Cranial neural crest and the building of the vertebrate head

806 | OCTOBER 2003 | VOLUME 4 www.nature.com/reviews/neuro

R E V I E W S

The craniofacial skeleton is a complex system ofinterconnected bones, the correct formation of whichis necessary for proper encasing of the brain and sensory organs, and for the normal functioning of thedigestive and respiratory tracts. It is derived largelyfrom one of the most versatile cell types of the embryo— the neural crest1.

The neural crest is a migratory cell population thatdetaches from the embryonic neural epithelium.Vertebrate species exhibit different patterns of neuralcrest cell (NCC) emigration. For example, in mammals,NCCs begin to emigrate from the tip or ‘crest’of the still-open neural folds2, whereas in birds NCCs arise only afterneural tube closure3. NCCs undergo an epithelial–mesenchymal transition and migrate away from theneural epithelium in streams to different regions ofthe embryo, where they contribute to the formation of various structures1. The processes of induction andmigration of NCCs have been extensively studied,and have been reviewed recently4–6. In this review, wewill focus on later aspects of neural crest specification,and in particular, on the involvement of NCCs in thedevelopment of the head and facial skeleton.

NCCs can differentiate into a broad range of celltypes once they reach their final destination1 (TABLE 1).Cranial NCCs arise from the DIENCEPHALON, the midbrainand the hindbrain, in distinct populations. They differ from trunk neural crest in that they have the potential to differentiate into cartilage, bone and

connective tissue1,7,8 (TABLE 1; FIG. 1). The rostral cranialNCCs give rise to the frontonasal skeleton, whereas themore posterior cranial NCCs fill the pharyngeal arches— also known as branchial (gill) arches in fish andamphibians — where they form the cartilage and bone ofthe jaw, middle ear and neck1,7,9–11. Cranial NCCs alsomake important contributions to the membranousbones of the skull vault7,10,12,13 (FIG. 1).

A recent series of experimental observations inmouse, chick and zebrafish has revived interest in specificaspects of head morphogenesis (REFS 14–16 and referencestherein). In this review, we discuss some of the mostrecent findings on molecular and cellular aspects ofNCC patterning and facial skeletal morphogenesis.We will present an integrated view of some seeminglycontradictory experimental observations and put themin the context of the molecular mechanisms that aredeployed to build the vertebrate head.

Establishing spatial identity in cranial NCCs To understand how cranial NCCs acquire their spatialidentity, it is pertinent to consider how spatial coordi-nates are established and interpreted along body axes17,18.The cranial neural crest might use a similar patterningstrategy to the developing nervous system, whereby agrid-like system of positional cues is created by gradientsof extracellular signals or morphogens and their intracel-lular molecular effectors. In the ventral spinal cord, forinstance, a gradient of sonic hedgehog (Shh) originates in

CRANIAL NEURAL CREST AND THEBUILDING OF THE VERTEBRATE HEADFabio Santagati and Filippo M. Rijli

Head development in vertebrates involves a complex series of molecular and morphogeneticevents that generate a coordinated pattern of cartilages, bones and nerves, and result inspecies-specific craniofacial morphologies. A specialized cell type of neural origin, the neuralcrest, is central to this process, as it provides the main source of craniofacial mesenchyme. The degree of patterning information that is intrinsic to the neural crest has been recentlydebated, and new advances have underscored the influence of environmental signalling on the transcriptional readout that coordinates craniofacial morphogenesis in space and time.

DIENCEPHALON

The more posterior of the twosubdivisions of the forebrain(the anterior subdivision beingthe telencephalon). Structuresthat are derived from thediencephalon include the retina,the pineal gland, the thalamusand the hypothalamus.

Institut de Génétique et deBiologie Moléculaire etCellulaire, Centre Nationalde la RechercheScientifique/InstitutNational de la Santé et de laRecherche Médicale/Université Lousis Pasteur,BP 10142-67404 IllkirchCedex, CU de Strasbourg,France.e-mail:[email protected]:10.1038/nrn1221

NATURE REVIEWS | NEUROSCIENCE VOLUME 4 | OCTOBER 2003 | 807

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FLOOR PLATE

The neural tube has beendivided into different regions.The ventral cells closest to themidline constitute the floorplate. The dorsal cells closest tothe midline correspond to theroof plate. The alar plate (dorsal)and the basal plate (ventral) liebetween these two cellpopulations, and are separatedby the sulcus limitans.

HOMEODOMAIN

A 60-amino-acid DNA-bindingdomain that comprises three α-helices and is found in manytranscription factors.

RETINOIC ACID

A derivative of vitamin A thatacts as a morphogen andregulator of differentiationduring embryogenesis.

between R1 and R2, but the NCCs that arise from R2 and migrate into the first arch are devoid of Hoxgene expression45,46 (FIG. 2).

The AP distribution of Hox expression patterns in theNCC-derived mesenchyme is important for determiningthe rostrocaudal identity of the pharyngeal arches. Thisis best illustrated by the outcome of the targeted muta-genesis of Hoxa2 in the mouse. Hoxa2 depletion causesthe second (hyoid) arch to lose its identity — instead ofmaking its own distinctive structures, it undergoes ahomeotic transformation into first (mandibular) archskeletal elements, albeit with reverse polarity47,48. As firstarch NCCs are devoid of Hox expression, Hoxa2 seems to superimpose a second-arch-specific mode of develop-ment on a first-arch-like default pathway47.

The role of Hoxa2 as a selector gene for second archdevelopment has been confirmed by complementarygain-of-function experiments in the chick and frog.Ectopic expression of Hoxa2 inhibited the formation offirst arch structures and resulted in a duplicated series of second arch elements49,50. Importantly, recent knock-down and gain-of-function approaches in zebrafishyielded second-to-first or first-to-second arch homeotictransformations, respectively, confirming the conservedrole of Hoxa2 (REF. 51). However, zebrafish hoxa2 actsredundantly with its paralogue hoxb2, and inactivation ofboth gene products is required to generate the homeoticphenotype. By contrast, Hoxb2 knockout in the mousedid not have any evident effect on skeletal NCC pattern-ing52,53, even in conjunction with the Hoxa2 mutation (F. M. R., unpublished data). This might be explained by the early downregulation of Hoxb2 expression in post-migratory NCCs in the mouse42, which does not occur in the zebrafish51. These results underscore some impor-tant regulatory differences for the Hox genes between vertebrate species.

Functional inactivations of Hox paralogous group 1and 3 genes (BOX 1) did not yield clear homeotic transfor-mations, but instead produced selective deletions or malformations of NCC skeletal derivatives of the secondarch, or of the third and fourth arches, respectively54–57.This confirms an important requirement for Hoxgenes for NCC rostrocaudal patterning. More severephenotypes were obtained in compound mutants, againindicating partial functional redundancy of Hox genes,as well as synergistic activities between paralogous members58–60.

The Hox-negative cranial NCCs that are rostral to the second arch are specified by other classes of homeo-domain transcription factors. Otx2 is expressed in theNCCs of the frontonasal region and in the first archNCCs that originate in the midbrain61 (FIG. 2). The Otx2-expressing NCCs of the first arch form the distal (ventral)jaw elements, whereas the rhombomeric Otx2-negativecells contribute to the proximal (dorsal) part of the arch.Accordingly, the Otx2 mutation mainly affects the devel-opment of frontonasal and distal mandibular elements62.It is noteworthy that in Hoxa2 mutants, the transforma-tion of the second arch into the first arch is limited to theproximal structures47,48, indicating that distal structuresrequire additional AP specification from Otx2.

the FLOOR PLATE, and in response to this gradient, cellsexpress various combinations of HOMEODOMAIN trans-cription factors, which define the progenitor domains for at least five different classes of neurons (reviewed inREFS 19,20).Along the anteroposterior (AP) axis, positionalidentity is determined by the nested expression ofanother class of homeodomain factors, the Hox genes(BOX 1). Early Hox expression in the neural tube is regulated by graded activities of RETINOIC ACID (RA) andlocalized sources of growth factors, such as FIBROBLAST

GROWTH FACTORS (FGFs) or WNT MOLECULES21–33. In the hindbrain, members of the Hox gene family specify the AP identity of transverse compartments of progenitorcells known as RHOMBOMERES (R1–R7; FIG. 1) (reviewed inREFS 34–36). By integrating the activities of homeodomaineffectors along the AP and dorsoventral (DV) axes, neuralprogenitors can acquire a specific developmental programme. Similarly, the cranial NCCs that colonize the pharyngeal arches could receive spatial informationthat defines their positional identity and restricts theirdevelopmental potential.

Anteroposterior positional identity. The NCCs thatarise from the hindbrain migrate in separatestreams37–39, resulting in the transposition of the Hoxgene expression code from the AP axis of the neuraltube to the AP axis of the head and pharyngeal archmesenchyme40,41. However, the Hox code of the NCCs isnot a simple replica of the neural tube expression pattern, but it can be respecified at the beginning ofmigration42–44. For example, Hoxa2 expression in theneural tube has its anterior limit at the boundary

Table 1 | Main derivatives of neural crest cells

Cranial Crest Trunk Crest

Sensory nervous system Ganglia of cranial nerves Spinal ganglia

Autonomic nervous Enteric nervous system Enteric nervous systemsystem (minor contribution)

Parasympathetic ganglia: Parasympathetic ciliary, pterygopalatine, otic ganglia: pelvic plexus.and submandibular

Sympathetic ganglia: superior cervical, stellate,celiac, superior and inferiormesenteric, aorticorenal.

Non-neuronal cells Satellite cells of ganglia. Satellite cells of ganglia.Schwann cells of cranial Schwann cells of peripheralnerves nerves

Pigment cells Melanocytes Melanocytes

Endocrine and Calcitonin-producing cells, Adrenal medullaparaendocrine cells type I cells of carotid body and

parafollicular cells of thyroid

Skeleton Face and skull bones, and Nonevisceral cartilages

Connective tissue Dermis, fat and smooth muscle Noneof skin; ciliary muscles; cornea;stroma of head and neck glands;dental papilla; walls of aortic andarch-derived arteries; meninges of prosencephalon and part of the mesencephalon

Modified, with permission, from REF. 58 © (1996) McGraw-Hill.


R E V I E W S

ventral domain is suppressed by Dlx5 and Dlx6, whoseexpression is ventrally restricted. Loss of Dlx5/Dlx6expression might cause a ventral gain-of-function ofDlx1/Dlx2, leading to a homeotic transformation intodorsal structures. It would be interesting to test whetherectopic expression of Dlx5/Dlx6 in the dorsal half of thefirst arch is sufficient to generate a reverse homeoticphenotype, namely an upper-to-lower-jaw transforma-tion. By analogy with the posterior prevalence model ofHox function69, which states that posteriorly expressedHox proteins are functionally dominant over those thatare expressed more anteriorly, a ‘ventral’ prevalencemodel involving Dlx genes might operate along the DVaxis of the pharyngeal arches.

The conserved regulation of the paralogous pair Dlx3and Dlx7 in the distal-most tips of the pharyngeal archesindicates that these genes might contribute to the specifi-cation of ventral elements70, although a role in providingDV positional identity is still in question. Interestingly, aseries of craniofacial malformations was observed inhumans carrying DLX3 mutations71. Because of the earlylethality of mouse Dlx3 mutants72, a conditional muta-genesis strategy will be necessary to address the preciserequirement for these genes in NCC patterning.

Integrating anteroposterior and dorsoventral cues. APpositional addresses and Hox codes seem to be providedat NCC premigratory stages11,46,73–76, whereas Dlx codes

The AP identity of each subpopulation of NCCs hasa strong influence on the development and assembly ofthe craniofacial skeleton. Cells of distinct AP origins donot generally mix, and although they can contribute tothe formation of the same structures, they remain segre-gated63. It will be interesting to address whether HDtranscription-factor expression is involved in keepingthe NCC populations segregated.

Dorsoventral positional identity. The Dlx genes areexpressed in a nested pattern along the DV axis of thepharyngeal arches (FIG. 2), implying that they providepositional identity to NCCs along this axis (reviewed inREF. 64). This was recently confirmed by the simultaneousinactivation of Dlx5 and Dlx6 in the mouse, which gener-ated a DV homeotic transformation. The ventral elements of the first arch were transformed into a mirrorimage of the dorsal (maxillary) elements, generating a mouse with two upper jaws facing each other, andrevealing an underlying default tendency of the first archto form upper-jaw-like structures65,66. This outcome isparticularly important from an evolutionary perspective,because it might recall an atavistic form of symmetricalmouth in primitive vertebrates65.

Dlx1 and Dlx2 are expressed homogeneouslythroughout the DV axis, yet they selectively control thepatterning of dorsal pharyngeal arch structures67,68.It is possible that the function of these genes in the

FIBROBLAST GROWTH FACTORS

(FGFs). Multifunctional factorsthat are involved in embryonicdevelopment. More than 20FGFs and 4 FGF receptors havebeen described. Theircoordinated activity controls cellproliferation, migration, survivaland differentiation. FGFsregulate growth andmorphogenesis by early actionon regional patterning, and alater effect on the growth ofprogenitor cells of the forebrain.

WNT PROTEINS

A family of highly conservedsecreted signalling moleculesthat regulate cell–cellinteractions duringembryogenesis. Wnt proteinsbind on the cell surface toreceptors of the Frizzled family.

RHOMBOMERES

Neuroepithelial segments foundtransiently in the embryonichindbrain that adopt distinctmolecular and cellularproperties, restrictions in cellmixing, and ordered domains ofgene expression.

Diencephalic + anterior mesencephalic NC

Posterior mesencephalic NC

R1 + R2 + R3 NC

R4 + R3 + R5 NC

R6 + R5 NC

FR

DE

MX

HY

SQ

MA

ST

IN

ZY

NA

ASFNP

R1R2

R3R4

mes

R5R6

R7

di

BA1

NA

NA

SO Embryo Human

PM

PMNC

MX

MX

FR

PL

FR

PA

PA

SQ

SQ

DE

DE

JU

ASPT

EN

BA

CO

PA

RPEB

QU

CBAR AN

IS

JU

Bird

Mouse

BA2BA3

Figure 1 | Skeletal fate of cranial neural crest cells in vertebrates. The embryo figure shows colonization of the head and pharyngeal arches by diencephalic,anterior and posterior mesencephalic, and rhombencephalic neural crest cells (NCCs), as indicated by the colour code. The diagram is representative of chick, mouse,and human embryos, although the NCC migratory pathways might differ slightly in different species. The skull drawings show comparative contributions of NCCpopulations to cranial skeletal elements of humans, mice and birds. Drawings are based on NCC fate-mapping studies and on extrapolation of avian and mouse datato known homologues in the human7,9–13,63,156,157. Some bones, including the squamosal (SQ), alisphenoid (AS), and pterygoid (PT), are shown with mixed contributionfrom different NCC populations. Note that in mammals the frontal (FR) and parietal (PA) bones have been reported to be of neural crest and mesodermal origin,respectively13. In birds, the frontal and parietal bones have been reported to be either entirely derived from NCCs12, as shown in the figure, or derived from a dual neuralcrest/mesodermal origin7,10. AN, angular bone; AR, articular bone; BA, basihyal; BA1–BA3, pharyngeal arches 1–3; CB, ceratobranchial; CO, columella; DE, dentarybone; di, diencephalon; EB, epibranchial; EN, entoglossum; FNP, frontonasal process; HY, hyoid bone; IN, incus; IS, interorbital septum; JU, jugal bone; MA, malleus;mes, mesencephalon; MX, maxillary bone; NA, nasal bone; NC, nasal capsule; PL, palatine bone; PM, premaxillary bone; QU, quadrate; RP, retroarticular process;R1–R7, rhombomeres 1–7; SO, scleral ossicles; ST, stapes; ZY, zygomatic bone.


R E V I E W S

NCC prepatterning versus plasticityThere has been a long-standing debate about cranialNCC prepatterning versus plasticity. One could considertwo extreme views — skeletogenic NCCs could be irreversibly committed before they migrate into the pha-ryngeal arches, or they could receive no patterninginformation in the neural tube and maintain a broadplasticity until they reach their final destination. Theaccumulating evidence challenges both of these models,and indicates that the truth lies somewhere in-between.

The field has been greatly influenced by the classicalneural crest grafting experiments in the avian embryoby Noden in the 1980s11. In his seminal work, Nodenpostulated that cranial NCCs are “specified with respectto their morphogenetic potential prior to their leavingthe neural tube”11. On the other hand, he also pointedout that patterning of NCC-derived arch components“is the result of a series of interactions between the crestpopulation and the surrounding tissue”11. Over the past 10 years, the amount of intrinsic morphogeneticinformation carried by the cranial neural crest has been further investigated in various vertebrate models,including mouse, chick and zebrafish. These analyseshave resulted in contrasting interpretations (reviewed

and DV positional identity seem to be established laterwithin the pharyngeal arch environment in response topolarizing signals from the surrounding epithelia (seelater text). The AP positional addresses provided by theHox genes distinguish the segmental identity of each archfrom that of its neighbours, whereas the Dlx-mediatedDV patterning operates within each arch without affect-ing AP segmental identity. An interesting parallel can bedrawn with AP and DV patterning mechanisms in thehindbrain, where AP and DV spatial coordinates are setindependently and sequentially, with DV positional values still being labile by the time AP values are fixed77.

After AP and DV positional values have been established, the molecular information provided at theirintersection is converted into a NCC differentiation pro-gramme that is specific for each axial level. It is conceivable that a third dimension of information isprovided along the mediolateral axis of the pharyngealarches, although there is little evidence for differentialgene expression along this axis. At present, it is presumed that signals from the underlying endodermand/or the axial mesoderm contribute to the inductionof midline or lateral structures. Further studies will berequired to resolve this issue.

Box 1 | Homeobox genes

The homeobox148 is a 180 base pair sequence that encodesa DNA-binding domain called the homeodomain, whichis shared by a large group of transcription factors. Thehomeobox was originally identified in Drosophila genes,mutation of which resulted in ‘homeotic’transformations149 whereby one structure wastransformed into the likeness of another; for example,antennae into legs. This class of homeobox genes isreferred to as the homeotic complex (HOM-C)150, due totheir clustered organization.

The Hox genes are the vertebrate homologues of theDrosophila HOM-C genes, and they share conserved rolesin patterning along the anteroposterior axis of theembryo151. In mammals, there are four Hox clusters —HOXA, HOXB, HOXC and HOXD in humans, Hoxa,Hoxb, Hoxc and Hoxd in mice — which are located ondifferent chromosomes. These clusters arose byduplications of a single ancestral Hox complex152. Hoxgenes that occupy the same position in different clustersare called paralogues; for example, Hoxa4, Hoxb4, Hoxc4and Hoxd4 represent paralogous group 4. Mammals have13 paralogous groups. The genes within a paralogousgroup share a high degree of homology and have similar expression patterns. During evolution, some paralogous groupslost one or two members, so that none of the four clusters now has a complete set of 13 genes.

The physical position of each gene in the cluster correlates with the rostral border of its expression domain along theanteroposterior axis. Those genes located closest to the 3′-end show the most rostral limits of expression (with theexception of the group 1 genes, which exhibit an anterior expression border between those of groups 2 and 3). Hox geneslocated further towards the 5′-end of the cluster are expressed in progressively more posterior domains. This feature iscalled spatial collinearity153,154.

The figure shows the four Hox clusters, with each paralogous group identified by a specific colour code. The position ofeach paralogous group corresponds approximately to the rostral border of its expression domain in the mammalianembryo. Detailed expression patterns are shown for Hox paralogous groups 1–4 in the rhombomeres. The colouring inthe hindbrain indicates the most rostral rhombomeres that express each gene — with the exception of the group 1 genes,the expression domains generally continue through the spinal cord to the posterior end of the neural tube.

Hoxa 3′Hoxb 3′

Hoxc 3′Hoxd 3′

5′ 5′ 5′ 5′

1 23

4

5

6

7

8

9

1011

12

13


R E V I E W S

transferred to a Hox-negative environment, they losetheir Hox code and reprogramme their differentiationcourse. However, if a large block of cells is transposed toan ectopic location, the original Hox code is maintained,indicating that cross-talk among like cells is necessary topreserve their identity46,74,84,86.

Whether a Hox-expressing environment is sufficientto induce Hox expression in Hox-negative NCCs dependson the experimental conditions46,74–76,84,86. In Noden’sexperiments, the transplantation of Hox-negative neuralfolds to the prospective second arch premigratory regionof the chick embryo resulted in a duplication of jaw elements in place of hyoid arch derivatives, similar to the outcome of the targeted inactivation of Hoxa2 in themouse11,47,48,74. However, it has recently been shown thatthis transformation is only generated when tissue fromthe Fgf8-expressing isthmus at the mid-hindbrainboundary is transplanted along with the premigratoryNCC precursors76 (FIG. 3). Fgf8 has Hoxa2 silencing activity24, and it might repress Hoxa2 expression in theNCCs that emerge from the region around the graft,resulting in an ectopic first arch fate. In a similar experi-ment where carry-over of isthmus tissue was avoided,a normal second arch skeleton was generated76. It will be important to assess whether Hoxa2 expression can be induced in Hox-negative grafted cells under theseconditions.

These results, together with the outcome of Hoxa2knockout in the mouse, indicate that Hoxa2 expressionin NCCs is necessary for the patterning of hyoid skeletalelements. Accordingly, selective deletion of Hoxa2in NCC precursors reproduces the same homeotictransformation as complete inactivation of Hoxa2 in themouse (S.-Y. Ren, M. Pasqualetti & F. M. R., unpublisheddata). But is Hoxa2 expression in Hox-negative NCCssufficient to promote second arch patterning? Hoxa2overexpression in presumptive first arch NCCs did notresult in first-to-second arch transformation49,80 unlessHoxa2 was also induced in the tissues surrounding theNCCs49,50. So, a Hox-expressing compatible environmentis required for the development of Hox-expressingNCCs. The pharyngeal arch mesoderm is devoid of Hoxexpression, but the second and posterior pharyngealarch ectoderm expresses Hox genes40, in addition to the neural tube, and the regulation of this expression is independent of the NCC mesenchyme74. So, Hox-dependent signals from the ectoderm and/or neuro-ectoderm might contribute to the patterning of the NCC mesenchyme in the second and posterior arches.Tissue-specific conditional deletions of Hox genesshould allow this question to be addressed.

Hox-negative NCCs. How much intrinsic morpho-genetic information is carried by the Hox-negativeNCCs that contribute to the frontonasal and facialskeleton, and how much patterning information dothey receive in the head or pharyngeal arch environ-ment? Most of the experimental data seem to reinforcean instructive role for epithelial tissues in the induction of mesenchyme outgrowth and morphogenesis fromthis NCC population. Signalling molecules from the

in REFS 41,44,78) (FIGS 3 and 4). To clarify the situation, it is necessary to revisit and attempt to integrate these results.

The first important issue is the degree of competenceof cranial NCCs to generate arch-specific skeletal ele-ments in relation to their Hox code. Hox-negative andHox-expressing NCCs differ in their competence to formpharyngeal arch skeletal derivatives. Hox-negative NCCshave broad potential to give rise to the whole frontonasaland facial skeletons79. By contrast, Hox expression hasbeen shown to be incompatible with the generation of first arch skeletal derivatives74,80. Nonetheless,Hox-expressing NCCs are required for the generation of second and posterior arch derivatives47,48,54–60,81. So,premigratory Hox codes seem to restrict the skeletogenicpotential of NCCs, possibly by modulating the responseof cranial NCCs to skeletogenic signals in the pharyngealarch environment82,83 (BOX 2).

Hox-expressing NCCs. Although the above observationsseem to provide evidence for prepatterning, the migrat-ing Hox-expressing NCCs might not be irreversiblycommitted. If the Hox code can be altered by the newenvironment, the prepatterning information could bereversed. During NCC migration, Hox gene expressionis maintained in the surrounding environment, both atthe level of the neural tube where the cells originate, andin the pharyngeal arches where they differentiate. Themesodermal component of the arch has been shown tohave a role in the maintenance of Hox gene expression84,and cell community effects85 also seem to beimportant84. If individual Hox-expressing NCCs are

BA2 BA1

Hoxa2Otx2Dlx1/2Dlx1/2 + Dlx5/6

Dlx1/2 + Dlx5/6 + Dlx3/7

D

VM

P A

L

Figure 2 | Molecular integration of anteroposterior and dorsoventral patterninginformation. Cross-section through the second (hyoid) arch (BA2), with partial removal of thesurface ectoderm of both BA2 and the first (mandibular) arch (BA1). Along the anteroposterior (AP)axis, Hoxa2 (yellow) and Otx2 (orange) are expressed in BA2 and BA1 neural crest cells (NCCs),respectively. Note that Hoxa2 is also expressed posteriorly in the BA2 ectoderm42,74. Along thedorsoventral (DV) axis, molecular identity is provided by the nested expression of Dlx genes (colour-coded dots in BA2 and BA1 NCCs). The model implies that, depending on its position, each cellexpresses a specific code of AP and DV patterning homeodomain factors. Arrows indicate theorientation of the three main body axes, matching the colour-coded expression of the indicatedgenes. ML, mediolateral axis.


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These observations seem to indicate a completecompliance of the Hox-negative skeletogenic NCCs withpatterning signals from the neighbouring epithelia.However, other experimental evidence challenges this standpoint of absolute plasticity, and indicates thatHox-negative NCCs have a more instructive function incraniofacial development. Schneider and Helms haveshown that when cranial neural crest precursors of thepresumptive beak region from quail and duck embryoswere exchanged, the hybrid forms appeared as duckswith a quail beak and quails with a duck beak91 (FIG. 4).Donor NCCs had provided patterning instructions for beak morphology within the host environment,indicating that they carried some intrinsic, independentmorphogenetic information. Furthermore, NCCs couldinfluence the molecular activity of the adjacent hostectoderm, indicating reversed hierarchical relationshipsbetween epithelial and mesenchymal tissues to thoseindicated by the experiments discussed earlier.

How can we reconcile these seemingly contrastingresults? It should be noted that FEZ ectoderm or foregutendoderm patterning activities are restricted, and thatthey depend on the specific NCC population withwhich they interact along the AP axis. Posterior hetero-topic ectoderm or endoderm grafts could not alter thefate of Hox-expressing NCC mesenchyme, indicatingthat the ectopic epithelial signalling could not prevailover intrinsic Hox-mediated NCC prepatterning79,89.Moreover, when the FEZ was transplanted onto firstarch mandibular mesenchyme, a supernumerary lowerbeak — but not an ectopic upper beak89 — was formed.Couly et al. described beak duplications only after

overlying ectoderm — including FGFs, BONE MORPHO-

GENETIC PROTEINS (BMPs) and Shh — are indispensablefor the outgrowth of maxillary and frontonasal skeletal elements, as well as for tooth and pharyngeal arch devel-opment (FIG. 5). These processes are severely restricted bymutations or manipulations that affect the ectodermalsignalling, and in the most extreme cases, total loss ofneural crest derivatives has been reported87,88. However,these data imply a requirement for an ectodermal con-tribution to NCC proliferation and/or differentiation,rather than a patterning role.

In a recent study, Hu et al. identified a polarizing zone— the frontonasal ectodermal zone (FEZ) — in the ecto-derm that overlies the frontonasal process (FNP) of chickembryos. The FEZ not only orientates the DV axis of themaxillary outgrowth, but can also induce an ectopicupper beak when transplanted onto a different region ofthe FNP89 (FIG. 4). This work implied a dominant pattern-ing role for the ectoderm over the NCC mesenchyme forthe specification of facial structures.

It has also been shown that removal of stripes offoregut endoderm in chick embryos prevents facial bonedevelopment in the adjacent NCC mesenchyme79. Somezebrafish mutants show failure of ventral head skeletonformation as a result of reduced or absent endoderm90.Moreover, Couly et al. have shown that the endodermdoes not simply have a sustaining function for the devel-opment of the facial and visceral skeleton, but that it hasa real patterning role during morphogenesis. Specificgrafted portions of chick endodermal tissue can inducethe formation of supernumerary jaw elements, and caneven determine their position and orientation79 (FIG. 4).

BONE MORPHOGENETIC

PROTEINS

Multifunctional secretedproteins of the transforminggrowth factor superfamily. In theearly embryo, they participate indorsoventral patterning.

Chick R1 – Is

R1 + Is

a b

Prepatterning Plasticity

BA1BA2

BA1BA1*

ANCBA1

BA2

R1 – Is

R1 + IsPNC

M

M

CB CQP

P Q

Q* Q*

P*M*

S

S

S* S*

P*M*

R4

Figure 3 | Cranial neural crest cell prepatterning versus plasticity (1). a | Noden’s heterotopic grafting of chick presumptive neural crest cells (NCCs)11.Presumptive anterior neural crest (ANC) — including the rostral hindbrain, the mid-hindbrain boundary (isthmus) and presumptive mesencephalic neural crest — whichnormally colonizes the first pharyngeal arch (BA1), replaces presumptive posterior hindbrain neural crest (PNC), which normally contributes to the second (BA2) andposterior arches. Ectopic ANC cells colonize BA2, generating a duplication of BA1 (BA1*) in place of BA2. Wild-type chick skull (top right), showing BA1 and BA2derivatives in blue and yellow, respectively. After grafting (bottom right), BA2 derivatives, like the columella (C) and the ceratobranchial (CB), are missing and arereplaced by a duplication of some BA1 derivatives (arrows). Colour codes are the same as in FIG. 1. M, Meckel’s cartilage; P, pterygoid; Q, quadrate; S, squamosal.Asterisk indicates ectopic duplicated elements. b | Role of mid-hindbrain isthmic tissue in cranial NCC patterning76. Grafts of rhombomere 1 with (R1+ Is) or without(R1 – Is) the adjacent isthmus, in place of rhombomere 4 (R4) (left). Effect on patterning of NCC-derived skeletal elements of BA2 (right). When R1 + Is is transplanted,a duplication of BA1 elements is obtained, similar to Noden’s experiment described in a. By contrast, the R1 – Is graft results in a normal BA2 phenotype.


R E V I E W S

The mesenchyme progressively loses its ability torespond to epithelial signalling and acquires an independent developmental course95.

Pharyngeal arch morphogenesisTo fully understand how various tissues and cell populations contribute to the patterning and morpho-genesis of craniofacial structures, we need to identify themolecular factors that are involved. Recently, there has been a considerable effort to search for genes thatare involved in craniofacial development15,96–99. Here,we will focus on just a few of the molecular mechanismsthat control craniofacial morphogenesis and differentia-tion (FIG. 5 and BOX 2).

Endothelin-1 signalling. The zebrafish sucker mutant hasprovided some interesting examples of how spatialinformation provided by locally secreted signalling factors can modulate the morphogenesis of NCC derivatives. The sucker mutant is defective for thesecreted peptide endothelin-1 (Et1), and it showsimpaired development of ventral pharyngeal arch elements100. A ventralizing gradient of Et1 signalling,which diffuses from the pharyngeal arch epithelium andmesoderm, indicates to the NCC mesenchyme where toform specific skeletal elements along the DV axis100–103

(FIG. 5). Different levels of the signal are interpreted by theNCCs as instructions to make different elements — in the second arch, a high Et1 concentration induces theformation of ventral dermal bones (the branchiostegalrays), whereas a low Et1 concentration specifies the dor-sal bones (the opercles). Gradual inhibition of Et1expression causes various effects, ranging from loss ofventral bones and concomitant expansion of the oper-cles, to complete loss or reduction of all the bones102.This implies that Et1 provides a general instruction for

implanting additional endoderm stripes underneath oraround positions that corresponded to their original APaxial level79. These findings indicate that the epithelial-mediated patterning instruction is not absolute, but isinterpreted by the NCCs according to their relative APpositional identity.

Epithelial–mesenchymal cross-talk. The processes that underlie the morphogenesis of the craniofacialskeleton seem therefore to be regulated by epithelial-mesenchymal bidirectional cross-talk, which is charac-terized by alternating ruling activity. Tooth developmentis a clear model for such a mechanism. Initially, signalsfrom the epithelium trigger a local response in theunderlying mesenchyme. In turn, the latter initiates anappropriate differentiation programme and signals backto the epithelium, providing specific instruction for tooth morphogenesis (reviewed in REF. 92). Neuralcrest transplants from mouse to chick embryos haveshown that mouse NCC mesenchyme can promotetooth development in a chick host93, indicating thatepithelia provide general signals that trigger tooth morphogenetic programmes, but which are interpretedaccording to the origin of the NCCs.

So, although the cranial NCC mesenchyme isendowed with an AP-restricted prepattern for the formation of skeletal elements, it also requires inducingsignals from the surrounding epithelia to obtain information about the position and orientation of thestructures that it will generate. The spatial organizationand axial polarity of the pharyngeal arch are determinedindependently of NCC contribution94, but once the spatial information has been conveyed by epithelial–mesenchymal signalling, the NCC mesenchyme takesover the leading role and continues with the process of morphogenesis on the basis of its own potential.

Box 2 | Skeletogenic potential in cranial neural crest cells

The cranial neural crest cells (NCCs) are unique among NCC populations, in that they can differentiate into skeletaltissue1. What confers this skeletogenic potential? Interestingly, recent work indicates that this potential is inverselyrelated to Hox expression status80,82. The anterior-most cranial neural crest is Hox-negative and generates most of thecartilage and membranous bones of the face and jaw, as well as the greatest number of skeletal elements. The NCCs ofthe second pharyngeal arch, which express only group 2 Hox genes, form a smaller number of skeletal elements, whichcontribute to the middle ear and hyoid bone in the neck region. The third arch NCCs, which express both group 2 and 3Hox genes, make an even less significant contribution to the neck skeleton. The NCCs of the trunk, which express severaladditional posterior Hox genes, do not generate any skeletal structures at all.

A skeletogenic fate can be acquired by trunk NCCs after long-term in vitro culture155, and interestingly, this correlateswith a loss of Hox gene expression82. Conversely, forced constitutive expression of Hoxa2, Hoxa3 or Hoxb4 in Hox-negative NCCs in the chick embryo gradually inhibits the development of the craniofacial skeleton80. In Hoxa2 nullmutants, cartilage and bone specification genes, such as Sox9 and CbfaI, respectively, are upregulated in a broaderpattern145. Moreover, overexpression of Sox9 in the second arch can lead to ectopic cartilaginous elements similar tothose observed in Hoxa2 mutants145.

Recent studies have provided molecular clues as to how Hoxa2 regulates skeletogenesis in the pharyngeal archenvironment. Hoxa2 locally represses the expression of first-arch-specific genes, such as Ptx1 and Lhx6, by participatingin a pathway that antagonizes epithelial FGF signalling83. On the other hand, Hoxa2 positively regulates the second-arch-specific expression of Gsc49,51. The levels of Hoxa2 protein might modulate the competence of the cranial NCCs torespond to FGFs by locally regulating the balance between survival, proliferation and specification into a skeletogenicfate82. From these data, the emerging picture is that to select for hyoid-specific patterning, Hoxa2 would act not just as aninhibitor of skeletogenic fate, but as a spatial and temporal modulator of local proliferative versus differentiativeresponses of cranial NCCs to environmental signals.


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receptor108) are expressed in the pharyngeal epitheliumand mesoderm, and in the NCC mesenchyme, respec-tively105,106. The bHLH transcription factor gene dHand(an orthologue of the zebrafish hand2) and the relatedgene eHand are downstream effectors of Et1 inNCCs109,110. dHand controls the expression of the HDtranscription factor Msx1, and complete disruption ofthis molecular pathway causes growth failure of the pharyngeal arches as a result of apoptosis111. Dlx6 hasbeen shown to mediate the Et1-dependent activation ofdHand in the ventral half of the arches110,112. This is inagreement with the proposed ventralizing activity of Et1signalling. However, Et1 and Eta mouse mutants alsoshow mild defects in the dorsal region of the pharyngealarches104,105, indicating that, as shown in zebrafish, a DV endothelin gradient might induce dorsal structuresat low concentrations and ventral structures at high concentrations.

bone differentiation, and its concentration range is translated into morphogenetic information that indicates which bone to make.

The Et1 concentration gradient is also involved in cartilage specification100 and joint formation103 in the visceral skeleton. At intermediate DV locations, low levels of Et1 are sufficient to activate the transcription factor gene bapx1, which regulates the expression of jointspecification genes. Ventrally, higher levels of Et1 turn on the expression of the BASIC HELIX–LOOP–HELIX (bHLH)transcription factor gene hand2, which represses bapx1and is required for pharyngeal cartilage formation. As aresult, the cells specified for joint formation (positive forbapx1) are located in the intermediate position where thejoint should form103.

Et1 signalling also has a key role in the ventral patterning of the first and second arches of mammals andbirds104–107. Et1 and its receptor Eta (a G-protein-coupled

BASIC HELIX–LOOP–HELIX

A structural motif present inmany transcription factors thatis characterized by two α-helicesseparated by a loop. The helicesmediate dimerization, and theadjacent basic region is requiredfor DNA binding.

Figure 4 | Cranial neural crest cell patterning versus plasticity (2). a | Contribution of NC to beak morphology revealed by interspecific grafting91. Prosencephalic,mesencephalic, R1 and R2 presumptive NCCs from a quail embryo (green) were transplanted to replace the same neural fold region of a stage-matched duck embryo(red). This generated a duck with a quail-like beak (arrow). The reciprocal approach (lower panel), in which anterior neural folds from a duck embryo were transplanted into aquail embryo, generated a quail with a duck-like beak (arrow). b | Patterning role for pharyngeal endoderm in cranial NCCs79. Homotopic transplantation of a super-numerary stripe of chick foregut endoderm (FE) at the level of the mandibular arch into a host embryo (left) results into outgrowth of an additional NCC-derived lower beak(LB*) (right). c | Patterning role for the frontonasal ectodermal zone (FEZ) in cranial NCCs89. Transposition of FEZ (left), defined by the juxtaposing expression of fibroblastgrowth factor 8 (red) and sonic hedgehog (blue). Colour codes are the same as in FIG. 5. The ectopic FEZ determines outgrowth of an additional upper beak (UB*) (right).

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CRE RECOMBINASE

Part of a site-specificrecombination system derivedfrom Escherichia colibacteriophage P1. Two shortDNA sequences (loxP sites) areengineered to flank the targetDNA. Activation of the Crerecombinase enzyme catalysesrecombination between the loxPsites, leading to excision of theintervening sequence.

caudal expression of the goosecoid (Gsc) transcriptionfactor gene116,117. Interestingly, Gsc is also downstream ofthe Et1 signalling pathway105,109. Targeted inactivation ofGsc in the mouse indicated that it is required for cranio-facial chondrogenesis118,119.

Similar mechanisms probably establish the AP polar-ity of more posterior arches. When forward and reversehomeotic transformations are induced by Hoxa2 loss- orgain-of-function in mouse, chick or zebrafish, theduplicated elements of the affected arch always appearas a mirror image of their orthotopic counterpart, indi-cating an inverted polarity of the first and secondarches. This might indicate the presence of a graded pat-terning signal that originates from the boundarybetween the two arches and operates in a bidirectionalmanner47,50. Fgf8 is a putative candidate, given itsexpression in the pharyngeal pouch endoderm and inthe ectoderm of the pharyngeal slit between the firstand second arches120,121 (FIG. 5). Detailed investigation ofsecond arch development in mouse mutants carrying CRE

RECOMBINASE-mediated deletions of Fgf8 in the archepithelia might provide more information.

Fgf8 marks the region of prospective oral ectodermbefore the oral cavity delineates, and before the mesen-cephalic NCC stream arrives to generate the oral mesenchyme121. The Fgf8 domain is delimited byanother signalling factor gene, Bmp4, which is expressedon both sides in the adjacent ectoderm (FIG. 5). Fgf8 andBmp4 characterize the incoming mesenchyme byinducing the expression of specific patterning genes.The NCCs exhibit a broad plasticity in response to thiskind of positional signalling; for example, expandingthe Fgf8-positive ectoderm before NCC migrationenlarges the embryonic oral region at the expense of the premandibular region. Conversely, inhibiting early ecto-dermal Fgf8 expression by ectopic applications of Bmp4causes loss of jaw structures121,122. The role of ectoder-mal Fgf8 seems to be limited to the initial induction ofthe target genes — their maintenance is independent ofFgf8, and seems to be intrinsic to NCCs95,123.

Members of the FGF family are also involved in signalling from the pharyngeal endoderm. In zebrafish,loss of fgf3 expression in the pharyngeal pouches resultsin early apoptotic elimination of posterior arch NCCs90,and failure to maintain fgf3 expression at later stagesleads to severe hypoplasia of the posterior arch cartilageelements124. The temporal regulation of Fgf3 activitymight provide a mechanism to establish the polarity ofstructures within an arch124. The duration of the signalcould directly influence the size of a particular element.Then, the interplay between FGF and the patternedactivity of FGF antagonist molecules, like sef or sproutyfamily members125, could influence cartilage shape bydirecting the survival of specific NCC subpopulationsin response to FGF, or by triggering death cues throughelimination of FGF activity.

FGF signalling can also influence cranial NCC fateby providing cues to their host environment. In FGFreceptor 1 (Fgfr1) mouse mutants, the prospective sec-ond arch NCCs fail to enter the arch during migration,and they accumulate in a region proximal to the arch126.

FGF signalling. Many different FGF molecules emanatefrom the facial epithelia113. Fgf8, which is expressed inspecific regions of the facial and pharyngeal ectoderm,as well as in the pharyngeal pouch endoderm, is a key fac-tor for NCC survival88,114. Conditional Fgf8 inactivationin the first arch ectoderm results in apoptosis of largeportions of NCC mesenchyme and consequent failureof facial skeleton development88. However, Fgf8 seems tobe more than a simple survival factor, as it can alsoinduce the expression of genes that are responsible fortissue-specific NCC differentiation95,115,116. This functionis associated with its ability to determine pharyngealarch polarity. In the first arch, the rostral (oral) pole,which will form teeth, can be distinguished from thecaudal (aboral) pole, which will form the jaw skeleton.Fgf8 signalling from the rostral epithelium has beenshown to be responsible for the specification oforal–aboral polarity116, and its polarizing activity isaccomplished by a signalling gradient along the AP axis,just as the Et1 gradient specifies the DV axis. High Fgf8concentrations induce rostral expression of Lhx tran-scription factor genes, and low concentrations induce

BA2

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Figure 5 | Molecular pathways involved in the specification of frontonasal, maxillary,mandibular and hyoid structures. The drawing shows an enlargement of the head region of avertebrate embryo (inset with rectangle). A section through an imaginary plane is shown, which ismid-sagittal at the level of the frontonasal process (FNP) and parasagittal (slightly more lateral thanthe midline) at the level of the first and second pharyngeal arches (BA1 and BA2, respectively). Theindicated genetic interactions are representative of data collected in mouse, chick or zebrafish (seetext for references). Some differences might exist in these pathways in different species. Theectodermal, endodermal and mesodermal localization of secreted signalling molecules is indicated,including bone morphogenetic protein 4 (Bmp4; green), sonic hedgehog (Shh; blue), fibroblastgrowth factor (Fgf8; red), endothelin-1 (Et1; violet), Fgf8/Fgf3 and Et1 (pink). It should be noted thatthe juxtaposition of expression domains in the surface ectoderm is arbitrary, but it is shown for thesake of simplicity, except at the level of the frontonasal ectodermal zone (FEZ). Receptor moleculesare expressed in the neural crest mesenchyme (yellow), though not exclusively, and they induce theactivation and/or maintenance of the indicated transcription factors. In turn, the activity oftranscription factors specifies the morphogenetic identity of structures both along theanteroposterior (inter-pharyngeal) and the dorsoventral (intra-pharyngeal) axes. Manipulation ofsignalling molecules and tissues leads to selective deletions or supernumerary head structures. HY, hyoid arch or BA2; MD, mandibular portion of BA1; Me, mesodermal core of pharyngeal arches;MX, maxillary portion of BA1; PP1, first pharyngeal pouch; PP2, second pharyngeal pouch.


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mesenchyme receives the patterning instructions for outgrowth of the FNP. Ablation of Shh-expressing ecto-derm leads to failure of development of the FNP, whereasapplication of Shh-expressing fibroblasts to the FNPinduces an ectopic upper beak in the chick129. Theexpression of Shh and Fgf8 is maintained by endogenousretinoid signalling87. Inhibition of retinoid signalling by the use of retinoic acid receptor (RAR) syntheticantagonists87, by the knockout of the RA-synthesizingenzyme Raldh2 (REF. 130), or by the compound targetedinactivations of Rarα (and Rarγ)131 leads to complete orpartial loss of the structures derived from the FNP.

BMPs also have a prominent role in the specificationof facial structures15,132,133. The maxillary processes, whichextend ventrolaterally on both sides of the FNP and fuseat the midline to form the palatal shelves, are specified byBMP expression. If BMP action is blocked by treatmentwith the specific inhibitor Noggin and concomitantapplication of RA, the maxillary processes are trans-formed into supplementary FNPs133. Skeletal structuresderived from other facial prominences are not duplicated,so BMP molecules might exert region- and stage-dependent effects on maxillary and mandibular morpho-genesis15,134, partly through antagonistic interactions withFGF molecules122,134,135. Early in development, themandibular mesenchyme seems to be under the balancedinfluence of death and survival signals provided by theectodermally expressed BMPs and FGFs, respectively134,whereas at later stages, BMPs promote mesenchyme differentiation into cartilage or bone135,136. The BMPsseem to exert different roles by regulating genes with opposite functions, such as Msx1, Msx2 and Sox9(REFS 121,135,136). Sox9 is a known regulator of cartilage formation137,138, whereas Msx1 and Msx2 are generallyregarded as negative regulators of differentiation, and areinvolved in apoptosis139,140. Mutations in these genes affectthe normal development of head skeletal structures inmice and humans141–144.

Conclusions and perspectivesThe building of the vertebrate head is an example ofa morphogenetic process that relies on a complex seriesof interactions between molecules and tissues. Some ofthese interactions have been conserved through millionsof years of evolution, whereas others are peculiar to dis-tinct species, generating a fascinating variety of facialmorphologies. Facial anomalies often correlate withbrain malformations (BOX 3) and, in some cases, disrup-tion of common signalling pathways is involved87. So,craniofacial development is of considerable interestfrom both neurobiological and clinical perspectives.

What are the next important questions to beaddressed by research into craniofacial development?Full comprehension of this process will rely on a com-plete dissection of the underlying molecular processes.Several important issues are still unexplored withrespect to the molecular mechanisms of patterning andspecification of skeletogenic NCCs. For instance, verylittle is known about the temporal requirement for keyHD transcription factors (for example, Hox and Dlx) in the regulation of pharyngeal arch morphogenesis.

This defect is not rescued by selective expression of Fgfr1in NCCs, implying that Fgfr1 influences the entry ofNCCs into the second arch by creating a permissiveenvironment for their migration. It is significant that themigration defect selectively affects the second archNCCs. So, specific combinations of FGF and/or FGFRmolecules might be involved in setting the compatibilityof a given arch with a positionally matched stream ofNCCs, possibly in relation to their Hox code.

In addition to their roles as survival factors, recentwork in vitro indicates that FGF molecules could regulatethe differentiation of NCCs in a dose-dependent manner.For example, low Fgf2 concentrations administered tocultured cranial NCCs support cell survival, whereashigher concentrations induce cartilage and bone differen-tiation82,127. It is tempting to speculate that a similar concentration-dependent response of NCCs occurs inthe pharyngeal arch, where a spatiotemporal gradient ofFGF signals from the epithelium might contribute to thesite-specific formation of cartilage aggregates. Indeed,it has been shown that Fgf2 and Fgf4 can promote theformation of cartilage elements at ectopic sites128.

Other signals. The FGFs operate in combination withother signalling molecules. In the frontonasal ectoderm,the FEZ is identified by a juxtaposed region of dorsalFgf8 and ventral Shh expression89, and at the interfacebetween these two signalling factors, the neural crest

Box 3 | Craniofacial defects

Anomalies of the face and skull bones account for approximately one-third of allcongenital defects. Some of them have been linked to distinct genetic disorders, and areeither confined to the craniofacial region or occur in association with a set ofabnormalities in other parts of the body. Other anomalies are caused by exposure of theembryo to teratogenic substances or environmental factors.

Some craniofacial anomalies, including those described here, also involve abnormaldevelopment of the brain. These diseases generally consist of malformations of the cranialvault and/or the frontonasal process (reviewed in REFS 146,147 and references therein).

Holoprosencephaly originates from an early disturbance of forebrain development,and encompasses a broad spectrum of phenotypes of variable severity, involving brainand facial malformations along the midline. The typical facial features include, in themost severe cases, short or absent nose, cleft upper lip, and a single central eye (cyclopia),often associated with the formation of an overlying proboscis. The brain is not subdividedinto hemispheres and the olfactory bulbs are missing. Holoprosencephaly can be causedby genetic factors, including mutations of SHH, GLI2, PTCH, ZIC2, SIX3, CRIPTO andDHCR7, or by teratogens, such as alcohol or retinoic acid.

Anencephaly is characterized by an open neural tube in the brain region, which resultsin exposure and degeneration of neural tissue and absence of the cranial vault. Thecerebral hemispheres can be completely missing or reduced in size. The skeletal defectscan extend to malformations of the skull floor and of the jaw and neck region.

Encephalocele is a neural tube defect caused by a gap in the skull, which results in theprotrusion of the meninges and brain tissue (herniation). It can occur in the occipital,parietal or frontonasal regions.

Craniosynostosis is defined as early closure of one or more of the cranial sutures. Thebrain is forced to grow in directions where the bone is not resisting, often resulting inmultiple neurological defects. The symptoms of craniosynostosis depend on which, andhow many, cranial sutures are involved. The symptoms range from aesthetic concerns toseizures, blindness, developmental delay and mental retardation. There are more than100 syndromes associated with craniosynostosis, including Apert, Crouzon, Pfeiffer andSaethre–Chotzen syndromes. Familial cases of craniosynostosis are associated withmutations in FGF receptor genes (FGFR1, 2, 3), TWIST and MSX2.


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In addition, recent work has drawn attention to theimportance of searching for target genes and under-standing how they integrate into functional networks,so that we can reconstitute the signalling mechanismsthat underlie epithelial–mesenchymal interactions during cellular differentiation and morphogenesis.Integration of the molecular data will provide impor-tant insights into the morphogenetic programme forskeletogenic NCCs. Eventually, this research shouldreveal how the structural organization of the NCCs atthe protein level changes at each stage of development,until the cells finally settle to form a definite structure.

Large-scale mutant analysis in zebrafish and targetedmutagenesis in the mouse are proving to be valuableapproaches for identifying new genes and elucidatingthe regulatory relationships between them6. Graftingand recombination experiments that take advantage ofthe accessibility of other embryonic systems will alsocontinue to be instrumental in unravelling the cell andtissue interactions that are involved in craniofacialdevelopment. We anticipate that future research intothis matter will be an exceptionally demanding but ultimately rewarding task.

Their expression is maintained up to late stages of cranio-facial development50,64,145, indicating that they might be involved in regulating the size and shape of skeletalstructures. By using available knockout technologies inthe mouse, temporally regulated inactivation will allow usto define the critical time window of HD factor functionthat is necessary for cranial NCC patterning. In addition, the analysis of conditional tissue-specific HDfactor mutants will allow us to dissect their specific rolesin the distinct tissues where they are expressed, includ-ing the NCCs, the neuroectoderm and the surface ectoderm of the pharyngeal arches. Analysis of com-pound mutants might reveal a requirement for geneticinteraction of HD factors in NCC patterning.

Another important question is how NCCs interpretand integrate the multiple extracellular signals that arepresent in the arch environment, so that they are specifiedin the correct proportions. It will be necessary to definethe control elements that direct the spatiotemporal regulation of HD factors in NCCs in response to specificsignalling molecules. This approach will provideinsights into the regulatory cascade that is required forspatially and temporally restricted HD factor activity.

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AcknowledgementsWe are grateful to S. Metz for help in the preparation of the figures.Work in the authors’ laboratory is supported by the EuropeanCommission grant Brainstem Genetics, the ARC Association pourla Recherche sur le Cancer, the Ministère pour le Recherche (ACIProgram) and by institutional funds from Centre National de laRecherche Scientifique, Institut National de la Santé et de laRecherche Médicale and Hôpital Universitaire de Strasbourg.

Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/bapx1 | Bmp4 | dHand | Dlx1 | Dlx2 | Dlx3 | DLX3 | Dlx5 | Dlx6 |eHand | Eta | Et1 | Fgf2 | fgf3 | Fgf4 | Fgf8 | Fgfr1 | GLI2 | Gsc |hand2 | hoxa2 | Hoxa2 | Hoxa3 | Hoxa4 | hoxb2 | Hoxb2 | Hoxb4 |Hoxc4 | Hoxd4 | Lhx6 | Msx1 | Msx2 | Noggin | Otx2 | PTCH | Ptx1| RAR | sef | Shh | SHH | SIX3 | Sox9 | sprouty | TWIST | ZIC2ZFIN: http://zfin.org/cgi-bin/webdriver?MIval=aa-ZDB_home.apgsuckerOMIM: http://www.ncbi.nlm.nih.gov/Omim/anencephaly | holoprosencephalyAccess to this interactive links box is free online.

O N L I N E

LocusLinkhttp://www.ncbi.nlm.nih.gov/LocusLink/Shhhttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=20423

Hoxa2http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=15399

hoxa2http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=30325

hoxb2http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=30338

Hoxb2http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=103889

Otx2http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=18424

Dlx5http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=13395





DLX3http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=1747

Fgf8http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=2253

SHHhttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=6469

Et1http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=1906

bapx1http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=337865

hand2http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=58150

Etahttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=1909

dHandhttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=64637

eHandhttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=59112

Msx1http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=81710

Gschttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=317715

Bmp4http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=12159

fgf3http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=30549

sefhttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=171474

sproutyhttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=114437

Fgfr1http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=14182



RARhttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=5914

Nogginhttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=9241

Msx2http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=17702

Sox9http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=20682

GLI2http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=2736

PTCHhttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=5727

ZIC2http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=7546

SIX3http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=6496

TWISThttp://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=7291


Hoxb4http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=15412

O N L I N E

Hoxc4http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=15423

Hoxd4http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=15436


Ptx1http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=18740

Lhx6http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=16874

ZFIN http://zfin.org/cgi-bin/webdriver?MIval=aa-ZDB_home.apg

suckerhttp://zfin.org/cgi-bin/webdriver?MIval=aa-locusview.apg&OID=ZDB-

LOCUS-990215-583

OMIMhttp://www.ncbi.nlm.nih.gov/Omim/

Holoprosencephaly http://www.ncbi.nlm.nih.gov/htbin-post/Omim/disp-

mim?236100

Anencephalyhttp://www.ncbi.nlm.nih.gov/htbin-post/Omim/disp-

mim?206500

At a glance• The craniofacial skeleton encases the brain and sensory organs, and is

important for the functioning of the digestive and respiratory tracts. Itis derived largely from the neural crest, a migratory cell populationthat detaches from the embryonic neural epithelium.

• The cranial neural crest might use a similar patterning strategy to thedeveloping nervous system, whereby a grid-like system of positionalcues is created by gradients of extracellular signals or morphogens andtheir intracellular molecular effectors.

• The anteroposterior identity of each subpopulation of neural crestcells (NCCs) has a strong influence on the development of the cranio-facial skeleton. Along this axis, positional identity is determined by thenested expression of Hox genes. Dlx genes seem to provide positionalidentity along the dorsoventral axis of the pharyngeal arches.

• There has been a long-standing debate about cranial NCC prepattern-ing versus plasticity. Skeletogenic NCCs could be irreversibly commit-ted before they migrate, or they might maintain a broad plasticity untilthey reach their final destination. The accumulating evidence indicatesthat the reality lies somewhere in-between these two models.

• The processes that underlie the morphogenesis of the craniofacialskeleton seem to be regulated by epithelial–mesenchymal bidirectionalcross-talk. Signals from the epithelium trigger a local response in theunderlying mesenchyme, which in turn initiates a differentiation pro-gramme and signals back to the epithelium.

• A gradient of endothelin-1 signalling, which diffuses from the pharyn-geal arch epithelium and mesoderm, provides a general instruction forbone differentiation, and its concentration range is translated into

morphogenetic information that indicates which bone to make.• Fibroblast growth factor 8, which is expressed in specific regions of the

facial and pharyngeal ectoderm, as well as in the pharyngeal pouchendoderm, is a key factor for NCC survival. It can also induce theexpression of genes that are responsible for tissue-specific NCC differ-entiation.

• Other signals, including sonic hedgehog, retinoids and bone morpho-genetic proteins, also have prominent roles in the specification of facialstructures.

• Very little is known about the temporal requirement for key transcrip-tion factors in the regulation of pharyngeal arch morphogenesis.Temporally regulated inactivation should allow us to define the criticaltime window of transcription factor function that is necessary for cra-nial NCC patterning.

• It will also be important to reconstitute the signalling mechanisms thatunderlie epithelial–mesenchymal interactions during cellular differen-tiation and morphogenesis. Future research should aim to reveal howthe structural organization of the NCCs at the protein level changes ateach stage of development, until the cells finally settle to form a defi-nite structure.

BiographiesFabio Santagati is a postdoctoral fellow in the laboratory of F. Rijli,where he is carrying out a molecular analysis of Hoxa2 function inneural crest cell patterning. He completed his studies in biological sci-ences at the University of Pavia, Italy, and obtained his Ph.D. on the reg-ulation of the Pax9 gene during mouse embryonic development in thelaboratory of R. Balling and K. Imai at the GSF-Research Centre inNeuherberg-Munich, Germany. His background is in molecular anddevelopmental biology.

Filippo Rijli is Directeur de Recherche at the Centre National de laRecherche Scientifique (CNRS), and group leader at the Institut deGénétique et de Biologie Moléculaire et Cellulaire in Strasbourg, France.His research has centred on the genetic and molecular analysis of hind-brain patterning and craniofacial morphogenesis, with a focus on thedevelopmental role of Hox genes. He generated the targeted inactivationof the mouse Hoxa2 gene — this study provided the first experimentaldemonstration of the role of a vertebrate Hox gene in patterning the cra-nial neural crest. He then accomplished the first targeted mutation of aHox retinoid regulatory element and studied its in vivo requirement forneural expression. More recently, work in his laboratory has focused onthe involvement of Hox genes in hindbrain neuronal patterning.

Documents

Cranial neural crest and the building of the vertebrate head