, the pha-ryngeal pouches generate several spe-cialized epithelial structures, such asthe thyroid, parathyroid, and thymus(Graham et al., 2005). The developmentof these organs involves the interactionbetween pharyngeal endoderm and itsflanking cranial neural crest cells. Com-promised retinoic acid signaling fromthe pharyngeal mesoderm affects thedevelopment of pharyngeal endoderm,which in turn causes defects in CNCmigration and development of pharyn-geal pouch-derived organs, such as thethymus and parathyroid glands (Nied-erreither et al., 2003). Clearly, the pha-ryngeal endoderm plays an importantrole in regulating the morphogenesis ofpharyngeal arch derivatives.

Mesoderm

The cranial paraxial mesoderm has anorganization different than the trunkparaxial mesoderm. The cranial parax-ial mesoderm can be roughly dividedinto (1) preotic head mesoderm, whichlacks any overt sign of segmentationand never forms somites; and (2) occip-ital somites, which are caudal to the oticvesicle and give rise to epaxial and hy-paxial muscles of the neck, the pharyn-geal and laryngeal muscles that developin the caudal branchial arches, and thetongue muscle (Fig. 3B; Noden, 1983;Couly et al., 1992; Huang et al., 1999;Mootoosamy and Dietrich, 2002).

There are profound morphologicaland molecular differences between thecranial and trunk paraxial mesoderm.In comparison to the clearly seg-mented somite development withinthe trunk mesoderm, studies havesuggested that head mesoderm has

become arrested evolutionarily assomitomeres (Jacobson, 1963; Pack-ard and Meier, 1983). Despite severalgene expression analyses suggestingsome degree of regionalization in thecranial paraxial mesoderm (such asParaxis, Tbx1, and Hoxb-1), there isno evidence supporting the existenceof metameric patterns. The impor-tance of somitomeric head mesodermis yet to be determined (Noden andTrainor, 2005). At the molecular level,genes that drive mesoderm segmenta-tion in the trunk are missing from thepre-otic mesoderm in the head. Whencranial paraxial mesoderm wasgrafted to the trunk region, activationof the myogenic program in this ec-topic position was inhibited, suggest-ing that there are distinct regulatorycascades acting in the development oftrunk and head muscles, possibly re-flecting their distinct function andevolution (Mootoosamy and Dietrich,2002). Of interest, however, hetero-topic grafting of paraxial mesodermcells to different regions of the cranialmesoderm in the mouse showed no re-striction in cell potency in the cranio-caudal axis, revealing considerableplasticity in the fate of the cranial me-soderm (Trainor et al., 1994). Signalsfrom surface ectoderm and endodermas well as CNC may influence the fateof mesoderm cells (Trokovic et al.,2003).

In terms of patterning capability,cranial paraxial mesoderm provides apermissive substratum for the migrat-ing CNC cells to populate thebranchial arch. Studies using chickembryos suggest that cranial paraxialmesoderm is able to direct CNC cellmovement independently of their epi-thelial or mesenchymal organization(Noden, 1986; Ferguson and Graham,2004). Of interest, recent cell fatemapping analysis has suggested thatmyoblast precursors contain posi-tional identity inherited from their so-matic mesenchymal stem cell precur-sors and can help to determineskeletal homologies that are based onmuscle attachments (Matsuoka et al.,2005). Conversely, CNC cells, whichprovide most of the connective tissuesand tendons in the head, may patternand shape the individual cranial mus-cle (Noden, 1986; Kontges and Lums-den, 1996). The intimate relationshipbetween cranial paraxial mesoderm

and the CNC is clearly establishedfrom the moment that CNC cells enterthe branchial arch and is maintainedthroughout craniofacial development.In parallel, studies have also demon-strated that cranial mesoderm is ca-pable of providing crucial signals forendoderm development.

To date, there is not a clear demon-stration of the patterning potential ofcranial paraxial mesoderm to regulatecraniofacial development in mam-mals. This finding is largely becauseof (1) lack of information on molecularsignals involved in the interaction be-tween cranial paraxial mesoderm andadjacent tissue and (2) our inability togenerate tissue-specific gene inactiva-tions. Recently, using the Cre/loxp re-combination approach, we have learnedthat Myf5-Cre- or Mesp1-Cre-mediatedrecombination can be used to generate atargeted gene inactivation in the cra-nial paraxial mesoderm-derived firstbranchial arch myogenic core (our un-published data). In Myf5-Cre;R26Rmouse embryos at E9.5, the firstbranchial arch myoblasts are lacZ-positive, accurately reflecting their ori-gin from the paraxial mesoderm (Fig.4). Targeted gene inactivation in thecranial paraxial mesoderm-derivedmesenchyme will reveal important in-formation on its patterning potential inregulating craniofacial development.

Collectively, it is clear that cranio-facial development requires contin-ued interaction and contribution bycell populations derived from the ec-toderm, the neural crest, the parax-ial mesoderm, and the endoderm.These interactions occur en routeand at the terminal site of tissue andorgan genesis. There is no exampleof any craniofacial developmentalevent that can occur in a totally cellautonomous or completely depen-dent manner. Such a dichotomousview can only impede our progresstoward a better understanding of theregulatory mechanism of craniofa-cial development. Instead, we needto focus our efforts to unveil the mo-lecular signals involved in tissue–tissue interactions at each criticaltime point and to gain a better under-standing of tissue boundary establish-ment, because a disturbed tissueboundary during early embryonic de-velopment can lead to craniofacial mal-formations.


SIGNALING INTERACTIONSIN THE PATTERNING ANDMORPHOGENESIS OFCRANIOFACIAL ORGANS

Recent studies have uncovered spe-cific signaling cascades that play cru-cial roles in regulating the patterningand morphogenesis of craniofacial or-gans. One of the best-studied modelsof craniofacial organogenesis is toothdevelopment, which involves contin-uous tissue–tissue interactions be-tween the ectoderm-derived enamelorgan epithelium and the cranialneural crest-derived ectomesen-chyme (Slavkin et al., 1968; Kollar,1972; Lumsden, 1988; Jernvall andThesleff, 2000; Tucker and Sharpe,2004, and references therein). Multi-ple growth and transcription factorsbelonging to several signaling fami-lies have been identified as criticalregulators at the initiation andthroughout all stages of tooth devel-opment (http://bite-it.helsinki.fi).Furthermore, most of the signalingnetworks are used reiterativelythroughout tooth development andare common to the regulatory sys-tems critical for governing the devel-opment of other organs (such as thedevelopment of feather, hair, mam-mary gland, salivary gland, and pan-creas; see reviews by Nuckolls et al.,1999; Shum et al., 2000). The grow-ing scientific evidence suggests ahighly conserved biological mecha-nism to regulate the patterning andmorphogenesis of craniofacial or-gans, but studies are now beginningto reveal the unique features of thesignaling network in regulatingtooth morphogenesis. This topic willbe the focus of our discussion here.

Patterning of theMammalian Dentition

The patterning of dentition dependson the proper development of the oralcavity, where maxillary and mandib-ular teeth are housed and involves thedetermination of location, shape, num-ber, and size of tooth development. Mul-tiple developmental decisions aremade in the patterning and develop-ment of mammalian dentition. Thedevelopment of the oral cavity be-gins with the establishment of thefrontonasal prominence and the firstbranchial arch. The migrating CNC

cells are the major driving forces forbranchial arch development. As thefirst branchial arch extends ventral–medially, it gives rise to both man-dibular and maxillary prominences(although recent study suggests thatthe maxillary prominence has a sep-arate origin from the mandibularprominence in chicken; Lee et al.,2004). The primitive oral cavityforms as a consequence of the fusionamong intermaxillary segments ofthe frontonasal prominence and thepaired maxillary and mandibularprominences. The oral epithelium, incontrast to the aboral (suboral) epi-thelium, becomes thickened to formthe dental lamina, marking the timeand location for tooth developmentto begin.

Before the initiation of tooth devel-opment, the mandibular ectoderm canbe roughly divided into the proximaldomain, which expresses FGF8 andgives rise to molars, and the distaldomain, which expresses BMP4 andgives rise to incisors (Fig. 5). Signifi-cantly, FGF and BMP act antagonis-tically to restrict Barx1 and Dlx2 ex-pression to the proximal domain of thefirst arch ectomesenchyme and Msx1and Msx2 expression to the distal do-main, respectively. The biological sig-nificance of such a regional molecularspecification has been demonstratedelegantly with inhibition of BMP sig-naling resulting in ectopic Barx-1 ex-pression in the distal, presumptive in-cisor mesenchyme and producingtransformation of tooth form from in-cisor-form to molar-form (Tucker etal., 1998b). Recently, study has shownthat Barx1 played a key role in thedevelopment of the dentition and di-gestive system, which was vital for theevolution of mammals (Miletich et al.,2005). Overall, FGF and BMP growthfactor gradients are critical determi-nants for specifying the initiationsites of tooth formations as well as forspecifying the CNC-derived dentalmesenchyme to become incisor-formvs. molar-form tooth organs.

Besides the proximal and distal do-mains within the oral ectoderm, themandibular arch mesenchyme canalso be divided into an oral and aboral(rostral–caudal) axis (Fig. 5). Signalsfrom the oral ectoderm appear to beresponsible for coordinating this divi-sion. Of interest, the postmigratory

CNC cells have an intimate associa-tion with the oral ectoderm (Chai etal., 2000). This CNC distribution pat-tern significantly overlaps with theexpression of two specific Lim-ho-meobox domain genes, Lhx6 andLhx7, within the CNC-derived ecto-mesenchyme, suggesting that theymay have critical functions in direct-ing CNC cells to reach their destina-tion (Grigoriou et al., 1998). In theaboral region, the homeobox geneGoosecoid (Gsc) is expressed whereLhx6 and Lhx7 are excluded (Tuckeret al., 1999). Apparently, FGF8 is re-sponsible for regulating the expres-sion of both Lhx and Gsc genes. Themandibular arch mesenchyme can beroughly divided into the Lhx-positiverostral (oral) domain and the Gsc-pos-itive caudal (aboral) domain (Fig. 5).

Clearly, patterning of the mamma-lian dentition is a three-dimensionalprocess. One aspect that is beginningto be explored is the determination oflingual–buccal (medial–lateral) den-tal cusp patterning (Fig. 5). Lingualand buccal cusps of the molar are dif-ferent (in number and shape), and leftand right first molars, for example,have a mirror image in patterning. Itwill be fascinating to learn how sig-nals are set up to achieve this har-mony during tooth development.

The development of each dentalcusp is under the control of a transientsignaling center known as the enamelknot, which is a dense population ofepithelial cells without any prolifera-tive activity (Fig. 6). The enamel knotcontains multiple signaling molecules(such as Shh, BMP, and FGF) andcontrols the size and shape of cuspformation. Thereafter, it is removedby programmed cell death (Jernvalland Thesleff, 2000; Tucker andSharpe, 2004). Unlike the incisortooth organ, molar development relieson additional enamel knots (second-ary and tertiary) to form multicusppatterning.

BMP signaling is involved in regu-lating the distance between adjacentsecondary enamel knots to control thepositioning of cusps. Two recentlypublished studies have provided im-portant information regarding thecontribution of BMP signaling inhibi-tion to the regulation of cusp pattern-ing. Ectodin is a secreted BMP inhib-itor with an inverse expression


pattern to p21, a hallmark cell cycleregulator expressed in the enamelknot during tooth development (Kas-sai et al., 2005). Loss of ectodin results

in enlargement of the enamel knot,highly altered cusp patterns, fusion ofmolars, and extra teeth. Interestingly,the most dramatic changes in cusp

patterning occurs along the buccalside of the cusps, suggesting that ec-todin functions in the evolution of lat-eral bias in teeth. Noggin is anotherwidely distributed BMP inhibitor (Chenet al., 2004). Overexpression of Nogginin the dental epithelium blocked the de-velopment of all mandibular and max-illary third molars at the bud stage andseverely altered the patterning of max-illary molars (Plikus et al., 2005). Thedifferential defects between mandibu-lar and maxillary molars suggest a re-quirement for varying thresholds ofBMP signaling.

Fig. 4.

Fig. 5.

Fig. 4. Two component genetic system for in-delibly marking the fate of cranial neural crest-(CNC), or mesoderm-, or ectoderm-derivedcells. A: Contribution of CNC cells during earlycraniofacial development. CNC-derived cells(blue) populate the frontonasal process (FN)and the first branchial arch (arrow). B: Cross-section of an embryonic day (E) 9.5 mouse em-bryo (Wnt1-Cre/R26R) shows CNC-derivedcells (blue) in the first branchial arch. The arrowindicates the paraxial mesoderm-derived myo-genic core in the first arch. The arrowhead in-dicates ectoderm. C: Contribution of paraxialmesoderm-derived cells during early craniofa-cial development. The arrow indicates theparaxial mesoderm-derived myogenic core(blue) in the first branchial arch of the Myf5-Cre/R26R sample. D: Cross-section of the first andsecond branchial arch contains paraxial meso-derm-derived myogenic cells (blue; arrow). No-tice these cells are pink in B. E: Contribution ofectodermal cells during craniofacial develop-ment. Ectoderm-derived cells (blue) cover thesurface of the K14-Cre/R26R embryo. The ar-row indicates the first branchial arch surfaceectoderm. F: Cross-section of the first branchialarch shows ectoderm-derived cells (arrow) onthe surface.

Fig. 5. Patterning of the first branchial arch.A: The frontal view of a scanning electronicmicroscopic image of an embryonic day (E) 9.5mouse embryo (25–29 somites) shows the fron-tonasal process (FN), the maxillary (max), andthe mandibular (mand) process. B: Schematicdrawing of A. C: The first branchial arch can bedivided into proximal/distal, oral/aboral, andbuccal/lingual (see D) domains. In the distaldomain, bone morphogenetic protein (BMP)signaling in the ectoderm regulates the expres-sion of Msx1 and Msx2. It also provides inhibi-tion for Barx1 expression in the cranial neuralcrest (CNC) -derived mesenchyme in the prox-imal domain. In the proximal domain, fibroblastgrowth factor (FGF) signaling regulates the ex-pression of multiple transcription factors (mod-ified from Tucker and Sharpe, 2004). D: Molarteeth (m1, m2, m3) show different cusp patternsalong buccal and lingual aspects of the jaw.


Control of Signaling Leveland Tooth Development

Recent studies show that local feed-back provides a tightly controlled FGFand BMP gradient to ensure propertooth development. For example, Is-let1 positively regulates the expres-sion of Bmp4, whereas a range ofPitx2 levels can differentially regulatethe expression of Fgf8 and Bmp4 dur-ing initial tooth development (Lu etal., 1999; Mitsiadis et al., 2003; Liu etal., 2003). Pitx2 is restricted to thedental epithelium throughout toothmorphogenesis (Fig. 6). Mutation ofthe human PITX2 gene results inRieger’s syndrome, an autosomaldominant disorder, that leads to theabsence of certain teeth and defects ofthe eye (Rieger, 1935; Semina et al.,1996). Loss of Pitx2 results in re-tarded tooth development at the initi-ation/early bud stage, clearly demon-strating the important function ofPitx2 gene in regulating tooth devel-opment (Lu et al., 1999). Significantly,loss of one copy of the Pitx2 gene canalso affect tooth development in mice(Gage et al., 1999). Therefore, Pitx2acts in a dosage-specific manner in hu-mans and mice. The expression ofPitx2 gene is sensitive to the level ofBMP4 or FGF8 signaling, indicating afeedback loop among these signalingmolecules (St. Amand et al., 2000).

Shh, another important epithelialsignaling molecule, controls the prolif-eration of enamel organ epithelialcells during initiation of tooth devel-opment (Fig. 6; Bitgood and McMa-hon, 1995; Dassule and McMahon,1998; Hardcastle et al., 1998; Co-bourne et al., 2001). The Wnt signal-ing pathway interacts with Shh signal-ing to establish boundaries during toothdevelopment. Specifically, Wnt-7b actsto repress the expression of Shh in non-dental oral ectoderm, whereas Shh ex-pression is restricted to the dental ecto-derm and can instruct, permit, orinduce tooth bud formations (Sarkar etal., 2000).

Determination of ToothNumber

Humans have a dental formula of3.2.1.2/2.1.2.3 in the permanent den-tition [two incisors, one canine, twopremolars (bicuspids) and three mo-

lars, with “/” marking the front mid-line]. Some rodents, such as mice,have a dental formula of 3.0.0.1/1.0.0.3. The initiation of each toothgerm is marked by the formation ofdental lamina. Ectodysplasin (EDA)signaling cascade molecules, whichbelong to the tumor necrosis factorfamily of ligands, are critical regula-tors for the determination of toothnumber during embryonic develop-ment (Fig. 6). Mice with a compro-mised EDA signaling cascade, suchas Tabby (EDA), Downless (EDAR,EDA receptor), and Crinkled (EDAR-ADD, EDA intracellular adaptorprotein) mutations, show abnormaltooth numbers and defects in the de-velopment of other ectodermal or-gans (hair follicles and exocrineglands; Sofaer, 1969, 1977; Headonet al., 2001; Pispa and Thesleff,2003; Tucker et al., 2004). On theother hand, overexpression of EDAsignaling molecules results in an ex-pansion of the molar tooth field andthe formation of a supernumerarytooth distal to the first molar (Mus-tonen et al., 2003; Tucker et al.,2004). Of interest, the supernumer-ary tooth has the cusp patterning ofa premolar, suggesting that dentalcusp patterning is highly dependenton the exact position in the oral cav-ity where tooth development occurs.

BMP signaling is another morpho-regulator for the number, size, andshape of tooth development. Loss ofBMP receptor 1a (Bmpr1a) causedretarded tooth development (Andl etal., 2004). Overexpression of BMPinhibitor Noggin in the oral ecto-derm resulted in miniaturization ofmaxillary first and second molars,and retarded maxillary third andmandibular molar development atthe lamina stage (Plikus et al.,2005). The selective loss of molars inNoggin-overexpressing mice, alongwith the abnormal buccal cusp pat-terning in ectodin null mutant mice,strongly suggest that various toothgerms and dental cusps have differ-ential requirements for the level ofBMP signaling. Modulation of BMPsignaling may account for one of themechanisms responsible for changesin tooth number, size, and shapethrough evolution.

ContinuedEpithelial–MesenchymalInteractions and the Successof Tooth Morphogenesis

After the initiation of tooth develop-ment, there is continued interactionbetween the dental epithelium andthe CNC-derived ectomesenchymethroughout tooth morphogenesis. Theinitial tooth generating potential re-sides within the epithelium and is ca-pable of inducing nontooth forming ec-tomesenchyme to develop teeth (Minaand Kollar, 1987; Jernvall and Thesleff,2000). Soon after development, thistooth-forming potential shifts to thedental mesenchyme. This shift oftooth-forming potential coincideswith a shift in BMP signaling fromthe epithelium to the mesenchyme.Morphologically, at this time, toothdevelopment advances into the budstage (Fig. 6). Multiple signalingmolecules within the enamel organepithelium have been implicated inthe regulation of transcription fac-tors in the surrounding ectomesen-chyme during the bud to the capstage transition (Fig. 6; Jernvall andThesleff, 2000; Tucker and Sharpe,2004). Interestingly, individual nullmutations of Msx1, Lef1, or Pax9 allresult in retarded tooth developmentat the bud stage, indicating the crit-ical roles of these transcription fac-tors during the transition from thebud to cap stage tooth development(Satokata and Maas, 1994; Kratoch-wil et al., 1996; Peters et al., 1998;Sasaki et al., 2005). Ectopic overex-pression and tissue recombinationexperiments have shown that thesetranscription factors are regulatedby epithelial signals, can control theexpression of growth factors andother transcription factors in the ec-tomesenchyme and provide instruc-tive feedback to the enamel organepithelium. Thus, they are essentialdetermining factors during the capstage tooth development.

To date, no null mutation experi-ments have resulted in failure of ini-tiation of tooth development (thicken-ing of the dental lamina). Some of thecompound null mutations, however,have resulted in retardation of toothdevelopment at an early bud stage,such as Msx1�/�/Msx2�/�, Dlx1�/�/Dlx2�/�, and Gli2�/�/Gli3�/�.


Clearly, there is functional redun-dancy among different members of thesame transcription factor family inregulating the advancement of toothmorphogenesis. Of interest, some ofthe null mutations reveal regionalspecificity of certain signaling mole-cules in regulating tooth morphogene-sis. For example, only maxillary molartooth organs were affected in Dlx1�/

�/Dlx2�/� double mutants. Further-more, loss of Dlx1 and Dlx2 genes re-sulted in a change of cell fate in thedental mesenchyme region from odon-togenic to chondrogenic (Thomas etal., 1997; Tucker and Sharpe, 1999).This finding also raises the possibilitythat a subpopulation of CNC cells car-rying Dlx1/Dlx2 genes migrates intothe maxillary molar region and, uponreceiving the proper instruction fromthe dental epithelium, can contributeto the formation of tooth organ. Byusing the two-component genetic sys-tem (Wnt1-Cre/R26R) for indeliblymarking the progenies of CNC cells(Fig. 4), it may be feasible to test thehypothesis that a subpopulation ofCNC cells designated for the maxil-lary molar region is affected in Dlx1/Dlx2 double-mutant mice (Chai et al.,2000; Han et al., 2003). The outcomeof such experiments may address thespeculation that a predetermined sub-population of CNC cells possesses in-structive functions in the induction oftooth development.

The activin �A null mutation re-veals another example of region-spe-cific signaling in regulating tooth de-velopment. All teeth, except themaxillary molars, are arrested at thebud stage in activin �A�/� mice, areverse phenotype compared with theDlx1/Dlx2 double mutant (Fergusonet al., 1998). Because activin �A isexpressed in the CNC-derived ecto-mesenchyme, it may help to specifythe fate of CNC cells during tooth de-velopment.

Overall, continuous and reciprocalepithelial–mesenchymal interaction isthe key for successful tooth organ mor-phogenesis. From initiation to odonto-blast/ameloblast differentiation to ma-trix formation, this interaction providesprecise communication between two ad-jacent tissue types and governs thenumber, size, and shape of tooth forma-tion. Our understanding of these biolog-ical processes may serve as a founda-

tion for the future design andfabrication of tooth regeneration.

SKULL DEVELOPMENTAND TISSUE BOUNDARY

The vertebral skull represents an ex-cellent model for the investigation ofdevelopment and evolution. In mam-mals, both CNC- and mesoderm-de-rived cells contribute to the develop-ment of the skull (Fig. 3D). Differentelements of the skull are establishedby the formation of tissue boundaries,which reflect an evolutionary contri-bution and are critical for the pre- andpostnatal dynamic development of thehead and face. Recent studies are nowbeginning to address the molecularregulation of patterning and size de-termination of different elements ofthe skull. Multiple excellent reviewshave addressed the regulation of skullvault development (Wilkie and Mor-riss-Kay, 2001; Santagati and Rijli,2003; Morriss-Kay and Wilkie, 2005),but there are very few reviews thataddress the molecular regulatorymechanism of facial bone develop-ment. Here, using mandible and pal-ate development as examples, wesummarize recent advancements to-ward the understanding of the regula-tory mechanism of craniofacial bonedevelopment.

The skull consists of the neurocra-nium and viscerocranium. The neuro-cranium (skull vault and base) sur-rounds and protects the brain. Inhumans, eight bones compose the neu-rocranium: the paired temporal andparietal bones and the singular fron-tal, sphenoid, ethmoid, and occipitalbones. Fourteen bones compose theviscerocranium (the jaws and otherpharyngeal arch derivatives): thepaired nasal bones, maxillae, palatinebones, lacrimal bones, zygoma and in-ferior nasal conchae, along with thesingular vomer and mandible. Theviscerocranium derives from the neu-ral crest, forms the face, and supportsthe functions of feeding and breath-ing.

Until recently, the tissue origin ofthe skull vault has been controversialbecause of conflicting reports usingquail–chick grafting. Noden (1978,1988) reported that the CNC only con-tributes to the rostral portion of thefrontal bones and that the remainder

of the skull vault is of mesoderm ori-gin. In contrast, Couly and coworkers(1993) concluded that the skull vaultis entirely neural crest derived. In themouse model, it has been demon-strated elegantly that frontal bonesare neural crest-derived and parietalbones are of mesoderm origin (Jiang etal., 2002). The posterior part of theskull vault (supraoccipital and exoc-cipital bones) derives from the occipi-tal somites (Fig. 3D). The dura materthat underlies the frontal and parietalbones is also neural crest derived(Jiang et al., 2002; Ito et al., 2003;Sasaki et al., 2006). The coronal andsagittal sutures are of mesoderm ori-gin (Morriss-Kay and Wilkie, 2005).Clearly, the skull vault elements aredeveloped at the boundary betweenCNC- and mesoderm-derived tissue.This boundary is of paramount impor-tance in mediating tissue–tissue in-teraction that control skull vault de-velopment. When there is mixing ofthe two cell populations as the resultof a gene mutation (such as in theephrin-B1 or Twist mutant), theboundary between CNC and the me-soderm is lost, resulting in the prema-ture fusion of cranial sutures knownas craniosynostosis (Twigg et al.,2004; Merrill et al., 2006).

MANDIBULARMORPHOGENESIS

Patterning of the branchial arches re-quires the establishment of both inter-branchial arch and intrabranchialarch identities (Depew et al., 2002a).It is well established that Hox, Pbx,and Otx homeobox genes are criticalfor the normal patterning of inter-branchial arch identities (Gendron-Maguire et al., 1993; Rijli et al., 1993;Matsuo et al., 1995; Selleri et al.,2001). For example, there clearly is aHox gene code that controls branchialarch development. To give rise to thederivatives of the first branchial arch,the structure needs to be Hox genenegative. Until recently, however, lessinformation has been available aboutthe genetic control of the establish-ment of the intrabranchial arch iden-tities. This is an important area incraniofacial development, because hu-man mandibular dysmorphogenesisappears to be a common malformationand appears in multiple congenital


birth defect syndromes, ranging fromagnathia (agenesis of the jaw) to mi-crognathia to patterning malforma-tions.

Based on the mouse model, we knownow that the first branchial arch(mandibular arch) becomes apparentat E8.0–E8.5 (6–8 somites) as smallswellings on the side of the developinghead. As CNC cells migrate into andproliferate within the mandibulararch, it develops rapidly toward theventral midline. The CNC-derivedcells are localized immediately subja-cent to the covering epithelium (Chaiet al., 2000). Mandibular developmentdepends upon the interaction betweenthe oral ectoderm and the CNC-de-rived mesenchyme within the firstbranchial arch. Within the oral ecto-derm, signaling molecules, such asBMP, TGF-�, and FGF, are expressedin a region-specific manner (Chai etal., 1994, 1997; Trumpp et al., 1999;Ito et al., 2002; Liu et al., 2005). Theymay regulate homeobox-containinggenes (such as Dlx, Lhx, and Gsc)within the CNC-derived mesenchymeto generate early polarity and are re-sponsible for patterning of the firstbranchial arch.

Ectodermal FGF signaling is criti-cal for CNC cell survival in the man-dibular arch as conditional inactiva-tion of Fgf8 in the ectoderm causedincreased apoptotic activity and a dra-matic loss of all first branchial archskeletal structures (except the most dis-tal portion of the mandible; Trumpp etal., 1999). During early embryonic de-velopment, however, FGF signaling isnot required for cell proliferation andsurvival, but instead is required for cellmigration. Therefore, FGF signalinghas differential functional specificity indifferent developmental contexts (Sunet al., 1999).

In the proximal domain of the firstbranchial arch, FGF signaling directlyor indirectly regulates the expressionof homeobox genes in the mesenchymeto control the development of mandi-ble (see previous discussion). In thedistal domain of the first branchialarch, BMP signaling appears to be acrucial regulator for mandibular mor-phogenesis. BMP4 is expressed withinthe distal ectoderm that covers thefirst branchial arch mesenchyme atE9.5. The postmigratory CNC cellsare exposed to the instructive signal-

ing (such as BMP) from the ectodermand become committed to give rise tostructures associated with the distalportion of the first arch. As embryonicdevelopment progresses, there is ashift of the instructive capability forpatterning the mandible from the ec-toderm to the CNC-derived mesen-chyme. The patterning of the proxi-mal–distal domain of the firstbranchial arch is achieved throughthe antagonistic interaction betweenBMP and FGF, which controls themesenchymal expression of signalingmolecules, such as Msx1 and Barx1,respectively (Tucker et al., 1998b;Tucker and Sharpe, 2004). BMP sig-naling clearly has an important role inregulating mandibular morphogene-sis. Specifically, loss of BMP signalingin the oral ectoderm and the pharyn-geal endoderm results in extreme phe-notypes, ranging from an almost com-pletely missing mandible to severedefects in the distal region (Liu et al.,2005). BMP target genes Msx1 andMsx2 have differential dose require-ments for BMP4 signaling, and theyact in a functionally redundant man-ner. Both Msx1 and Msx2 are ex-pressed in the midline region, and lossof BMP4 signaling results in compro-mised Msx gene expression. Mutationof Msx1 and Msx2 genes results inmidline cleft of the first arch and se-vere defects in mandibular morpho-genesis (Satokata et al., 2000; Ishii etal., 2005; our unpublished data).

Mandibular development requiresproper modulation of BMP and FGFsignaling in the ectoderm. This modu-lation is achieved through the antag-onistic interaction between BMP andFGF signaling, the inhibition of BMPsignaling by Noggin or Chordin and bythe feedback from the underlying CNC-derived mesenchyme (Stottmann et al.,2001; Wilson and Tucker, 2004; Liu etal., 2005). For example, BMP signalingis required for maintaining FGF signal-ing in the proximal domain of the man-dibular arch, but it represses FGF sig-naling in the distal domain (Liu et al.,2005). Furthermore, BMP signaling issubject to multiple points of regulation,at the ligand, receptor, Smad, and tran-scription complex level (Massague etal., 2005). Clearly, tightly controlledBMP and FGF signaling is of para-mount importance for mandibular mor-phogenesis.

Similar to the proximal–distal do-mains within the ectoderm of the firstbranchial arch, there are discrete pop-ulations within the underlying CNC-derived mesenchyme. Dlx genes areknown to play important roles in reg-ulating the patterning of the develop-ing jaw. Six Dlx genes (Dlx1, Dlx2,Dlx3, Dlx5, Dlx6, and Dlx7) have beendescribed in mice (Dolle et al., 1992;Robinson and Mahon, 1994; Simeoneet al., 1994; Qiu et al., 1995, 1997;Stock et al., 1996). During the devel-opment of the branchial arches, Dlxgenes show nested expression patternsand play important roles in establishingthe identity along the proximodistalaxis within each branchial arch (Depewet al., 2002a, 2005). Specifically, Dlx1/2are expressed in the CNC-derived ecto-mesenchyme both in the proximal anddistal region of the branchial arch,whereas Dlx5/6 and Dlx3/7 show pro-gressively restricted expression do-mains toward the distal region of thebranchial arch.

Recent studies have shown that acombinatorial Dlx code regulates theestablishment of the distinct skeletalelements within a given branchialarch unit (Qiu et al., 1995, 1997; De-pew et al., 2002a,b; Cobourne andSharpe, 2003). Loss of Dlx5 and Dlx6results in a homeotic transformationof the lower jaw into an upper jaw,supporting a model of patterningwithin the branchial arch that relieson a nested pattern of Dlx gene ex-pression (Depew et al., 2002b). In ad-dition, jaw development is sensitive tothe dosage of Dlx genes, as haploinsuf-ficiency of single or multiple Dlx geneshas a gradient effect on mandibulardevelopment (Depew et al., 2005).This observation clearly suggests thatthe expression of Dlx genes must betightly regulated. To date, little infor-mation is available about the directregulation on Dlx gene expression. Al-though FGF8-soaked beads placed inthe first arch epithelium are able toinduce Dlx2 and Dlx5 expression inthe mandibular mesenchyme, loss ofFgf8 in the ectoderm shows unalteredDlx2 and Dlx5 expression in the firstbranchial arch (Trumpp et al., 1999).Similarly, BMP-soaked beads are ableto induce Dlx gene expression, but theendogenous BMP and Dlx expressionpatterns do not suggest a direct regu-latory relationship. Overall, Dlx genes


are clearly important for intrabranchialarch patterning, and further studies inthis area will advance our understand-ing of their role and the regulatorymechanism in this process.

PALATOGENESIS AND THEMOLECULAR MECHANISMOF CLEFT PALATE

The mammalian palate develops fromtwo primordia: the primary palate andthe secondary palate. The primarypalate represents only a small part ofthe adult hard palate. The secondarypalate is the primordium of the hardand soft parts of the palate. Palatedevelopment is a multistep process that

Fig. 6.

Fig. 7.

Fig. 6. Schematic drawing of reciprocal andreiterative signaling in regulating epithelial–mesenchymal interactions throughout sequen-tial stages of tooth morphogenesis. A precisespatial and temporal orchestration of multiplegrowth and transcription factors (as listedwithin each box) is critical for the initiation aswell as patterning of tooth germ development.These signaling molecules tightly regulate boththe enamel organ epithelia and the cranial neu-ral crest-derived dental mesenchyme. Specifi-cally, when tooth development is initiated withformation of dental lamina, its underlying mes-enchyme is almost entirely populated with cra-nial neural crest (CNC) -derived cells (darkblue). As tooth germ develops from the bud tothe cap stage, CNC-derived cells are concen-trated (dark blue) at the interface with theenamel organ epithelium, whereas the periph-eral portion of the dental sac is populated withboth CNC- and non–CNC-derived cells (lightblue). Molecular signaling residing within theenamel organ epithelium and the CNC-deriveddental mesenchyme are engaged in a constantand reciprocal dialogue to mediate tooth mor-phogenesis. The dark red dot representsenamel knot-a signaling center within theenamel organ epithelia, whereas light pink rep-resents enamel organ epithelium (modified fromJernvall and Thesleff, 2000; Chai et al., 2000).

Fig. 7. Anatomy of palatogenesis and five cat-egories of palatal shelf defects that result incleft palate. A: Mouse palatogenesis starts atembryonic day (E) 11.5. By E13.5, palatalshelves (P) are on both sides of the tongue (T).Each palatal shelf can be divided into the me-dial (m) and lateral (l) aspects. t, tooth germ.B,C: Between E13.5 and E14.0, palatal shelvesturn horizontally above the tongue and faceeach other along the midline. D: At E14.5, pal-atal fusion begins to take place. The arrow in-dicates the midline epithelial seam. n, nasalepithelium; o, oral epithelium. E,F: From E15.5to E16.5, palatal fusion is completed through-out the entire palate. Five different categories ofpalatal shelf defects that result in cleft palate(also see Zhang et al., 2002). P, palatal shelf.


involves palatal shelf growth, elevation,midline fusion of palatal shelves, andthe disappearance of midline epithelialseam (Fig. 7). The palatal structuresare composed of the CNC-derived ecto-mesenchyme and pharyngeal ectoderm(Ferguson, 1988; Shuler, 1995). The ep-ithelia that cover the palatal shelvesare regionally divided into oral, nasal,and medial edge epithelium (Fig. 7D).The nasal and oral epithelia differenti-ate into pseudostratified and squamousepithelia, respectively, whereas the me-dial edge epithelium (MEE) is removedfrom the fusion line by means of pro-grammed cell death and cell migration(Martinez-Alvarez et al., 2000; VaziriSani et al., 2005).

Mouse palatogenesis initiates atE11.5 as marked by the formation ofpalatal shelves extending from the in-ternal aspects of maxillae. After initi-ation, the palatal shelves projectdownward on each side of the tonguebetween E12.5 and E13.5 (Fig. 7A,B).As the jaws develop, the tongue de-scends, thus providing space to accom-modate the horizontal apposition ofpalatal shelves above the tongue (Fig.7C). The fusion of palatal shelves oc-curs at E14.5, resulting in the forma-tion of continuous palate (Fig. 7D).Both the elevation and fusion of thepalatal shelves occur in an anterior toposterior sequence. The complete fu-sion of palatal shelves results in theseparation of the oral cavity from thenasal cavity (Fig. 7E,F).

Fate of Medial EdgeEpithelial Cells and theContribution of CranialNeural Crest Cells DuringPalatogenesis

There has been a tremendous interestin the fate of medial edge epithelialcells during palatal fusion. Apoptosisis clearly one of the important mecha-nisms for eliminating MEE cells dur-ing palatal fusion (Farbman, 1968;Saunders, 1966; Dudas and Kaarti-nen, 2005). At the molecular level,TGF-� and RA are critical inducers forapoptosis of the MEE. The dosage ofthese signaling molecules may differ-entially control the progression of thecell cycle in the MEE (Cuervo et al.,2002; Martinez-Alvarez et al., 2000;Cuervo and Covarrubias, 2004). An al-

ternative fate for the MEE is migra-tion along the midline toward the na-sal and oral epithelia, resulting in theloss of MEE (Carette and Ferguson,1992, Hilliard et al., 2005). Studiesfrom multiple laboratories have sug-gested that epithelial–mesenchymaltransformation (EMT) is an importantcellular mechanism for the disappear-ance of MEE (Fitchett and Hay, 1989;Griffith and Hay, 1992; Shuler et al.,1992). These studies were largelybased on cell lineage tracing usingmembrane-intercalating dye and epi-thelial and mesenchymal cellularmarkers. Recently, however, studiesusing genetic cellular markers havechallenged the theory of EMT duringpalatal fusion. Both in vivo and invitro studies show that EMT does notoccur during palatal fusion (Cuervo etal., 2002; Vaziri Sani et al., 2005; Xuet al., 2006). Apoptosis and cell migra-tion may be sufficient to account forthe cellular mechanism for the disap-pearance of MEE cells.

CNC cells are critical for palatogen-esis. Until recently, however, littlehas been known about the fate of theCNC-derived palatal mesenchyme orthe molecular mechanisms that regu-late the epithelial–mesenchymal in-teractions during palate development.The lack of information is largelydue to the difficulty in CNC cell la-beling and fate analysis in the palate.Significantly, using the Wnt1-Cre/R26R model, we have systematicallyfollowed the migration, proliferation,and differentiation of CNC cellsthroughout embryogenesis. We showthat the CNC-derived ectomesen-chyme contribute significantly to thepalatal mesenchyme and that there isa dynamic distribution of these CNCcells during palatogenesis (Chai et al.,2000; Ito et al., 2003). This two-component genetic system providesthe opportunity to integrate analysisof the fate and function of the mam-malian neural crest with mouse mo-lecular genetics in both normal andabnormal embryonic development.

Mouse Models forInvestigating the MolecularRegulatory Mechanism ofPalatogenesis

Multiple genetically mutated mousemodels have made significant contri-

butions to our understanding of thegene pathways involved in palate de-velopment and the nature of signalingmolecules that act in a tissue-specificmanner at critical stages of embryonicdevelopment. Interestingly, however,most of genetic mutations cause mul-tiple structural and functional defectsin the developing embryo. Conse-quently, assessment of the role of aparticular gene in regulating palato-genesis has been a challenge. To fur-ther complicate the issue, cleft lipand/or palate is a complex trait causedby multiple genetic and environmen-tal factors (Murray, 2002). Neverthe-less, these animal models have ad-vanced our understanding of someimportant gene functions in humanpalate development.

One of the important discoverieshas been the existence of genetic het-erogeneity along the anterior–poste-rior and medial–lateral axes of the de-veloping palate (for review, seeHilliard et al., 2005). This heterogene-ity may provide differential regula-tory mechanisms for the fusion of theanterior vs. posterior region of the pal-ate. For example, MEE cells begin toundergo apoptosis at different timesduring palatal fusion, depending ontheir location. It has been shown thatapoptosis of MEE cells is triggered bypalatal shelf contact in the anteriorregion, whereas it is initiated beforeany contact between the opposingshelves in the posterior region (Cu-ervo et al., 2002). This may be theresult of differential molecular signalsin the palatal mesenchyme along theanteroposterior (A-P) axis that in-struct different fates to the palatal ep-ithelium (Ferguson and Honig, 1984).More recent studies have demon-strated that constant and reciprocalinteractions between the palatal epi-thelium and the CNC-derived mesen-chyme are responsible for setting upthis genetic heterogeneity along theA-P axis and are crucial for normalpalatal development and fusion(Zhang et al., 2002; Murray andSchutte, 2004; Rice et al., 2004).

Multiple genes have been found tobe critical for the development of theanterior region of the palate. For ex-ample, Msx1, Bmp4, Bmp2, Fgf10,and Shox2 show restricted expressionpatterns in the anterior region of thepalate (Rice et al., 2004; Hilliard et al.,


2005). Loss of Msx1 results in a cellproliferation defect in the CNC-de-rived palatal mesenchyme in the an-terior region of secondary palate.BMP4 functions downstream of Msx1and controls the expression of Shh inthe palatal epithelium. Shh in turnregulates the expression of Bmp2 inthe mesenchyme to promote cell prolif-eration (Zhang et al., 2002). Meanwhile,FGF10 is expressed in the anterior pal-atal mesenchyme and functions in aparacrine manner through its receptorFGFR2 in the palatal epithelium to me-diate Shh expression, which in turn reg-ulates Bmp2 expression in the mesen-chyme to promote cell proliferation. Sothe BMP and FGF signaling pathwaysconverge on Shh signaling in the epithe-lium to control the growth of the ante-rior region of the palatal shelf. TheShox2 gene is exclusively expressed inthe anterior palatal mesenchyme. Lossof Shox2 results in an incomplete cleftof the anterior hard palate, whereas fu-sion of the posterior palate is normal(Yu et al., 2005). Significantly, thisstudy clearly demonstrates that thereare different regulatory mechanismsthat control the development and fusionof the anterior and posterior parts of thepalate and that a successful fusion ofthe posterior part of the palate can oc-cur, despite that there is a failure ofanterior palate fusion.

We know less about the specificgene expression patterns in the poste-rior region of the palate. Fgfr2 is ex-pressed in the epithelium and theCNC-derived mesenchyme in the mid-dle and posterior palate. FGF8 signal-ing selectively induces the expressionof Pax9 in the posterior region of pal-atal mesenchyme. Loss of Pax9 resultsin a palatal shelf development defectand cleft palate (Peters et al., 1998;Hilliard et al., 2005). To date, there islittle known about the regulation ofPax9 expression in the developing pal-ate. Therefore, the biological signifi-cance of FGF8-mediated Pax9 expres-sion during palatogenesis remains tobe determined. In addition to the dif-ferential gene expression patternsalong the A-P axis of the developingpalate, there is also mesenchymal het-erogeneity between the medial andlateral regions of the palatal shelf(Fig. 7A). For example, the oddskipped-related genes Osr1 and Osr2are expressed in a medial–lateral gra-

dient in the palatal shelf. Signifi-cantly, mutation of the Osr2 gene re-sults in the compromised developmentof the medial aspect of palatal shelfdevelopment and retards palatal shelfelevation (Lan et al., 2004). The ex-pression of Fgfr2 is focused on the me-dial aspect of the developing palatalshelf, suggesting a possible functionalsignificance in regulating the develop-ment and elevation of palatal shelf.

In analyzing different mutant ani-mal models with cleft palate, we pro-pose to divide the palatal shelf devel-opment defects into the following fivecategories. (1) Failure of palatal shelfformation (Fig. 7). Although this is asevere type of palatal shelf develop-ment defect, it has a rare occurrence.Mutation of activin-�A causes a se-vere facial primordia development de-fect, which is likely responsible for theretardation of palatal shelf develop-ment and complete cleft palate (Mat-zuk et al., 1995). The Fgfr2 mutationalso affects the initial development ofthe palatal shelf and results in com-plete cleft palate (Rice et al., 2004). (2)Fusion of the palatal shelf with thetongue or mandible (Fig. 7). For exam-ple, loss of function mutation of Fgf10results in anterior palatal shelf fusionwith the tongue, whereas the middleand posterior part of the palatal shelfadheres to the mandible, thus pre-venting the elevation of the palatalshelf (Alappat et al., 2005). In hu-mans, mutations in TBX22 have beenreported in families with X-linkedcleft palate and ankyloglossia (Bray-brook et al., 2001). Similarly, Tbx22 isexpressed in the developing palateand tongue in mice, suggesting an im-portant role of Tbx22 in regulatingpalate and tongue development (Bushet al., 2002). (3) Failure of palatalshelf elevation (Fig. 7). Studies haveshown that mutations of Pax9, Pitx1,or Osr2 can lead to failed palatal shelfelevation and cleft palate defect (Pe-ters et al., 1998; Szeto et al., 1999; Lanet al., 2004). The cellular defect ismainly associated with the CNC-de-rived palatal mesenchyme, suggestingimportant functions of these tran-scription factors in regulating the fateof CNC cells during palatogenesis. (4)Failure of palatal shelves to meet af-ter elevation (Fig. 7). By far, this is themost common type of cleft palate de-fect documented in animal studies.

For example, mutations in Msx1 andLhx8 and conditional inactivation ofTgfbr2 in CNC cells or Shh in the ep-ithelium all result in retarded palatalshelf development (Satokata andMaas, 1994; Zhao et al., 1999; Ito etal., 2003; Rice et al., 2004). (5) Persis-tence of medial edge epithelium (Fig.7). In Tgfb3 or Egfr mutant mice,there is an alteration of the fate ofMEE cells (Kaartinen et al., 1995;Proetzel et al., 1995; Miettinen et al.,1999). In Tgfb3 null mutant mice,MEE cells fail to undergo apoptosisand persist along the midline to pre-vent normal fusion. In addition to itsfunction in regulating the fate ofMEE, TGF-�3 is also critical forproper proliferation of the CNC-de-rived palatal mesenchyme (our un-published data). Furthermore, muta-tions in TGF-�3 have been associatedwith cleft palate in humans, under-scoring the crucial function of TGF-�signaling in regulating palatogenesis(Lidral et al., 1998).

PROSPECTUS

Each year, approximately 250,000 in-fants born in the United States havesome mental or physical defects.Three fourths of all malformationsseen at birth involve craniofacial dys-morphogenesis, affecting the develop-ment of head, face, or neck. Thesemalformations are particularly devas-tating, as our faces are our identity—they are how we see ourselves andhow others see us. In recent years,research has progressed so that weknow the precise genetic error thatleads to many craniofacial birth de-fects.

At the conclusion of a recent GordonResearch Conference on CraniofacialMorphogenesis and Tissue Regenera-tion (Ventura, CA), it was clear thatthere has been tremendous interestand development in recent years to-ward a better understanding of themolecular regulatory mechanism ofcraniofacial development. Develop-mental and evolutionary biologists aswell as tissue engineers are workingtogether to investigate and comparethe tissue origin, patterning, andgrowth of various craniofacial organsin an effort to reproduce and/or repairdefective tissue in the craniofacial re-gion.


To date, only approximately onethird of mouse mutations associatedwith craniofacial malformations havebeen linked to mutations of the or-thologous human genes with similardysmorphogenesis (Wilkie and Mor-riss-Kay, 2001). It is not known whatdefects might arise from mutations ofthe other two thirds as some of thesemutations in mice result in early em-bryonic lethality. This lethality dem-onstrates the important function ofthese genes in early embryonic devel-opment but makes it impossible to in-vestigate the functional significance ofthat particular gene in regulatingcraniofacial development. It is con-ceivable that the equivalent muta-tions in human will also result in alethal phenotype. Nevertheless, animportant conclusion from studies us-ing various animal models suggeststhat there are no “craniofacial genes.”The same morphogen (BMP, TGF-�,FGF, Shh, and so on) that controlslimb development, for example, alsoplays a crucial role in regulatingcraniofacial morphogenesis. Clearly,the functional specificity and the de-velopmental outcome of a signalingmolecule is determined by the cellupon which the morphogen acts, asdifferent cell types are exposed to adifferent combination of growth andtranscription factors and may responddifferently to the same growth factorsignaling (“Ask not what TGF-� cando for the cell, ask what the cell can dowith TGF-�,” Joan Massague, per-sonal communication). Another im-portant lesson from animal studies isthe available dosage of an importantregulatory gene, as haploinsufficiencyor gain of function of a critical gene isoften associated with congenital mal-formations in humans. Overall, stud-ies using mutant mice have signifi-cantly improved our understanding ofhuman embryogenesis in general andcraniofacial development in particu-lar. It is of paramount importancethat there are constant interactionsamong researchers involved in bothhuman and animal studies as this willhelp to ensure the rapid progress to-ward a better understanding, treat-ment, and prevention of human con-genital malformations.

Tissue regeneration is another areawith many exciting new developmentsbased on research advancements in

developmental and evolutionary biol-ogy (Chai and Slavkin, 2003). The re-cent convergence of the human ge-nome project and scientific advancestoward understanding the molecularregulation of craniofacial morphogen-esis, stem cell biology, and biotechnol-ogy offer unprecedented opportunitiesto realize craniofacial tissue regener-ation. One of the next critical stepswill be to apply our knowledge of mo-lecular regulation of tooth morpho-genesis, for example, to manipulateadult stem cells toward an odonto-genic phenotype.

Significant progress has been madein stem cell biological research, whichhas advanced our understanding inthe area of hematopoiesis, tissue engi-neering, and biomaterials (Lovell-Badge, 2001). Recently, studies haveshown that adult stem cells have amuch higher degree of developmentalpotential than previously thought,and this has prompted considerationsto explore the potential of stem cellmediated muscle, bone, cartilage, anddentin regeneration (Gronthos et al.,2000; Bianco et al., 2001; Bianco andRobey, 2001). Stem cells are truly re-markable. They have the potential togrow into an array of specialized cellsand hold great promise for treatingmedical and dental conditions. Sys-temically injected mouse bone marrowderived cells have given rise to mus-cle, cartilage, bone, liver, heart, brain,lung, alveolar epithelium, intestine,and, of course, hematocytes (Ferrari etal., 1998; Lagasse et al., 2000; Wood-bury et al., 2000; Kotton et al., 2001).Although most of these animal studiesserve now as precursors for future hu-man clinical trials in treating certainmedical and dental conditions, the ba-sic scientific principles learned fromthese current analyses certainly haveadvanced our understanding of the bi-ological regulation of tissue engineer-ing. The 21st century provides us withtremendous opportunities to enablebiological solutions to biological prob-lems.

ACKNOWLEDGMENTSWe thank Pablo Bringas, Jr., JunHan, Ryoichi Hosokawa, Julie Mayo,Kyoko Oka, and Xun Xu for their as-sistance with this manuscript. Weapologize to those colleagues whose

publications were not cited due tospace limitations. Both Dr. Chai’s laband Dr. Maxson’s lab were supportedby the NIH.

REFERENCES

Abzhanov A, Tzahor E, Lassar AB, TabinCJ. 2003. Dissimilar regulation of celldifferentiation in mesencephalic (cra-nial) and sacral (trunk) neural crest cellsin vitro. Development 130:4567–4579.

Abzhanov A, Protas M, Grant BR, GrantPR, Tabin CJ. 2004. Bmp4 and morpho-logical variation of beaks in Darwin’sfinches. Science 305:1462–1465.

Alappat SR, Zhang Z, Suzuki K, Zhang X,Liu H, Jiang R, Yamada G, Chen Y.2005. The cellular and molecular etiol-ogy of the cleft secondary palate in Fgf10mutant mice. Dev Biol 277:102–113.

Andl T, Ahn K, Kairo A, Chu EY, Wine-LeeL, Reddy ST, Croft NJ, Cebra-ThomasJA, Metzger D, Chambon P, Lyons KM,Mishina Y, Seykora JT, Crenshaw EBIII, Millar SE. 2004. Epithelial Bmpr1aregulates differentiation and prolifera-tion in postnatal hair follicles and is es-sential for tooth development. Develop-ment 131:2257–2268.

Aoki TO, David NB, Minchiotti G, Saint-Etienne L, Dickmeis T, Persico GM,Strahle U, Mourrain P, Rosa FM. 2002.Molecular integration of casanova in theNodal signalling pathway controllingendoderm formation. Development 129:275–286.

Baker CV, Bronner-Fraser M. 2001. Verte-brate cranial placodes I. Embryonic in-duction. Dev Biol 232:1–61.

Basch ML, Garcia-Castro MI, Bronner-Fraser M. 2004. Molecular mechanismsof neural crest induction. Birth DefectsRes C Embryo Today 72:109–123.

Beddington RS, Robertson EJ. 1999. Axisdevelopment and early asymmetry inmammals. Cell 96:195–209.

Bianco P, Robey PG. 2001. Stem cells intissue engineering. Nature 414:118–121.

Bianco P, Riminucci M, Gronthos S, RobeyPG. 2001. Bone marrow stromal stemcells: nature, biology, and potential ap-plications. Stem Cells 19:180–192.

Bitgood MJ, McMahon AP. 1995. Hedge-hog and Bmp genes are coexpressed atmany diverse sites of cell–cell interac-tion in the mouse embryo. Dev Biol 172:126–138.

Braybrook C, Doudney K, Marcano AC, Ar-nason A, Bjornsson A, Patton MA, Good-fellow PJ, Moore GE, Stanier P. 2001.The T-box transcription factor geneTBX22 is mutated in X-linked cleft pal-ate and ankyloglossia. Nat Genet 29:179–183.

Bronner-Fraser M. 1993. Neural crest cellmigration in the developing embryo.Trends Cell Biol 3:392–397.

Brown CB, Wenning JM, Lu MM, EpsteinDJ, Meyers EN, Epstein JA. 2004. Cre-mediated excision of Fgf8 in the Tbx1expression domain reveals a critical role


for Fgf8 in cardiovascular developmentin the mouse. Dev Biol 267:190–202.

Brugger SM, Merrill AE, Torres-VazquezJ, Wu N, Ting MC, Cho JY, Dobias SL, YiSE, Lyons K, Bell JR, Arora K, WarriorR, Maxson R. 2004. A phylogeneticallyconserved cis-regulatory module in theMsx2 promoter is sufficient for BMP-de-pendent transcription in murine andDrosophila embryos. Development 131:5153–5165.

Bush JO, Lan Y, Maltby KM, Jiang R.2002. Isolation and developmental ex-pression analysis of Tbx22, the mousehomolog of the human X-linked cleft pal-ate gene. Dev Dyn 225:322–326.

Carette MJ, Ferguson MW. 1992. The fateof medial edge epithelial cells during pal-atal fusion in vitro: an analysis by DiIlabelling and confocal microscopy. Devel-opment 114:379–388.

Chai Y, Slavkin HC. 2003. Prospects fortooth regeneration in the 21st century: aperspective. Microsc Res Tech 60:469–479.

Chai Y, Mah A, Crohin C, Groff S, BringasP Jr, Le T, Santos V, Slavkin HC. 1994.Specific transforming growth factor-betasubtypes regulate embryonic mouseMeckel’s cartilage and tooth develop-ment. Dev Biol 162:85–103.

Chai Y, Sasano Y, Bringas P Jr, Mayo M,Kaartinen V, Heisterkamp N, Groffen J,Slavkin H, Shuler C. 1997. Characteriza-tion of the fate of midline epithelial cellsduring the fusion of mandibular promi-nences in vivo. Dev Dyn 208:526–535.

Chai Y, Jiang X, Ito Y, Bringas P Jr, Han J,Rowitch DH, Soriano P, McMahon AP,Sucov HM. 2000. Fate of the mammaliancranial neural crest during tooth andmandibular morphogenesis. Develop-ment 127:1671–1679.

Chai Y, Ito Y, Han J. 2003. TGF-beta sig-naling and its functional significance inregulating the fate of cranial neuralcrest cells. Crit Rev Oral Biol Med 14:78–88.

Chen Y, Bei M, Woo I, Satokata I, Maas R.1996. Msx1 controls inductive signalingin mammalian tooth morphogenesis. De-velopment 122:3035–3044.

Chen D, Zhao M, Mundy GR. 2004. Bonemorphogenetic proteins. Growth Factors22:233–241.

Clouthier DE, Hosoda K, Richardson JA,Williams SC, Yanagisawa H, Kuwaki T,Kumada M, Hammer RE, YanagisawaM. 1998. Cranial and cardiac neuralcrest defects in endothelin-A receptor-deficient mice. Development 125:813–824.

Cobourne MT, Sharpe PT. 2003. Tooth andjaw: molecular mechanisms of pattern-ing in the first branchial arch. Arch OralBiol 48:1–14.

Cobourne MT, Hardcastle Z, Sharpe PT.2001. Sonic hedgehog regulates epithe-lial proliferation and cell survival in thedeveloping tooth germ. J Dent Res 80:1974–1979.

Couly GF, Coltey PM, Le Douarin NM.1992. The developmental fate of the ce-

phalic mesoderm in quail-chick chime-ras. Development 114:1–15.

Couly GF, Coltey PM, Le Douarin NM.1993. The triple origin of skull in highervertebrates: a study in quail-chick chi-meras. Development 117:409–429.

Couly G, Creuzet S, Bennaceur S, VincentC, Le Douarin NM. 2002. Interactionsbetween Hox-negative cephalic neuralcrest cells and the foregut endoderm inpatterning the facial skeleton in the ver-tebrate head. Development 129:1061–1073.

Creuzet S, Couly G, Le Douarin NM. 2005.Patterning the neural crest derivativesduring development of the vertebratehead: insights from avian studies. J Anat207:447–459.

Crump JG, Maves L, Lawson ND, Wein-stein BM, Kimmel CB. 2004. An essen-tial role for Fgfs in endodermal pouchformation influences later craniofacialskeletal patterning. Development 131:5703–5716.

Cuervo R, Covarrubias L. 2004. Death isthe major fate of medial edge epithelialcells and the cause of basal lamina deg-radation during palatogenesis. Develop-ment 131:15–24.

Cuervo R, Valencia C, Chandraratna RAS,Covarrubias L. 2002. Programmed celldeath is required for palate shelf fusionand is regulated by retinoic acid. DevBiol 245:145–156.

Dassule HR, McMahon AP. 1998. Analysisof epithelial-mesenchymal interactionsin the initial morphogenesis of the mam-malian tooth. Dev Biol 202:215–227.

Dahmann C, Basler K. 1999. Compartmentboundaries: at the edge of development.Trends Genet 15:320–326.

Depew MJ, Tucker AS, Sharpe PT. 2002a.Craniofacial development. In: Mouse de-velopment. Orlando: Academic Press. p421–498.

Depew MJ, Lufkin T, Rubenstein JL.2002b. Specification of jaw subdivisionsby Dlx genes. Science 298:381–385.

Depew MJ, Simpson CA, Morasso M,Rubenstein JL. 2005. Reassessing theDlx code: the genetic regulation ofbranchial arch skeletal pattern and de-velopment. J Anat 207:501–561.

Dolle P, Price M, Duboule D. 1992. Expres-sion of the murine Dlx-1 homeobox geneduring facial, ocular and limb develop-ment. Differentiation 49:93–99.

Dorsky RI, Moon RT, Raible DW. 2000.Environmental signals and cell fatespecification in premigratory neuralcrest. Bioessays 22:708–716.

Dudas M, Kaartinen V. 2005. Tgf-beta su-perfamily and mouse craniofacial devel-opment: interplay of morphogenetic pro-teins and receptor signaling controlsnormal formation of the face. Curr TopDev Biol 66:65–133.

Echelard Y, Vassileva G, McMahon AP.1994. Cis-acting regulatory sequencesgoverning Wnt-1 expression in the devel-oping mouse CNS. Development 120:2213–2224.

Evans DJ, Noden DM. 2006. Spatial rela-tions between avian craniofacial neural

crest and paraxial mesoderm cells. DevDyn.

Farbman AI. 1968. Electron microscopestudy of palate fusion in mouse embryos.Dev Biol 18:93–116.

Ferguson MW. 1988. Palate development.Development 103(Suppl):41–60.

Ferguson MW. 1994. Craniofacial malfor-mations: towards a molecular under-standing. Nat Genet 6:329–330.

Ferguson CA, Graham A. 2004. Redefiningthe head-trunk interface for the neuralcrest. Dev Biol 269:70–80.

Ferguson MW, Honig LS. 1984. Epithelial-mesenchymal interactions during verte-brate palatogenesis. Curr Top Dev Biol19:138–164.

Ferguson CA, Tucker AS, Christensen L,Lau AL, Matzuk MM, Sharpe PT. 1998.Activin is an essential early mesenchy-mal signal in tooth development that isrequired for patterning of the murinedentition. Genes Dev 12:2636–2649.

Ferrari G, Cusella-De Angelis G, ColettaM, Paolucci E, Stornaiuolo A, Cossu G,Mavilio F. 1998. Muscle regeneration bybone marrow-derived myogenic progeni-tors. Science 279:1528–1530.

Fitchett JE, Hay ED. 1989. Medial edgeepithelium transforms to mesenchymeafter embryonic palatal shelves fuse.Dev Biol 131:455–474.

Gage PJ, Suh H, Camper SA. 1999. Dosagerequirement of Pitx2 for development ofmultiple organs. Development 126:4643–4651.

Gavalas A, Trainor P, Ariza-McNaughtonL, Krumlauf R. 2001. Synergy betweenHoxa1 and Hoxb1: the relationship be-tween arch patterning and the genera-tion of cranial neural crest. Development128:3017–3027.

Gendron-Maguire M, Mallo M, Zhang M,Gridley T. 1993. Hoxa-2 mutant mice ex-hibit homeotic transformation of skeletalelements derived from cranial neuralcrest. Cell 75:1317–1331.

Graham A, Lumsden A. 1993. The role ofsegmentation in the development of thebranchial region of higher vertebrateembryos. Birth Defects Orig Artic Ser29:103–112.

Graham A, Okabe M, Quinlan R. 2005. Therole of the endoderm in the developmentand evolution of the pharyngeal arches.J Anat 207:479–487.

Griffith CM, Hay ED. 1992. Epithelial-mesenchymal transformation duringpalatal fusion: carboxyfluorescein tracescells at light and electron microscopiclevels. Development 116:1087–1099.

Grigoriou M, Tucker AS, Sharpe PT, Pach-nis V. 1998. Expression and regulation ofLhx6 and Lhx7, a novel subfamily of LIMhomeodomain encoding genes, suggestsa role in mammalian head development.Development 125:2063–2074.

Gronthos S, Mankani M, Brahim J, RobeyPG, Shi S. 2000. Postnatal human dentalpulp stem cells (DPSCs) in vitro and invivo. Proc Natl Acad Sci U S A 97:13625–13630.

Gross JB, Hanken J. 2005. Cranial neuralcrest contributes to the bony skull vault


in adult Xenopus laevis: insights fromcell labeling studies. J Exp Zool B MolDev Evol 304:169–176.

Hagedorn L, Floris J, Suter U, Sommer L.2000. Autonomic neurogenesis and apo-ptosis are alternative fates of progenitorcell communities induced by TGFbeta.Dev Biol 228:57–72.

Hall BD, Cadle RG, Golabi M, Morris CA,Cohen MM Jr. 1992. Beare-Stevensoncutis gyrata syndrome. Am J Med Genet44:82–89.

Han J, Ito Y, Yeo JY, Sucov HM, Maas R,Chai Y. 2003. Cranial neural crest-de-rived mesenchymal proliferation is regu-lated by Msx1-mediated p19(INK4d) ex-pression during odontogenesis. Dev Biol261:183–196.

Hardcastle Z, Mo R, Hui CC, Sharpe PT.1998. The Shh signalling pathway intooth development: defects in Gli2 andGli3 mutants. Development 125:2803–2811.

Haworth KE, Healy C, Morgan P, SharpePT. 2004. Regionalisation of early headectoderm is regulated by endoderm andprepatterns the orofacial epithelium. De-velopment 131:4797–4806.

Headon DJ, Emmal SA, Ferguson BM,Tucker AS, Justice MJ, Sharpe PT, Zo-nana J, Overbeek PA. 2001. Gene defectin ectodermal dysplasia implicates adeath domain adapter in development.Nature 414:913–916.

Helms JA, Cordero D, Tapadia MD. 2005.New insights into craniofacial morpho-genesis. Development 132:851–861.

Hilliard SA, Yu L, Gu S, Zhang Z, Chen YP.2005. Regional regulation of palatalgrowth and patterning along the anteri-or-posterior axis in mice. J Anat 207:655–667.

Holland LZ, Laudet V, Schubert M. 2004.The chordate amphioxus: an emergingmodel organism for developmental biol-ogy. Cell Mol Life Sci 61:2290–308.

Hu G, Lee H, Price SM, Shen MM, Abate-Shen C. 2001. Msx homeobox genes in-hibit differentiation through upregula-tion of cyclin D1. Development 128:2373–2384.

Huang R, Zhi Q, Izpisua-Belmonte JC,Christ B, Patel K. 1999. Origin and de-velopment of the avian tongue muscles.Anat Embryol (Berl) 200:137–152.

Imai H, Osumi-Yamashita N, Ninomiya Y,Eto K. 1996. Contribution of early-emi-grating midbrain crest cells to the dentalmesenchyme of mandibular molar teethin rat embryos. Dev Biol 176:151–165.

Ishii M, Merrill AE, Chan YS, Gitelman I,Rice DP, Sucov HM, Maxson RE Jr.2003. Msx2 and Twist cooperatively con-trol the development of the neural crest-derived skeletogenic mesenchyme of themurine skull vault. Development 130:6131–6142.

Ishii M, Han J, Yen HY, Sucov HM, Chai Y,Maxson RE Jr. 2005. Combined deficien-cies of Msx1 and Msx2 cause impairedpatterning and survival of the cranialneural crest. Development 132:4937–4950.

Ito Y, Bringas P Jr, Mogharei A, Zhao J,Deng C, Chai Y. 2002. Receptor-regu-lated and inhibitory Smads are critical inregulating transforming growth factorbeta-mediated Meckel’s cartilage devel-opment. Dev Dyn 224:69–78.

Ito Y, Yeo JY, Chytil A, Han J, Bringas PJr, Nakajima A, Shuler CF, Moses HL,Chai Y. 2003. Conditional inactivation ofTgfbr2 in cranial neural crest causescleft palate and calvaria defects. Devel-opment 130:5269–5280.

Jacobson AG. 1963. The determination andpositioning of the nose, lens and ear. I.Interactions within the ectoderm, andbetween the ectoderm and underlyingtissues. J Exp Zool 154:273–283.

Jeffery WR, Strickler AG, Yamamoto Y.2004. Migratory neural crest-like cellsform body pigmentation in a urochordateembryo. Nature 431:696–699.

Jernvall J, Thesleff I. 2000. Reiterative sig-naling and patterning during mamma-lian tooth morphogenesis. Mech Dev 92:19–29.

Jerome LA, Papaioannou VE. 2001. Di-George syndrome phenotype in mice mu-tant for the T-box gene, Tbx1. Nat Genet27:286–291.

Jiang X, Iseki S, Maxson RE, Sucov HM,Morriss-Kay GM. 2002. Tissue originsand interactions in the mammalian skullvault. Dev Biol 241:106–116.

Jollie M. 1981. Segment theory and thehomologizing of cranial bone. Am Nat118:785–802.

Kaartinen V, Voncken JW, Shuler C, War-burton D, Bu D, Heisterkamp N, GroffenJ. 1995. Abnormal lung development andcleft palate in mice lacking TGF-beta 3indicates defects of epithelial-mesenchy-mal interaction. Nat Genet 11:415–421.

Kassai Y, Munne P, Hotta Y, Penttila E,Kavanagh K, Ohbayashi N, Takada S,Thesleff I, Jernvall J, Itoh N. 2005. Reg-ulation of mammalian tooth cusp pat-terning by ectodin. Science 309:2067–2070.

Kee Y, Bronner-Fraser M. 2005. To prolif-erate or to die: role of Id3 in cell cycleprogression and survival of neural crestprogenitors. Genes Dev 19:744–755.

Kollar EJ. 1972. Histogenetic aspects ofdermal-epidermal interactions. In: De-velopmental aspects of oral biology. NewYork: Academic Press. p 126–149.

Kontges G, Lumsden A. 1996. Rhombence-phalic neural crest segmentation is pre-served throughout craniofacial ontogeny.Development 122:3229–3242.

Kotton DN, Ma BY, Cardoso WV, Sander-son EA, Summer RS, Williams MC, FineA. 2001. Bone marrow-derived cells asprogenitors of lung alveolar epithelium.Development 128:5181–5188.

Kratochwil K, Dull M, Farinas I, GalceranJ, Grosschedl R. 1996. Lef1 expression isactivated by BMP-4 and regulates induc-tive tissue interactions in tooth and hairdevelopment. Genes Dev 10:1382–1394.

Kuratani S. 2004. Evolution of the verte-brate jaw: comparative embryology andmolecular developmental biology reveal

the factors behind evolutionary novelty.J Anat 205:335–347.

Kuratani S. 2005. Developmental studiesof the lamprey and hierarchical evolu-tionary steps towards the acquisition ofthe jaw. J Anat 207:489–499.

Kuratani S, Nobusada Y, Horigome N,Shigetani Y. 2001. Embryology of thelamprey and evolution of the vertebratejaw: insights from molecular and devel-opmental perspectives. Philos Trans RSoc Lond B Biol Sci 356:1615–1632.

LaBonne C, Bronner-Fraser M. 1999. Mo-lecular mechanisms of neural crest for-mation. Annu Rev Cell Dev Biol 15:81–112.

Lagasse E, Connors H, Al-Dhalimy M, Re-itsma M, Dohse M, Osborne L, Wang X,Finegold M, Weissman IL, Grompe M.2000. Purified hematopoietic stem cellscan differentiate into hepatocytes invivo. Nat Med 6:1229–1234.

Lan Y, Ovitt CE, Cho ES, Maltby KM,Wang Q, Jiang R. 2004. Odd-skipped re-lated 2 (Osr2) encodes a key intrinsicregulator of secondary palate growth andmorphogenesis. Development 131:3207–3216.

Le Douarin NM, Ziller C, Couly GF. 1993.Patterning of neural crest derivatives inthe avian embryo: in vivo and in vitrostudies. Dev Biol 159:24–49.

Le Douarin NM, Creuzet S, Couly G, DupinE. 2004. Neural crest cell plasticity andits limits. Development 131:4637–4650.

Le Lievre CS. 1978. Participation of neuralcrest-derived cells in the genesis of theskull in birds. J Embryol Exp Morphol47:17–37.

Lee SH, Bedard O, Buchtova M, Fu K,Richman JM. 2004. A new origin for themaxillary jaw. Dev Biol 276:207–224.

Lidral AC, Romitti PA, Basart AM,Doetschman T, Leysens NJ, Daack-Hir-sch S, Semina EV, Johnson LR, MachidaJ, Burds A, Parnell TJ, Rubenstein JL,Murray JC. 1998. Association of MSX1and TGFB3 with nonsyndromic cleftingin humans. Am J Hum Genet 63:557–568.

Light W, Vernon AE, Lasorella A, IavaroneA, LaBonne C. 2005. Xenopus Id3 is re-quired downstream of Myc for the forma-tion of multipotent neural crest progeni-tor cells. Development 132:1831–1841.

Lindsay EA, Vitelli F, Su H, Morishima M,Huynh T, Pramparo T, Jurecic V, Ogun-rinu G, Sutherland HF, Scambler PJ,Bradley A, Baldini A. 2001. Tbx1 haplo-insufficieny in the DiGeorge syndromeregion causes aortic arch defects in mice.Nature 410:97–101.

Liu W, Selever J, Lu MF, Martin JF. 2003.Genetic dissection of Pitx2 in craniofa-cial development uncovers new functionsin branchial arch morphogenesis, lateaspects of tooth morphogenesis and cellmigration. Development 130:6375–6385.

Liu W, Selever J, Murali D, Sun X, BruggerSM, Ma L, Schwartz RJ, Maxson R, Fu-ruta Y, Martin JF. 2005. Threshold-spe-cific requirements for Bmp4 in mandib-ular development. Dev Biol 283:282–293.


Lovell-Badge R. 2001. The future for stemcell research. Nature 414:88–91.

Lu MF, Pressman C, Dyer R, Johnson RL,Martin JF. 1999. Function of Rieger syn-drome gene in left-right asymmetry andcraniofacial development. Nature 401:276–278.

Lumsden A. 1984. Tooth morphogenesis:contributions of the cranial neural crestin mammals. In: Tooth morphogenesisand differentiation. Paris: Colloq. IN-SERM 125.

Lumsden AG. 1988. Spatial organization ofthe epithelium and the role of neuralcrest cells in the initiation of the mam-malian tooth germ. Development103(Suppl):155–169.

Lumsden A, Krumlauf R. 1996. Patterningthe vertebrate neuraxis. Science 274:1109–1115.

Martinez-Alvarez C, Tudela C, Perez-Miguelsanz J, O’Kane S, Puerta J, Fer-guson MW. 2000. Medial edge epithelialcell fate during palatal fusion. Dev Biol220:343–357.

Massague J. 2000. How cells read TGF-beta signals. Nat Rev Mol Cell Biol 1:169–178.

Massague J, Seoane J, Wotton D. 2005.Smad transcription factors. Genes Dev19:2783–2810.

Matsuo I, Kuratani S, Kimura C, TakedaN, Aizawa S. 1995. Mouse Otx2 func-tions in the formation and patterning ofrostral head. Genes Dev 9:2646–2658.

Matsuoka T, Ahlberg PE, Kessaris N,Iannarelli P, Dennehy U, RichardsonWD, McMahon AP, Koentges G. 2005.Neural crest origins of the neck andshoulder. Nature 436:347–355.

Matzuk MM, Kumar TR, Vassalli A, Bick-enbach JR, Roop DR, Jaenisch R, Brad-ley A. 1995. Functional analysis of ac-tivins during mammalian development.Nature 374:354–356.

Merrill AE, Bochukova EG, Brugger SM,Ishii M, Pitz DT, Wall SA, Lyons KM,Wilkie AM, Maxson RE. 2006. Cell mix-ing at a neural crest-mesoderm bound-ary and deficient ephrin-Eph signalingin the pathogenesis of craniosynostosis.Hum Mol Genet 15:1319–1328.

Meulemans D, Bronner-Fraser M. 2004.Gene-regulatory interactions in neuralcrest evolution and development. DevCell 7:291–299.

Miettinen PJ, Chin JR, Shum L, SlavkinHC, Shuler CF, Derynck R, Werb Z.1999. Epidermal growth factor receptorfunction is necessary for normal cranio-facial development and palate closure.Nat Genet 22:69–73.

Miletich I, Sharpe PT. 2004. Neural crestcontribution to mammalian tooth forma-tion. Birth Defects Res C Embryo Today72:200–212.

Miletich I, Buchner G, Sharpe PT. 2005.Barx1 and evolutionary changes in feed-ing. J Anat 207:619–622.

Mina M, Kollar EJ. 1987. The induction ofodontogenesis in non-dental mesen-chyme combined with early murine man-dibular arch epithelium. Arch Oral Biol32:123–127.

Mitsiadis TA, Angeli I, James C, LendahlU, Sharpe PT. 2003. Role of Islet1 in thepatterning of murine dentition. Develop-ment 130:4451–4460.

Mootoosamy RC, Dietrich S. 2002. Distinctregulatory cascades for head and trunkmyogenesis. Development 129:573–583.

Morriss-Kay GM, Wilkie AO. 2005. Growthof the normal skull vault and its alter-ation in craniosynostosis: insights fromhuman genetics and experimental stud-ies. J Anat 207:637–653.

Murray JC. 2002. Gene/environment causesof cleft lip and/or palate. Clin Genet 61:248–256.

Murray JC, Schutte BC. 2004. Cleft palate:players, pathways, and pursuits. J ClinInvest 113:1676–1678.

Mustonen T, Pispa J, Mikkola ML, Pum-mila M, Kangas AT, Pakkasjarvi L,Jaatinen R, Thesleff I. 2003. Stimulationof ectodermal organ development by Ec-todysplasin-A1. Dev Biol 259:123–136.

Niederreither K, Subbarayan V, Dolle P,Chambon P. 1999. Embryonic retinoicacid synthesis is essential for earlymouse post-implantation development.Nat Genet 21:444–448.

Niederreither K, Vermot J, Le Roux I,Schuhbaur B, Chambon P, Dolle P. 2003.The regional pattern of retinoic acid syn-thesis by RALDH2 is essential for thedevelopment of posterior pharyngealarches and the enteric nervous system.Development 130:2525–2534.

Noden DM. 1975. An analysis of migratorybehavior of avian cephalic neural crestcells. Dev Biol 42:106–130.

Noden DM. 1978. The control of avian ce-phalic neural crest cytodifferentiation. I.Skeletal and connective tissues. Dev Biol67:296–312.

Noden DM. 1983. The role of the neuralcrest in patterning of avian cranial skel-etal, connective, and muscle tissues. DevBiol 96:144–165.

Noden DM. 1986. Origins and patterningof craniofacial mesenchymal tissues. JCraniofac Genet Dev Biol Suppl 2:15–31.

Noden DM. 1988. Interactions and fates ofavian craniofacial mesenchyme. Devel-opment 103(Suppl):121–140.

Noden DM. 1991. Cell movements and con-trol of patterned tissue assembly duringcraniofacial development. J CraniofacGenet Dev Biol 11:192–213.

Noden DM, Trainor PA. 2005. Relationsand interactions between cranial meso-derm and neural crest populations. JAnat 207:575–601.

Nomura M, Li E. 1998. Smad2 role in me-soderm formation, left-right patterningandcraniofacialdevelopment.Nature393:786–790.

Northcutt RG. 2005. Taste bud develop-ment in the channel catfish. J CompNeurol 482:1–16.

Northcutt RG, Gans C. 1983. The genesisof neural crest and epidermal placodes: areinterpretation of vertebrate origins. QRev Biol 58:1–28.

Nuckolls GH, Shum L, Slavkin HC. 1999.Progress toward understanding craniofa-

cial malformations. Cleft Palate CraniofacJ 36:12–26.

Nugent P, Greene RM. 1998. MSX-1 geneexpression and regulation in embryonicpalatal tissue. In Vitro Cell Dev BiolAnim 34:831–835.

Osumi-Yamashita N, Noji S, Nohno T,Koyama E, Doi H, Eto K, Taniguchi S.1990. Expression of retinoic acid recep-tor genes in neural crest-derived cellsduring mouse facial development. FEBSLett 264:71–74.

Packard DS Jr, Meier S. 1983. An experi-mental study of the somitomeric organi-zation of the avian segmental plate. DevBiol 97:191–202.

Peters H, Neubuser A, Kratochwil K, Ball-ing R. 1998. Pax9-deficient mice lackpharyngeal pouch derivatives and teethand exhibit craniofacial and limb abnor-malities. Genes Dev 12:2735–2747.

Pispa J, Thesleff I. 2003. Mechanisms ofectodermal organogenesis. Dev Biol 262:195–205.

Plikus MV, Zeichner-David M, Mayer JA,Reyna J, Bringas P, Thewissen JG,Snead ML, Chai Y, Chuong CM. 2005.Morphoregulation of teeth: modulatingthe number, size, shape and differentia-tion by tuning Bmp activity. Evol Dev7:440–457.

Proetzel G, Pawlowski SA, Wiles MV, YinM, Boivin GP, Howles PN, Ding J, Fer-guson MW, Doetschman T. 1995. Trans-forming growth factor-beta 3 is requiredfor secondary palate fusion. Nat Genet11:409–414.

Qiu M, Bulfone A, Martinez S, Meneses JJ,Shimamura K, Pedersen RA, RubensteinJL. 1995. Null mutation of Dlx-2 resultsin abnormal morphogenesis of proximalfirst and second branchial arch deriva-tives and abnormal differentiation in theforebrain. Genes Dev 9:2523–2538.

Qiu M, Bulfone A, Ghattas I, Meneses JJ,Christensen L, Sharpe PT, Presley R,Pedersen RA, Rubenstein JLR. 1997.Role of the Dlx homeobox genes in proxi-modistal patterning of the branchialarches: mutations of Dlx-1, Dlx-2, andDlx-1 and -2 alter morphogenesis ofproximal skeletal and soft tissue struc-tures derived from the first and secondarches. Dev Biol 185:165–184.

Rice R, Spencer-Dene B, Connor EC, Gritli-Linde A, McMahon AP, Dickson C,Thesleff I, Rice DPC. 2004. Disruption ofFgf10/Fgfr2b-coordinated epithelial-mes-enchymal interactions causes cleft palate.J Clin Invest 113:1692–1700.

Rieger HH. 1935. Beitrage zur kenntnisseltener Missbildungen der Iris. GraefesArch Clin Exp Ophthalmol 133:602–635.

Rijli FM, Mark M, Lakkaraju S, Dierich A,Dolle P, Chambon P. 1993. A homeotictransformation is generated in the ros-tral branchial region of the head by dis-ruption of Hoxa-2, which acts as a selec-tor gene. Cell 75:1333–1349.

Robinson GW, Mahon KA. 1994. Differen-tial and overlapping expression domainsof Dlx-2 and Dlx-3 suggest distinct rolesfor Distal-less homeobox genes in cranio-


facial development. Mech Dev 48:199–215.

Ruhin B, Creuzet S, Vincent C, BenouaicheL, Le Douarin NM, Couly G. 2003. Pat-terning of the hyoid cartilage dependsupon signals arising from the ventral fo-regut endoderm. Dev Dyn 228:239–246.

Sanford L, Ormsby I, Gittenberger-deGroot A, Sariola H, Friedman R, BoivinG, Cardell E, Doetschman T. 1997. TG-Fbeta2 knockout mice have multiple de-velopmental defects that are non-over-lapping with other TGFbeta knockoutphenotypes. Development 124:2659–2670.

Santagati F, Rijli FM. 2003. Cranial neuralcrest and the building of the vertebratehead. Nat Rev Neurosci 4:806–818.

Santagati F, Minoux M, Ren SY, Rijli FM.2005. Temporal requirement of Hoxa2 incranial neural crest skeletal morphogen-esis. Development 132:4927–4936.

Sarkar L, Cobourne M, Naylor S, SmalleyM, Dale T, Sharpe PT. 2000. Wnt/Shhinteractions regulate ectodermal bound-ary formation during mammalian toothdevelopment. Proc Natl Acad Sci U S A97:4520–4524.

Sasaki T, Ito Y, Xu X, Han J, Bringas P Jr,Maeda T, Slavkin HC, Grosschedl R,Chai Y. 2005. LEF1 is a critical epithe-lial survival factor during tooth morpho-genesis. Dev Biol 278:130–143.

Sasaki T, Ito Y, Bringas P Jr, Chou S,Urata MM, Slavkin H, Chai Y. 2006.TGF{beta}-mediated FGF signaling iscrucial for regulating cranial neuralcrest cell proliferation during frontalbonedevelopment.Development133:371–381.

Satokata I, Maas R. 1994. Msx1 deficientmice exhibit cleft palate and abnormali-ties of craniofacial and tooth develop-ment. Nat Genet 6:348–356.

Satokata I, Ma L, Ohshima H, Bei M, WooI, Nishizawa K, Maeda T, Takano Y,Uchiyama M, Heaney S, Peters H, TangZ, Maxson R, Maas R. 2000. Msx2 defi-ciency in mice causes pleiotropic defectsin bone growth and ectodermal organformation. Nat Genet 24:391–395.

Saunders JW Jr. 1966. Death in embryonicsystems. Science 154:604–612.

Schneider RA, Helms JA. 2003. The cellu-lar and molecular origins of beak mor-phology. Science 299:565–568.

Selleck MA, Scherson TY, Bronner-FraserM. 1993. Origins of neural crest cell di-versity. Dev Biol 159:1–11.

Selleri L, Depew MJ, Jacobs Y, Chanda SK,Tsang KY, Cheah KS, Rubenstein JL,O’Gorman S, Cleary ML. 2001. Require-ment for Pbx1 in skeletal patterning andprogramming chondrocyte proliferationand differentiation. Development 128:3543–3557.

Semina EV, Reiter R, Leysens NJ, AlwardWL, Small KW, Datson NA, Siegel-Bar-telt J, Bierke-Nelson D, Bitoun P, ZabelBU, Carey JC, Murray JC. 1996. Cloningand characterization of a novel bicoid-related homeobox transcription factorgene, RIEG, involved in Rieger syn-drome. Nat Genet 14:392–399.

Serbedzija GN, Bronner-Fraser M, FraserSE. 1992. Vital dye analysis of cranialneural crest cell migration in the mouseembryo. Development 116:297–307.

Shah NM, Groves AK, Anderson DJ. 1996.Alternative neural crest cell fates are in-structively promoted by TGFbeta super-family members. Cell 85:331–343.

Shigetani Y, Sugahara F, Kawakami Y,Murakami Y, Hirano S, Kuratani S.2002. Heterotopic shift of epithelial-mes-enchymal interactions in vertebrate jawevolution. Science 296:1316–1319.

Shimamura K, Rubenstein JL. 1997. In-ductive interactions direct early region-alization of the mouse forebrain. Devel-opment 124:2709–2718.

Shuler CF. 1995. Programmed cell deathand cell transformation in craniofacialdevelopment. Crit Rev Oral Biol Med 6:202–217.

Shuler CF, Halpern DE, Guo Y, Sank AC.1992. Medial edge epithelium fate tracedby cell lineage analysis during epithelial-mesenchymal transformation in vivo.Dev Biol 154:318–330.

Shum L, Takahashi K, Takahashi I, Na-gata M, Tan DP, Semba I, Tanaka O,Bringas P, Nuckolls G, Slavkin HC.2000. Embryogenesis and the classifica-tion of craniofacial dysmorphogenesis.In: Oral and maxillofacial surgery. Phil-adelphia: WB Saunders Co. p 149–194.

Simeone A, Acampora D, Pannese M,D’Esposito M, Stornaiuolo A, GulisanoM, Mallamaci A, Kastury K, Druck T,Huebner K. 1994. Cloning and charac-terization of two members of the verte-brate Dlx gene family. Proc Natl AcadSci U S A 91:2250–2254.

Slavkin HC, Beierle J, Bavetta LA. 1968.Odontogenesis: cell–cell interactions invitro. Nature 217:269–270.

Sofaer JA. 1969. The genetics and expres-sion of a dental morphological variant inthe mouse. Arch Oral Biol 14:1213–1223.

Sofaer JA. 1977. The teeth of the “sleek”mouse. Arch Oral Biol 22:299–301.

Spemann H. 1938. Embryonic develop-ment and induction. New Haven: YaleUniversity Press.

St. Amand TR, Zhang Y, Semina EV, ZhaoX, Hu Y, Nguyen L, Murray JC, Chen Y.2000. Antagonistic signals betweenBMP4 and FGF8 define the expression ofPitx1 and Pitx2 in mouse tooth-forminganlage. Dev Biol 217:323–332.

Stock DW, Ellies DL, Zhao Z, Ekker M,Ruddle FH, Weiss KM. 1996. The evolu-tion of the vertebrate Dlx gene family.Proc Natl Acad Sci U S A 93:10858–10863.

Stottmann RW, Anderson RM, Klingen-smith J. 2001. The BMP antagonistsChordin and Noggin have essential butredundant roles in mouse mandibularoutgrowth. Dev Biol 240:457–473.

Sun X, Meyers EN, Lewandoski M, MartinGR. 1999. Targeted disruption of Fgf8causes failure of cell migration in thegastrulating mouse embryo. Genes Dev13:1834–1846.

Szeto DP, Rodriguez-Esteban C, Ryan AK,O’Connell SM, Liu F, Kioussi C, Gleiber-

man AS, Izpisua-Belmonte JC, Rosen-feld MG. 1999. Role of the Bicoid-relatedhomeodomain factor Pitx1 in specifyinghindlimb morphogenesis and pituitarydevelopment. Genes Dev 13:484–494.

Thomas BL, Tucker AS, Qui M, FergusonCA, Hardcastle Z, Rubenstein JL,Sharpe PT. 1997. Role of Dlx-1 and Dlx-2genes in patterning of the murine denti-tion. Development 124:4811–4818.

Thyagarajan T, Totey S, Danton MJ,Kulkarni AB. 2003. Genetically alteredmouse models: the good, the bad, and theugly. Crit Rev Oral Biol Med 14:154–174.

Trainor PA, Tan SS, Tam PP. 1994. Cra-nial paraxial mesoderm: regionalisationof cell fate and impact on craniofacialdevelopment in mouse embryos. Devel-opment 120:2397–2408.

Trainor PA, Krumlauf R. 2000. Patterningthe cranial neural crest: hindbrain seg-mentation and Hox gene plasticity. NatRev Neurosci 1:116–124.

Trainor PA, Ariza-McNaughton L, Krum-lauf R. 2002. Role of the isthmus andFGFs in resolving the paradox of neuralcrest plasticity and prepatterning. Sci-ence 295:1288–1291.

Trokovic N, Trokovic R, Mai P, Partanen J.2003. Fgfr1 regulates patterning of thepharyngeal region. Genes Dev 17:141–153.

Trokovic N, Trokovic R, Partanen J. 2005.Fibroblast growth factor signalling andregional specification of the pharyngealectoderm. Int J Dev Biol 49:797–805.

Trumpp A, Depew MJ, Rubenstein JLR,Bishop JM, Martin GR. 1999. Cre-medi-ated gene inactivation demonstratesthat FGF8 is required for cell survivaland patterning of the first branchialarch. Genes Dev 13:3136–3148.

Tucker AS, Lumsden A. 2004. Neural crestcells provide species-specific patterninginformation in the developing branchialskeleton. Evol Dev 6:32–40.

Tucker AS, Sharpe PT. 1999. Moleculargenetics of tooth morphogenesis and pat-terning: the right shape in the rightplace. J Dent Res 78:826–834.

Tucker AS, Sharpe P. 2004. The cutting-edge of mammalian development; howthe embryo makes teeth. Nat Rev Genet5:499–508.

Tucker AS, Al Khamis A, Sharpe PT.1998a. Interactions between Bmp-4 andMsx-1 act to restrict gene expression toodontogenic mesenchyme. Dev Dyn 212:533–539.

Tucker AS, Matthews KL, Sharpe PT.1998b. Transformation of tooth type in-duced by inhibition of BMP signaling.Science 282:1136–1138.

Tucker AS, Yamada G, Grigoriou M, Pach-nis V, Sharpe PT. 1999. Fgf-8 determinesrostral-caudal polarity in the firstbranchial arch. Development 126:51–61.

Tucker AS, Headon DJ, Courtney JM,Overbeek P, Sharpe PT. 2004. The acti-vation level of the TNF family receptor,Edar, determines cusp number and toothnumber during tooth development. DevBiol 268:185–194.


Twigg SR, Kan R, Babbs C, Bochukova EG,Robertson SP, Wall SA, Morriss-KayGM, Wilkie AO. 2004. Mutations of eph-rin-B1 (EFNB1), a marker of tissueboundary formation, cause craniofronto-nasal syndrome. Proc Natl Acad Sci U SA 101:8652–8657.

Vaziri Sani F, Hallberg K, Harfe BD, Mc-Mahon AP, Linde A, Gritli-Linde A.2005. Fate-mapping of the epithelialseam during palatal fusion rules out ep-ithelial-mesenchymal transformation.Developmental Biology 285:490–495.

Veitch E, Begbie J, Schilling TF, SmithMM, Graham A. 1999. Pharyngeal archpatterning in the absence of neural crest.Curr Biol 9:1481–1484.

Wendling O, Dennefeld C, Chambon P,Mark M. 2000. Retinoid signaling is es-sential for patterning the endoderm ofthe third and fourth pharyngeal arches.Development 127:1553–1562.

Wilkie AO, Morriss-Kay GM. 2001. Genet-ics of craniofacial development and mal-formation. Nat Rev Genet 2:458–468.

Wilson J, Tucker AS. 2004. Fgf and Bmpsignals repress the expression of Bapx1in the mandibular mesenchyme and con-trol the position of the developing jawjoint. Dev Biol 266:138–150.

Woodbury D, Schwarz EJ, Prockop DJ,Black IB. 2000. Adult rat and humanbone marrow stromal cells differentiateinto neurons. J Neurosci Res 61:364–370.

Wu P, Jiang TX, Suksaweang S, WidelitzRB, Chuong CM. 2004. Molecular shap-ing of the beak. Science 305:1465–1466.

Xu X, Han J, Ito Y, Bringas P Jr, Chai Y.2006. Cell autonomous requirement forTgfbr2 in the disappearance of MedialEdge Epithelium during palatal fusion.Dev Biol. (in press).

Yelick PC, Schilling TF. 2002. Moleculardissection of craniofacial developmentusing zebrafish. Crit Rev Oral Biol Med13:308–322.

Yu JK, Holland ND, Holland LZ. 2002. Anamphioxus winged helix/forkhead gene,AmphiFoxD: insights into vertebrate

neural crest evolution. Dev Dyn 225:289–297.

Yu L, Gu S, Alappat S, Song Y, Yan M,Zhang X, Zhang G, Jiang Y, Zhang Z,Zhang Y, Chen Y. 2005. Shox2-deficientmice exhibit a rare type of incompleteclefting of the secondary palate. Develop-ment 132:4397–4406.

Zhang Z, Song Y, Zhao X, Zhang X, FerminC, Chen Y. 2002. Rescue of cleft palate inMsx1-deficient mice by transgenic Bmp4reveals a network of BMP and Shh sig-naling in the regulation of mammalianpalatogenesis. Development 129:4135–4146.

Zhao Y, Guo YJ, Tomac AC, Taylor NR,Grinberg A, Lee EJ, Huang S, WestphalH. 1999. Isolated cleft palate in micewith a targeted mutation of the LIM ho-meobox gene lhx8. Proc Natl Acad Sci US A 96:15002–15006.

Zhao H, Bringas P Jr, Chai Y. 2006. An invitro model for characterizing the post-migratory cranial neural crest cells ofthe first branchial arch. Dev Dyn 235:1433–1440.


Chai & Maxson [2006] Recent Advances in Craniofacial Morpholgenesis

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