REVIEWS–A PEER REVIEWED FORUM

Significance of the Cranial Neural CrestAnthony Graham,* Jo Begbie, and Imelda McGonnell

The cranial neural crest has long been viewed as being of particular significance. First, it has been held that the cranialneural crest has a morphogenetic role, acting to coordinate the development of the pharyngeal arches. By contrast,the trunk crest seems to play a more subservient role in terms of embryonic patterning. Second, the cranial crest notonly generates neurons, glia, and melanocytes, but additionally forms skeletal derivatives (bones, cartilage, and teeth,as well as smooth muscle and connective tissue), and this potential was thought to be a unique feature of the cranialcrest. Recently, however, several studies have suggested that the cranial neural crest may not be so influential interms of patterning, nor so exceptional in the derivatives that it makes. It is now becoming clear that themorphogenesis of the pharyngeal arches is largely driven by the pharyngeal endoderm. Furthermore, it is nowapparent that trunk neural crest cells have skeletal potential. However, it has now been demonstrated that a key rolefor the cranial neural crest streams is to organise the innervation of the hindbrain by the cranial sensory ganglia. Thus,in the past few years, our views of the significance of the cranial neural crest for head development have been altered.Developmental Dynamics 229:5–13, 2004. © 2003 Wiley-Liss, Inc.

Key words: neural crest; cranial; endoderm; pharyngeal; sensory

Received 22 July 2003; Accepted 22 August 2003

INTRODUCTION

The established view of the cranialneural crest was that it played a piv-otal role in vertebrate head devel-opment. It was thought that the cra-nial neural crest carried the cues fororganising the morphogenesis of thehead and, uniquely, was the sourceof much of the head skeleton. How-ever, it is now becoming apparentthat we must reassess our views ofthe significance of the cranial neuralcrest, and it is with this reassessmentthat this review is concerned.

MIGRATION OF THE CRANIALNEURAL CREST

The migration of the cranial neuralcrest is quite distinct from that of thetrunk. In the trunk, the neural crest

cells are subjected to the alternatingpermissive and inhibitory halves ofsomites, which result in their segmen-tal organisation (Rickmann et al.,1985). In the head, the neural crestcells pour out from the developingbrain into a periphery that is devoidof somites. Yet, the cranial crest cellsdo not migrate as a single mass butare organised into streams, three ofwhich can be identified in the devel-oping head of all vertebrate embry-os: trigeminal, hyoid, and post-otic(Fig. 1). The trigeminal crest arisesfrom the midbrain and rhom-bomeres 1 and 2 of the hindbrainand forms neurons within the trigem-inal ganglion and the componentsof the orofacial prominences andmandibular arch—the skeleton ofthe lower and upper jaw (Lumsden

et al., 1991; Schilling and Kimmel,1994). The second crest stream, thehyoid, arises primarily from rhom-bomere 4 of the hindbrain and formsneurons of the proximal facial gan-glion as well as the constituents ofthe second pharyngeal arch—thehyoid skeleton (Lumsden et al., 1991;Schilling and Kimmel, 1994). Finally,the post-otic crest is generated byrhombomeres 6 and 7 of the hind-brain and forms the neurons of theproximal and jugular ganglia, andthe skeletal components of the pos-terior pharyngeal arches (Lumsdenet al., 1991; Schilling and Kimmel,1994).

In the trunk, the first migrating neu-ral crest cells move ventrally fromthe neural tube and migratethrough the anterior half sclerotome.

MRC Centre for Developmental Neurobiology, New Hunts House, Guys Campus, Kings College London, London, United KingdomGrant sponsor: The Medical Research Council (UK); Grant sponsor: The Wellcome Trust.*Correspondence to: Anthony Graham, MRC Centre for Developmental Neurobiology, New Hunts House, Guys Campus, Kings CollegeLondon, London SE1 1UL, UK. E-mail: anthony.graham@kcl.ac.uk

DOI 10.1002/dvdy.10442

DEVELOPMENTAL DYNAMICS 229:5–13, 2004

© 2003 Wiley-Liss, Inc.

Fig. 1. Cranial neural crest migrates in three streams. The trigeminalstream (red) emanates from the midbrain (mb), rhombomeres (r) 1 and2. It migrates into the upper jaw primordia, underneath the eye, and intothe first pharyngeal arch (PA1), which forms the lower jaw. The hyoidstream (green), from rhombomere 4, migrates into the second pharyn-geal arch, forming the jaw support. The post-otic stream (purple)emerges from rhombomeres 6 and 7. It populates the caudal pharyn-geal arches, of which there are three in the chick embryo depictedhere.

Fig. 2. Regionalisation of the pharyn-geal endoderm. Endodermal pharyngealpouches (pp) sit between the pharyngealarches and express a range of markersthat demonstrate distinct regionalisation.Pax-1 (green) is expressed at the dorsaltip of each pouch. BMP-7 (blue) is ex-pressed in the posterior endodermal mar-gin, while Fgf8 (red) is expressed in theanterior endodermal margin. Shh (yel-low) is expressed at the posterior marginof pouches 2 and 3 only. D, dorsal; V,ventral; P, posterior; A, anterior; mb, mid-brain; r, rhombomere.

Fig. 3. Segregation of cranial neural crest establishes correctafferent innervation in the vertebrate head. Whole-mount chickembryo labeled with neurofilament antibody to visualise axonalprojections. The trigeminal axons (T) extend toward the hindbrainalong the first or trigeminal crest stream (red), entering at rhom-bomere (r) 2. Axons of the geniculate (G) extend along the hyoidcrest stream (green), into rhombomere 4. Axons from the petrosal(P) and nodose (N) extend along the post-otic crest stream (pur-ple) into rhombomeres 6 and 7, respectively. Mb, midbrain.

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These cells have a neuronal fate.Those that move furthest ventrallyform the sympathetic ganglia, whilethe remainder cease migration inthe anterior sclerotome and formthe dorsal root ganglia. The later mi-grating neural crest cells, however,move dorsolaterally between thedermamyotome and the overlyingectoderm, and these cells will formthe melanocytes (Weston and But-ler, 1966; Serbedzija et al., 1990;Erickson et al., 1992). In the head,there is also a correlation betweenthe timing of crest migration and thefates the crest cells follow, but it issignificantly different. Here, the earlymigratory cells populate the pharyn-geal arches and facial prominences,and these groups will generate ecto-mesenchymal derivatives: bone, car-tilage, and connective tissue, whilethe later migrating cells stay closer tothe developing central nervous sys-tem and generate neurons and gliaof the cranial ganglia (Baker et al.,1997).

SEGREGATION OF THE CRANIALCREST STREAMS

The segregation of the cranial crestis evident as soon as they are gen-erated by, and emerge from, theneural tube, and it is events withinthe developing hindbrain that areresponsible for establishing this seg-regation. Studies in chick haveshown that one mechanism thathelps enforce the segregation of thecrest is the focal depletion of crestcells from the two territories that liebetween the streams, rhombomeres3 and 5. These two rhombomeres doproduce some neural crest cells.However, these cells do not migratelaterally but rather move anteriorlyand posteriorly to join the streams ofcrest migrating from the adjacenteven-number rhombomeres (Sechristet al., 1993; Birgbauer et al., 1995;Kulesa and Fraser, 2000). The majorityof the neural crest cells produced byrhombomeres 3 and 5, however, arelost by means of apoptosis, which isinduced in these crest cells by signalsfrom the neighbouring segments(Graham et al., 1993). One of themain consequences of this inductiveinteraction is to promote the expres-sion of the signalling molecules Bmp-4

in the crest primordia of rhombomeres3 and 5, which in turn acts to sponsorthe apoptotic elimination of thesecells (Graham et al., 1994). In vitro ap-plication of Bmp-4 can also promotecrest depletion of rhombomeres 2and 6, which correlates with the find-ing that much of the hindbrain neuralcrest expresses the necessary recep-tors and intracellular effectors to re-spond to this factor (Farlie et al., 1999;Smith and Graham, 2001). Bmp-4 willnot induce, however, cell death inrhombomere 4 neural crest, whichcorresponds with the finding thatthese crest cells express the Bmp-4 an-tagonist Noggin in addition to theBmp receptors and intracellular trans-ducers (Smith and Graham, 2001).Thus, the neural crest cell death is lo-calised to rhombomeres 3 and 5 bothby restricting the expression of theBmp-4 ligand to these segments as aresult of an inductive interaction fromthe flanking segments and throughthe expression of Noggin in the neuralcrest cells of rhombomere 4, whichare juxtaposed to sites of Bmp-4 ex-pression.

Studies in other species have sug-gested that the establishment of thecranial neural crest streams may besomewhat different to that observedin chick. In mammalian embryos, cra-nial neural crest migration com-mences before neural tube closureand occurs over a protracted periodof time (Morriss-Kay and Tucket, 1991).In the chick, however, cranial neuralcrest cells only begin migrating beforeneural tube closure at mesence-phalic levels, with most cranial crestcells migrating after tube closure(Tosney, 1982). Another difference be-tween chick and mammals is that ithas long been established that, inmammals, the cranial crest do not mi-grate in a strict rostrocaudal se-quence. Indeed, there is also varia-tion in the timings and patterns ofmigration between mammalian em-bryos (Tan and Morris-Kay, 1985). Re-cent studies have also suggested thatthe mechanisms underlying thestreaming of the cranial crest in themouse differ significantly from thoseuncovered in the chick, although theprecise nature of these mechanismshas yet to be elucidated (Trainor etal., 2002). Similarly, we know littleabout the mechanism by which cra-

nial neural crest streaming is main-tained in other species where focalcell death in rhombomeres 3 and 5 isnot observed, such as in zebrafish(Schilling and Kimmel, 1994) and Xe-nopus embryos (Hensey and Gautier,1998).

Other work has shown that thereare additional mechanisms thatcould contribute to the streaming ofthe neural crest. Several studiesseem to suggest that there are inhib-itory influences emanating fromrhombomeres 3 and 5, which restrictthe movement of cells across thesesegments. For example, if the neuralcrest primordium of rhombomere 4 isgrafted dorsally in rhombomere 3,even though many neural crest cellsare now produced they are de-flected anteriorly and posteriorly(Niederlander and Lumsden, 1996).This effect could be due to the ex-pression in both rhombomeres 3 and5 of Sema3A, a molecule which isknown to inhibit neural crest migra-tion (Eickholt et al., 1999). Similarly, ithas been suggested that the mes-enchyme opposite these two seg-ments is inhibitory to neural crest mi-gration (Farlie et al., 1999) and thatthis finding is due to the function ofErbB4 in the hindbrain (Golding etal., 2000). These studies help explainwhy the few crest cells generated byrhombomeres 3 and 5 do not movelaterally from these rhombomeresbut migrate anterior and posterior.Furthermore, work in Xenopus hasalso suggested that interactions be-tween the ephrins and their recep-tors play a role in streaming the crestin the periphery (Smith et al., 1997).Given these results, it seems likelythat the segregation of the neuralcrest into streams is in part due toevents in the hindbrain, including, inchick, the induction of focal neuralcrest apoptosis in rhombomeres 3and 5, as well the action of subse-quent cues in the periphery, whichact to reinforce and stabilise thissegregation.

MORPHOGENETIC ROLE FORTHE CRANIAL NEURAL CREST

It has in the past been argued thatthe segregation of the cranial neuralcrest into these streams was essentialfor ensuring correct morphogene-

CRANIAL NEURAL CREST 7

sis—creating skeletal elements ofthe correct size, shape, and orienta-tion in the appropriate location. Akey piece of evidence supportingthis viewpoint comes from studies inwhich neural crest cells were forcedto populate an inappropriate pha-ryngeal arch (Noden, 1983). Thus, ifpresumptive first arch crest is graftedin place of presumptive secondarch crest, then the grafted crestcells fill the second arch, rather thanthe first, and, furthermore, althoughthese transposed crest cells gener-ate skeletal derivatives, they differ-entiate as components of the lowerjaw, i.e., first arch, rather than sec-ond arch elements. These trans-posed neural crest cells also causedthe musculature of the second archto form skeletal attachments typicalof the first arch (Noden, 1983). Theconclusion that emerged from thesestudies was that it was the neuralcrest cells that were acting to organ-ise the development of the pharyn-geal arches. Furthermore, as thesegrafts were of neural tube origin be-fore the emergence of the neuralcrest, it was also suggested that theneural crest cells acquired theirsense of identity when they werewithin the neural primordium. Thus,the segregation of the cranial crestinto stream would act to ensure thatfirst arch crest, carrying cues for jawdevelopment, did not interminglewith hyoid crest, carrying cues forthe patterning of the second arch;the streaming of the crest would actto guarantee the faithful transfer ofpatterning information from the neu-ral primordium to the periphery.

The idea that the segregation ofthe cranial crest into streams was im-portant for the morphogenesis of thehead was further enforced by along-term fate map of the cranialcrest, which revealed a highly con-strained pattern of skeletomuscularconnectivity in the head (Kontgesand Lumsden, 1996). In this study, itwas found that head muscle con-nective tissues derived from neuralcrest from a particular axial levelwere always exclusively anchoredto skeletal domains derived from thesame origin. This finding occurredeven within skeletal elements, suchas the jaw or the tongue skeleton,which are compounds of crest cells

populations from different axial lev-els. Furthermore, this even holds trueif one of the attachment points is onthe mesodermally derived basalcranial capsule; these attachmentpoints were shown to derive fromdiscrete clusters of crest cells em-bedded on the skull.

In many ways, the hypothesis thatit is the neural crest cells that patternthe pharyngeal arches is very attrac-tive. First, although this patterningmechanism is seemingly differentfrom that observed in the rest of theembryo, there is a commonality inthat, in all cases, the skeletogenictissue is the repository of the pattern-ing information. In the trunk, theparaxial mesoderm-derived somites,which generate the vertebra, directmotor neuron patterning in the ad-jacent spinal cord (Ensini et al.,1998). The lateral plate mesoderm,which forms the appendicular skele-ton, acts to attract muscle precur-sors from adjacent somites and sub-sequently directs their patterninginto specific muscle types (Cheval-lier and Kieny, 1982; Francis-West etal., 2003). In the head, the neuralcrest is the prime source of skeletaltissue, and here these cells arethought to pattern the arches. Sec-ond, if the neural crest cells do pat-tern the arches, then this also pro-vides an explanation of how thedifferent tissues of the developinghead can be coordinated. For ex-ample, the neural crest cells thatpopulate and organise the first archarise from the same axial level as thetrigeminal motor neurons that inner-vate that arch, and thus one canimagine how a correspondence isachieved between sensorimotor in-nervation, centered on the hind-brain, and the musculoskeletal ef-fectors in the periphery.

ENDODERM PLAYS A KEY ROLEIN PHARYNGEALMORPHOGENESIS

Several studies, however, have nowsuggested that this crest-centricview of pharyngeal patterningneeds to be reassessed. More spe-cifically, it is now apparent that thepharyngeal endoderm plays a keyrole in organising the developmentof the pharyngeal arches. The for-

mation of endodermal pharyngealpouches is the first indication of pha-ryngeal arch development (Veitchet al., 1999). Furthermore, the post-otic crest that fills the posteriorarches is split by the formation of thepharyngeal pouches. In keepingwith this view, it has been shownthat, if the neural crest is ablated,through removal of the neural tubebefore crest production, pharyngealarches do form and are regionalised(Veitch et al., 1999). In normal pha-ryngeal arches, Bmp-7 is expressedat the posterior endodermal margin,FGF-8 in the anterior endoderm, andPax-1 in the most dorsal endodermof the pharyngeal pouches, andthese expression domains are un-changed in arches that are devoidof neural crest cells (Veitch et al.,1999; Fig. 2). Also, these crest-freearches have a sense of individualidentity. Normally, sonic hedgehog isa prominent early feature of the pos-terior endoderm of the second arch,following later in the posteriorendoderm of the third arch, andagain in the absence of crest thespatial and temporal dynamics ofsonic hedgehog is unchanged inthe absence of crest (Veitch et al.,1999; Fig. 2). Thus, the endodermcan form a segmented series that isappropriately regionalized in the ab-sence of neural crest.

That pharyngeal segmentation isnot dependent upon the crest is alsosignificant, because it reflects theevolutionary history of the pharyn-geal arches. Endodermal pharyn-geal segmentation is a feature of allchordates (Schaeffer, 1987) and,thus, originated before the evolutionof the neural crest. The regionalisa-tion of the pharyngeal endodermalso preceded the emergence ofthe neural crest, and it has beenshown in Amphioxus that the Pax-1/9gene is expressed within the pharyn-geal pouches in a similar manner tovertebrates (Holland and Holland,1996). Thus, the evolution of the ver-tebrate pharynx must have involvedan integration between the neuralcrest cells, which generate the pha-ryngeal skeleton, and a more an-cient segmentally organised pha-ryngeal endoderm.

Further evidence for a role forendoderm in directing pharyngeal

8 GRAHAM ET AL.

arch development has come fromthe zebrafish mutant vgo in whichthe pharyngeal pouches fail to form(Piotrowski and Nusslein-Volhard,2000). In this mutant, arch develop-ment is severely perturbed and theneural crest-derived pharyngealcartilages are disorganised, oftenfusing with each other, or in themore posterior arches, failing toform altogether. This failure is notdue to defects in the crest but re-sults from the failure of pouch for-mation. Other mutants that displayendoderm defects, such as bonand cas which do not produceendoderm, also show defects inthe formation of the pharyngealarches, and in these animals, thepharyngeal cartilages fail to format all. It has also now been demon-strated that the neural crest cellsare induced to form cartilage bymeans of the action of FGF-3 em-anating from the endoderm (Davidet al., 2002). Furthermore, it has beendefinitively demonstrated, in chickembryos, that neural crest cells re-spond to patterning cues producedby the pharyngeal endoderm to gen-erate distinct skeletal components(Couly et al., 2002). The ablation ofspecific domains of the pharyngealendoderm at early stages of develop-ment results in the specific loss of par-ticular neural crest-derived skeletal el-ements. Moreover, if rostral neuralcrest cells are exposed to an ectopicpiece of pharyngeal endoderm, thenthey will generate a supernumeraryjaw. Of interest, the orientation of theectopic strip of endoderm determinesthe orientation of the skeletal ele-ments of the additional jaw.

PHARYNGEAL PATTERNING ISCONSENSUAL

These studies clearly highlight thatthe endoderm plays a pre-eminentrole in the patterning of the pharyn-geal arches; however, the neuralcrest cells are not completely pas-sive in this process. Rather, severalstudies suggest that the response ofdifferent populations of the neuralcrest to endodermal cues is depen-dent upon the transcription factorsthat they express, most notably theirHox gene repertoire. During normaldevelopment, each of the cranial

neural crest streams expresses differ-ent Hox genes (Hunt et al., 1991). Thetrigeminal stream is Hox negative,the hyoid expresses Hox-a2, and thepost-otic crest express members ofthird paralogous Hox group—Hox-a3, Hoxb3, Hox-d3. Of interest, inmice lacking Hox-a2, the secondarch exhibits a transformation, andjaw elements form within it (Gend-ron-Maguire et al., 1993; Rijli et al.,1993). These duplicated jaw ele-ments form as a mirror image to thefirst arch elements, which would beconsistent with these cells also re-sponding to cues emanating fromthe first pharyngeal pouch. How-ever, if Hox-a2 expression is forced inthe neural crest cells of the first arch,then jaw development is inhibitedand an ectopic hyoid forms, andagain this is mirror imaged (Gram-matopoulos et al., 2000; Pasqualettiet al., 2000). That Hox genes allowneural crest cells to respond differ-entially to endoderm cues is alsosupported by the observation that, ifHox-positive crest were exposed toan ectopic strip of rostral pharyn-geal endoderm, then, unlike the ros-tral crest which do not express Hoxgenes and form a jaw in this situa-tion, these cells do not form a jaw(Couly et al., 2002; Creuzet et al.,2002).

It has become clear that othertranscription factors, in addition toHox genes, can similarly act to mod-ulate the way in which crest cellsinterpret the peripheral patterningcues. In particular, the Dlx family,which display nested expression do-mains along the proximodistal axis ofthe arches seems to function in thisway (Simeone et al., 1994; Qiu et al.,1997a). Normally, the crest-derivedmesenchyme of the maxillary pri-mordia (forming the upper jaw) andthe proximal mandibular primordiaonly express Dlx-1 and -2. The rest ofthe mandibular mesenchyme ex-presses these genes and also Dlx-5and -6. Additionally, the distal-mostmesenchyme expresses Dlx-7 and -3.In mice mutant for both Dlx-5 and -6,several mandibular primordia skele-tal elements are missing and in theirplace skeletal elements characteris-tic of the maxillary primordia form(Depew et al., 2002). In these mu-tants, most of the mandibular mes-

enchyme only expresses Dlx-1 and-2, thus these cells behave as ifthey were maxillary. Thus, the pat-terning of the pharyngeal arches isa consensual process in which pat-terning cues emanating from theendoderm, or other pharyngeal tis-sues, are differentially interpretedby the crest cells of each of thearches, and this process is depen-dent upon the transcription factorsthey express.

IMPORTANCE OF THESTREAMING OF THE CRANIALNEURAL CREST

The demonstration that it is theendoderm that is the prime mover inorganising the development of thepharyngeal arches diminishes theidea that the key role of creststreaming is for the patterning thearches, segregating the first streamcarrying cues for the morphogenesisof the jaw from the second for thehyoid arch. This idea has becomeeven less attractive with the findingthat lamprey embryos also displaythree obvious crest streams, eventhough these animals lack a jawand hyoid (Horigome et al., 1999).We have demonstrated recentlythat the streaming of cranial neuralcrest, in fact, is necessary for a com-pletely different role: the organisa-tion of the afferent innervation of thehindbrain.

The majority of the neurons of thecranial sensory ganglia derive fromneurogenic placodes; focal thicken-ings of the embryonic ectoderm thatgenerate neuronal cells (Grahamand Begbie, 2000). The neurogenicplacodes form in stereotypical posi-tions in all vertebrates. The trigeminalplacodes emerge opposite the ante-rior hindbrain, the otic placodes at themiddle hindbrain level, and theepibranchial placodes—geniculate,petrosal, and nodose—close to thetips of the clefts between the pharyn-geal arches. The neuronal cells thatare formed within these placodesthen move internally to the site ofganglion formation wherein they dif-ferentiate and send out axons towardtheir central and peripheral targets.Importantly, these ganglia innervatethe hindbrain at specific axial levels(Lumsden and Keynes, 1989). The ax-

CRANIAL NEURAL CREST 9

ons of the trigeminal ganglion enterthe hindbrain at rhombomere 2, thoseof the geniculate and vestibuloac-coustic, which is derived from the oticplacode, at rhombomere 4, and theaxons of the petrosal and the nodoseganglia at rhombomeres 6 and 7.Rhombomeres 3 and 5 do not receiveany afferent innervation. There is, thus,a clear relationship between therhombomeres that receive sensory in-puts and those that generate thecrest streams (Fig. 3).

The neural crest streams that ex-tend from the hindbrain into the pe-riphery are generated in advance ofthe production of any of the pla-codal neuronal cells. Importantly,the neuronal cells that delaminatefrom the placodes migrate internallyalong the neural crest streams (Beg-bie and Graham, 2001). For exam-ple, the cells of the geniculate pla-code, which is associated with thesecond arch, move inward alongthe hyoid crest stream (Fig. 3). If thehindbrain is ablated before the pro-duction of the neural crest, the cellsleaving the placode enter a crest-free environment and do not mi-grate internally. Instead, they formdisconnected subectodermal gan-glia with aberrant projections thatdo not reach the hindbrain and of-ten extend toward other ganglia(Begbie and Graham, 2001). It couldbe argued that this failure in the mi-gration of the placodal cells is dueto the absence of the neural tubeand not specifically the neural crest.However, if the hindbrain is removedafter the production of the crest, theplacodal cells do move inward andestablish appropriate projections(Begbie and Graham, 2001). Thus,the presence of a neural creststream is essential for the projectionof sensory axons to the correct loca-tion in the developing hindbrain.

This integrative role for the neuralcrest further clarifies the phenotypicalterations seen in several mutantmice in which one or more of theepibranchial ganglia fail to connectto the hindbrain, even though noneof the genes in question are ex-pressed in the epibranchial ganglia:Sox-10 (Britsch et al., 2001), COUP-TFI(Qiu et al., 1997b), ErbB4 (Golding etal., 2000), CRKL (Guris et al., 2001),AP-2 (Zhang et al., 1996). It can now

be appreciated that these defectsare likely to be a secondary conse-quence of alteration in the behav-iour of the neuroglial neural crestcells. Further insights into this processcan be gained from another mousemutant in which �-catenin is mu-tated at the sites of wnt-1 expressionand which, thus, lacks �-catenin in itsmigratory crest cells (Brault et al.,2001). In this animal, the neural crestcells migrate normally but are subse-quently eliminated by means of apo-ptosis, and again the epibranchialganglia fail to connect to the hind-brain. Thus, the epibranchial neuro-nal cells are dependent upon thecrest cells themselves, rather thansome common earlier pathway thatis used by both the crest and theplacodal neuronal cells.

This evidence suggests that theprime function of the cranial creststreaming is to organise the afferentinnervation of the hindbrain. The realclue to this comes with the fact thatcrest streaming occurs in lampreys,which lack a jaw but have basicallythe same relationship between theircranial sensory ganglia and thehindbrain as all other vertebrates.That is not to say, however, that withthe evolution of the gnathostomes,existing crest streaming has beenco-opted to help pattern the jawsand jaw-support, and indeed tohelp organise the constrained pat-tern of skeletomuscular connectivityin the head. Rather, it is likely thatthese functions of crest streamingare built upon a more ancestralfunction of helping to organise thesensory innervation of the hindbrain.

CRANIAL NEURAL CREST HASSKELETOGENIC POTENTIAL

Besides any morphogenetic role, themajor difference between the cra-nial and the trunk neural crest lies inthe fact that the cranial crest gener-ates ectomesenchymal derivatives:bone, cartilage, dentine, muscle,and connective tissue. Indeed, fatemaps of the cranial crest con-structed in chick have suggestedthat the majority of the head skele-ton is crest derived (Noden, 1988;Couly et al., 1993). These elementsinclude dermal bones of the skulland all of the cartilages and bones

of the oro-pharyngeal region. Fur-thermore, it has also been shownthat the cranial crest generates theconnective tissues of the muscles,the dermis of the head, and thesmooth muscle cells associated withthe feathers and the aortic arches(Le Douarin and Kalcheim, 1999).Similar fate maps of the chick trunkcrest have suggested that thesecells do not generate bone and car-tilage. Although studies in zebrafishand Xenopus have shown that trunkneural crest cells do contribute to finconnective tissue (Collazo et al.,1993; Smith et al., 1994), these stud-ies did not find trunk crest formingskeletal derivatives.

The different fates of the cranialand the trunk neural crest cells werealso thought to represent differ-ences in potential. Thus, while cra-nial crest can form all ectomesen-chymal derivatives, trunk crest is onlycapable of forming connective tis-sues and smooth muscle, but notcartilage or bone. The supportingevidence for this comes from both invitro and in vivo studies. Clonal anal-ysis of cranial crest cells found thatthese would generate cartilage inaddition to neurons, glia, and mela-nocytes when cultured in rich me-dium on a 3T3 feeder layer, whichoffered support for a range of differ-entiation options (Baroffio et al.,1991). Yet, trunk crest cultured underthe same conditions were only everobserved to produce, neurons, glia,and melanocytes (Baroffio et al.,1988). Correspondingly, it was foundthat when trunk neural tube wasgrafted into the head, the neuralcrest cells produced by this graftnever contributed to cranial skeletalelements (Nakamura and Ayer-leLievre, 1982). Rather, these trans-posed cells tended to stay close tothe neural tube and differentiate asneurons and Schwann cells. It wasfound that these cells could formconnective tissue and muscle, butthey were never found to contributeto cranial skeletal elements (Naka-mura and Ayer-le Lievre, 1982).

Although these experimental ap-proaches yielded results that wereconsistent with trunk neural crestcells lacking the potential to gener-ate skeletal derivatives, they did notdirectly test its ability to do so. There-

10 GRAHAM ET AL.

fore, we explicitly assessed the skel-etogenic potential of trunk crest byculturing both chick cranial andtrunk crest cells in media commonlyused for growing bone and carti-lage cells (McGonnell and Graham,2002). As expected, bone and carti-lage differentiation occurred in cul-tures of cranial crest. Importantly,this was also found to be true of trunkcrest cultures, which under theseconditions generated both chon-

drocytes and osteocytes. The abilityof trunk crest to contribute to cranialskeletal elements was also tested invivo. As described, previous studieshad found that grafted trunk crestcells did not contribute to skeletalelements, yet this of course couldhave resulted from the failure ofthese cells to migrate from the trans-posed piece of neural tissue to thesites of skeletal differentiation. To cir-cumvent this potential difficulty, we

grafted trunk neural crest cells di-rectly into the facial primordia(McGonnell and Graham, 2002).These cells dispersed, and as ex-pected formed neurons, Schwanncells, and melanocytes; however,they also contributed to the formingskeletal elements, such as Meckel’scartilage. Another much older studyhas also shown that mammalian trunkneural crest cells can participate intooth formation when recombined

Fig. 4. Trunk neural crest has skeletogenicpotential. Neural crest fates are restrictedalong the anteroposterior axis of the embryo.Crest-derived parasympathetic (green) andsensory neurons (blue) are differentially dis-tributed along the whole embryo, while sym-pathetic neurons (grey) are restricted to thetrunk and ectomesenchyme (red) to the cra-nial region. Other fates not shown, such ascardiac and enteric crest, are similarly re-stricted. However, all neural crest has the po-tential to make the full range of possible crestderivatives, indicating that fate decisions aredriven by the environment that crest migratesthrough and differentiates in.

Fig. 5. Evolution of the trunk skeleton. Fos-sil evidence shows that early vertebrates,such as the Heterostracan, possessed ex-tensive postcranial dermal exoskeleton(red), likely derived from the trunk neuralcrest. Trunk exoskeleton became much re-duced with the appearance of the somite-derived endoskeleton. The zebrafish has apredominantly endoskeletal trunk supportbut possesses a remnant of exoskeleton inthe dermal fin rays, which is also likely de-rived from the trunk neural crest. Higher ver-tebrates, such as the chick, have an exclu-sively endoskeletal trunk support derivedsolely from the sclerotome of the somite.

CRANIAL NEURAL CREST 11

with oral ectoderm (Lumsden, 1988).Thus, trunk crest cells do have skeleto-genic potential.

In the past, the generation of dif-ferent derivatives by the neural crestof distinct axial levels was thoughtnot to reflect a lack of potential, butto be a consequence of environ-mental cues restricting these fates.The only exception to this scenariowas thought to apply to skeleto-genic potential, which was believedto be intrinsic to the cranial crest (LeDouarin and Kalcheim, 1999). Ourresults demonstrate that, in fact,there is no difference in potentialbetween the cranial and the trunkneural crest; both can generate thefull repertoire of crest derivatives(Fig. 4). Thus, with regard to poten-tial, there is nothing special aboutthe cranial crest.

During normal development theskeletogenic potential of trunk crestmust be suppressed. However, theskeletogenic potential of the trunkcrest was likely realized in early ver-tebrates (Fig. 5). There are many fos-sil fish that display extensive postcra-nial exoskeletal coverings of dermalbone and dentine, two cells typesthat derive from neural crest cells(Maisey, 1988; Smith and Hall, 1990;Donoghue and Sansom, 2002). Theskeletogenic potential of trunk crestis also expressed, albeit to a limitedextent, in some extant vertebrates. Astudy in zebrafish has suggested thatthe lepidotrichia, the distal miner-alised portions of the fins rays, areneural crest derived (Smith et al.,1994). Thus, it would seem that, dur-ing vertebrate evolution, the sup-pression in the skeletogenic poten-tial of the trunk crest mirrors thereduction in the extent of the trunkexoskeleton. Contrastingly, duringthis process, the endoskeleton hasemerged to be the major axial skel-etal support. This has undoubtedlyinvolved an enhancement of therole of the somites, and in particularof the sclerotome. It is likely, there-fore, that the environmental cuesthat suppress trunk neural crest skel-etal differentiation, in amniotes, areeither expressed in or related to theformation of the sclerotome.

It is also important to appreciatethat, from an evolutionary perspec-tive, the separation of the neural

crest into cranial versus truncal pop-ulations is likely to be a derived fea-ture, with what we term cranial crestrepresenting the ancestral state. In-deed, in many ways it’s not that thedevelopment of the head is distinctbecause of the role played by thecrest, but rather it’s the trunk thatdiffers, because in this region of theembryo, the mesoderm has be-come dominant.

CONCLUDING REMARKS

Five years ago, many people, our-selves included, would have arguedstrongly that the cranial neural crestplayed a pivotal role in the develop-ment of the vertebrate head. The cra-nial crest generates the head skele-ton, and these cells were believed tocoordinate the developed of this re-gion. Now, however, it is apparentthat this view must be reassessed. Theability of the cranial crest to generateskeletal derivatives is not unique to thisaxial level, but is shared by all crestcells. Rather, as with all other crest po-tentials, skeletal potential is restrictedby environmental cues. Furthermore,it is now clear that the crest does notplay the key role in patterning thehead and that, for the pharyngealarches, this patterning is fulfilled by thepharyngeal endoderm. However, it isnow evident that the streaming of thecranial neural crest is fundamental tothe organisation of the afferent inner-vation of the hindbrain. Thus, today,our views of the significance of thecranial neural crest have changed.

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