Neural crest cell formation and migration in the developing

embryoMARIANNE BRONNER-FRASER

Developmental Biology Center,University of California, Irvine, California 92717,USA

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0892-6638/94/0008-0699/$01 .50. © FASEB 699

ABSTRACT Neural crest cells arise from the neuraltube shortly after its closure and migrate extensivelythrough prescribed regions of the embryos, where theydifferentiate into most of the peripheral nervous systemas well as the facial skeleton and pigment cells. Along theembryonic axis, several distinct neural crest populationsdiffer both in their migratory pathways and range ofderivatives. Whereas those cells arising from the mid-brain migrate as a uniform sheet of cells, neural crest cellsemerging from the hindbrain and trunk regions migratein a segmented manner. For example, trunk neural crestcells move preferentially through the rostral, but not cau-dal, half of each somite. Interactions with tissues encoun-tered during migration strongly influence this segmentalmigratory pattern. For example, the mesodermal somitesdictate the segmental migration of trunk neural crest cellsand the otic placode appears to attract hindbrain neuralcrest cells. Although little is known about the molecularbasis underlying migration, patterns of gene expressionin the hindbrain are thought to contribute to the segmen-tal arrangement of neural crest cells. Furthermore, neuralcrest cells possess integrmn receptors that may be impor-tant for interacting with extracellular matrix molecules intheir surroundings.- Bronner-Fraser, M. Neural crestcell formation and migration in the developing embryo.FASEBJ. 8: 699-706; 1994.

Key Words: dye labeling . cell movement ext racellular matrixhindbrain . lineage

THE NEURAL CREST

The vertebrate neural crest forms during neurulation, whenthe flattened neural epithelium, or the neural plate, thickensand subsequently invaginates to form the neural tube. Initia-tion of neural crest migration from the dorsal midline of theneural tube occurs in a head-to-tailward (rostrocaudal) se-quence, shortly after tube closure. The neural tube gives riseto the central nervous system, consisting of the brain and thespinal cord. Neural crestcellsemerge from the neural tube,

migrate extensively,and form most elements of the peripheralnervous system, as well as facialcartilage,pigment cells,and

neuroendocrine cells.Upon departing from the dorsal surface of the neural tube,

neural crest cells enter a cell-free zone that is rich in extracel-lular matrix molecules. Although they are readily detectablein this cell-free zone, they soon intermix with and becomemorphologically indistinguishable from tissues throughwhich they migrate. To follow their migration as a functionof time, it has been necessary to label neural crest cells usinga variety of cell marking techniques. Successfully appliedmethodologies for following neural crest migration include:1) transplantions of neural tubes labeled with either radioac-

tive or species-specific markers into unlabeled host embryos(1, 2); 2) staining with antibodies such as HNK-1/NC-1 (3,

4) that recognize migrating neural crestcells;and 3) labelingwith the lipophiic dye Dil into the lumen of the neural tubeor directly into the neural folds (5, 6).

All three methods have yielded similar results regarding

the pathways and derivatives of the neural crest. The follow-ing picture has emerged. After leaving the neural tube, neu-ral crest cells move in a highly patterned fashion throughneighboring tissues. Subsequently, they become localized indiverse and characteristic sites within the embryo, wherethey differentiate into a wide array of derivatives. The exactpatterns of migration and types of derivatives formed varyaccording to the axiallevelof origin within the neural tube.For example, cranial neural crest cells from the midbrainregion migrate as a broad, unsegmented sheet of cells underthe ectoderm. In contrast, neural crest cells in the hindbrainand trunk regions migrate in a segmental fashion throughneighboring tissues. As a consequence of the differences inmigratory pathways and derivatives, neural crest populationsalong the rostrocaudal length of neural axis have been desig-nated as cranial, vagal, trunk, and lumbosacral (2).

FORMATION OF THE NEURAL CREST ANDESTABLISHMENT OF DORSOVENTRAL POLARITYIN THE NEURAL TUBE

Before the emergence of neural crest cells, the neural tube isa single cell layer thick and contains morphologically similarcells.Injectionof lineage tracer into individual trunk neuraltube cellsrevealsthat a cellin the dorsal neural tube can

form multiple types of neural crest derivatives, includingsensory and sympathetic ganglion cells, pigment cells, andadrenomedullary cells (7, 8). In tissue culture, some neuralcrestcellshave been shown to have stem cell properties (9).Furthermore, neural crest and dorsal neural tube cells canarisefrom the same progenitor.This suggests that the neuralcrestisnot a segregated population within the neural tube.

Once formed, the neural tube has a characteristic polarityalong the rostrocaudal as well as the dorsoventral axes.Regionalization along the rostrocaudal axis is manifested bythe formation of subdivisions in the neural tube, such as theforebrain, midbrain, hindbrain, and spinal cord. Along thedorsoventral axis,differentcell types arise from different por-tions of the neural tube. The dorsal neural tube forms theroof plate, commissural neurons, and neural crest cells,which subsequently emigrate (Fig. 1A). The ventral neuraltube forms the floor plate in the ventral midline and the mo-tor neuron columns that form lateral to the floor plate andproject their processes into the periphery.

The notochord is thought to play an important role in es-tablishing the dorsoventral polarity of the neural tube. Anotochord grafted lateral to the neural tube induces an extrafloor plate and motor neurons (10-13). Neural tube cells ad-

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C,

NOTOONORD ABLATION

NEURAL CRESTCOMMISSURAL NEURONSROOF PLATE

MOTORNEURONS (delay.d)FLOOR PLATE (d.Iay.d)

Figure 1. Schematic diagram showing the cell types that arise in the

dorsal and ventral neural tube in unoperated embryos (A), embryos

with a dorsally implanted notochord (B), and embryos in which theendogenous notochord is ablated (C). A) Neural crest cells normally

emerge from the dorsal neural tube. The roof plate and commis-sural neurons differentiate dorsally, whereas motor neurons and

floor plate cells differentiate ventrally (13). B) After implantation ofa notochord into the dorsal midline, neural crest cells continue to

emerge dorsally. Commissural neurons and motor neurons coexistin the dorsal portion of the neural tube (16) and a floor plate de-velops in the dorsal midline when the operation is performed before

neural tube closure (13). C) After ablation of the endogenous

notochord, the pattern of neural crest migration and commissural

neuron formation occurs in a delayed fashion. The floor plate andmotor neurons differentiate ventrally approximately 1 day behind

their normal schedule, being absent 2 days after the operation, but

present by 3 to 4 days postablation (17).

jacent to the region of contact assume a wedge-shaped mor-phology characteristic of the floor plate (14, 15) and expressfloor plate-specific markers (13).

Although a dorsally grafted notochord can induce ventralproperties, it does not suppress formation of neural crestcellsor commissural neurons (Fig. 1B; 16). Thus, some dorsalproperties may be established before neural tube closure and

cannot be subverted by the presence of a notochord. Simi-larly, ablation of the notochord, originally thought to lead toabsence of the floor plate, recently has been shown to delaybut not prevent floor plate and motor neuron formation (Fig.1C; 17). Cumulatively, these experiments suggest that forma-tion of both the neural crest dorsally and the floor plate yen-trallymay be caused by signals that occur early in embryo-genesis. Consistent with this possibility,tissue cultureexperiments reveal that the isolated ventral neural plate hasthe ability to form some neural crest derivatives autono-mously well before neural tube closure. This ability to formneural crest may be mediated by interactions between theneural plate and epidermis (M. A. J. Selleck and M.Bronner-Fraser, unpublished results).

INITIATION OF NEURAL CREST MIGRATION

Neural crest cells emerge at or near the dorsal midline of theneural tube shortly after its closure and continue to emigratefor 24 to 36 h. Little is known about the factors that causedelamination of neural crest cells from the neuroepithelium.Perhaps those neural tube cells closest to the dorsal midlinelose cell-cell contacts by virtue of their position, emigrate,and by definition, form neural crest cells. Several propertiesof the dorsal midline make it a logical site of egress for neuralcrest cells. For example, the basement membrane surround-

ing the neural tube is discontinuous over its dorsal aspect(18). Before neural crest cell emigration, there appear to bechanges in cell-cell adhesiveness among neuroepithelial cellsin the midline region. N-cadherin immunoreactivity, con-tained in adherens junctions that interconnect adjacent neu-ral tube cells, decreases in the dorsal midline region (19).Furthermore, the site of neural crest exit appears to be deter-mined by the time of tube closure. Even after the neural tubeis rotated 180#{176}dorsoventrally, neural crest cells emerge nor-mally relative to the neural tube and independent of theirrelationship to other embryonic tissue (1, 20).

MIGRATION PATTERNS OF CRANIAL NEURALCREST CELLS

Perhaps the most complicated patterning of the neural crestoccurs in the head region. The cranial neural crest consistsof several populations that differ in their migratory path-ways, patterns of gene expression, and types of derivatives.For functional purposes, the cranial neural crest cells can besubdivided into regions designated here as caudal forebrain,midbrain, rostral hindbrain, and caudal hindbrain (whichoverlaps with rostral vagal) neural crest cells. Each popula-tion has a somewhat different pattern of migration andprospective derivatives. Avian neural crest cells do not emergefrom the forebrain neural tube except for the caudal-mostportion. At the midbrain level, neural crest cells emerge andmigrate primarily as a broad, unsegmented sheet of cells un-der the ectoderm. These cells contribute to a wide range ofderivatives that include the periocular skeleton, connectivetissue of the eye, membrane bones of the face, ciliary gan-glion, part of the trigeminal ganglion, and Schwann cells (2).

The hindbrain is a unique region of the neural tube, be-cause it is the only portion of the developing brain that isovertly segmented. It is subdivided into eight segments orrhombomeres. Rhombomeres represent compartments oflineage restriction such that clones of cells do not cross rhom-bomere boundaries (21). Production of neurons in the hind-brain occurs first in the even-numbered rhombomeres, fol-lowed by odd-numbered ones (22). Furthermore, even andodd rhombomeres have different adhesive properties, whichresult in the formation of boundaries when they are juxta-posed (23, 24).

Unlike the more rostral neural crest populations, hind-brain neural crest cells migrate in a segmental fashion, asthree broad streams emanating laterally adjacent to rhombo-meres (r) rl/2, r4, and r6, with no neural crest cells apparentat the level of r3 or r5. The first of these streams populatesthe trigeminal ganglion and mandibular arch; the secondpopulates the hyoid arch, as well as the geniculate and yes-tibular ganglia; the third stream populates the third andfourth branchial arches and associated peripheral ganglia(25-27).

The segmental pattern of neural crest migration in thehindbrain could arise from a segmentally restricted origin ofneural crest cells within the neural tube or from a segmentalmigratory pattern imposed by the environment of the rhom-bencephalic neural crest. The DiI-labeling experiments ofLumsden and colleagues (27) suggest that r3 and r5 do notform neural crest cells, consistent with the possibility that thesegmental migratory pattern reflects a patterned origin wi-thin the neural tube. They proposed that r3 and r5 fail toproduce neural crest cell, perhaps because of cell death (24).In contrast, the grafting experiments of Couly and LeDoua-nfl (28) suggested that neural crest cells did arise at leastfrom r5.

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NEURAL CREST MIGRATION 701

To resolve the discrepancies between these results, we haveexamined cranial neural crest migratory pathways using thecombination of neurofilament immunocytochemistry, whichrecognizes early hindbrain neural crest cells, and labelingwith the vitaldye, Dii (6).Neurofilament-positive cellswiththe appearance of premigratory and early-migrating neuralcrest cells were noted at all axial levels of the hindbrain, sug-gesting a uniform origin for neural crest cells within allrhombomeres. At slightly later stages, neural crest cellmigration in this region appeared segmented, with no neuralcrest cells obvious in the mesenchyme lateral to r3 and be-tween the neural tube and the otic vesicle lateral to r5 (6).

Potential problems associated with using antibodies asmarkers for migrating cells are that they may not recognize allcells in the population and that their expression may be tran-

sient. To circumvent these difficulties and use an alternativemarker, individual rhombomeres were labeled with small fo-cal injections of Dil. Injections at the levels of r3 and r5demonstrate that both of these rhombomeres generate neu-ral crest cells (Fig. 2). The segmental distribution of neuralcrest cells results from the Dil-labeled cells that originate inr3 and r5 deviating rostrally or caudally and failing to enterthe adjacent preotic mesoderm or otic vesicle region (6). Wi-thin a few hours after injection, individual neural crest cellscan be viewed migrating along the r3 neural tube and shift-ing caudally toward the border between r3/4 or betweenr2/3, at which points they leave the surface of the neural tube(unpublished observation). The finding that neural crestcells originating from r3 and r5 avoid specific neighboringdomains raises the intriguing possibility that, as in the trunk,

Figure 2. Confocal microscope images of embryos after receiving focal injections of Dii into individual rhombomeres. A) An embryo fixed2 h after receiving a focal injection of Dii into r3. The injection was performed at the 9-ss and the embryo was fixed at the lO-ss. Theinjection site is visible within the confines of r3 (between arrowheads). A few Dil-labeled cells already have dispersed from the initial injec-tion site, and are seen migrating both rostrally and caudally but not laterally. B) An embryo injected into r3 at the 8-ss, in which Dii-labeledcellshave moved from r3 in a caudolateralstream rostral to the otic vesicle (OT), fusing with the stream emanating from r4. C)An embryo injected in r4 at the 12-ss; many Dil-labeled neural crest cells coursed ventrolaterally into the second branchial arch. D) Anembryo injected into r5 at the li-ss, in which the Dil-labeled cells (arrows) have moved both rostrally and caudally around the ode vesicle,fusing with neuralcrestcellsderivedfrom r4 and r6 (modified from Sechrist et al., ref 6).

a R4*_ R3

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the periphery plays a major role in the axial patterning of thecranial neural crest and the neural crest-derived peripheralnervous system.

Two possible explanations could account for the segmentalpattern of neural crestmigration in the hindbrain. First,itmay be established by signals within the neural tube, beforeemigration. Second, the pattern may depend on interactionswithin the migratory environment. Distinguishing betweenthese possiblemechanisms requires challenging neural crestmigratory pathways. To do so, we performed rostrocaudal ro-tations of either the cranial neural tube or adjacent ecto-derm/mesoderm, as illustrated in Fig. 3, as well as grafts ofthe otic placode. Neural crest migration is assessed using Diias a cell marker. Rotation of the neural tube does not alterthe segmental pattern of neural crest cell migration. Rather,neural crest cells migrate in a manner generally appropriatefor their new location. For example, when r3 and r4 aretransposed, labeled r4 cells primarily deviate caudally

toward the second arch, with some cells moving rostrallytoward the first (Fig. 3B). This pattern looks similar to thatseen from unrotated r3. In contrast, labeled neural crest cellsarising from rotated r3 cells migrate laterally to the secondbranchial arch, as would cells normally emerging from r4.However, the patterns of neural crest cell migration are notalways identical to those observed in unoperated embryos.There are two consistent differences: 1) rotated r3 cells leavethe neural tube surfacenear the r3/4 border, whereas normal

r4 cells exit the neural tube uniformly; and 2) cells from ro-tated r4 enter the mesenchyme adjacent to r3 whereas nor-mal r3 neural crest cells do not invade this mesoderm.

One consequence of grafting the rhombomeres is that asmall, ectopic otic vesicle often forms adjacent to rotated r4

after grafting it to a new position (see Fig. 5B). In most em-bryos containing such an extra veside, Dil-labeled neuralcrest cells move directionally toward these otic vesicles. Simi-larly, they move toward a grafted (Fig. 3C) or caudally dis-placed otic vesicle. On the other hand, rotation of the meso-derm adjacent to r3 and r4 has no obvious effect on thepattern of neural crest migration. These results demonstratethat signals from another tissue, the otic vesicle, caninfluence the pattern of neural crest migration in the hind-brain (29). This influence could be rendered either by selec-tive attraction or pathway-derived cues.

GENE EXPRESSION IN THE HINDBRAIN

Some understanding of the molecular basis of hindbrain andneural crest specification has come from analyses of the ex-

pression and function of Hox homeobox genes, some of thehomologs of the homeotic genes of Drosophila (reviewed byHunt et al., ref 30). The vertebrate Hox genes are organizedin four paralogous clusters, with all genes within each clusterhaving the same orientation relative to the 5-3 direction oftranscription. With one exception, successive genes from5-3’ are expressed in domains that extend from the caudalend to a progressively more rostral limit in the spinal cordor hindbrain. Those genes expressed in the hindbrain havelimits of expression at rhombomere boundaries. Hox gene ex-pression in some early migrating crest is identical to that inthe rhombomere of origin and persists during migration intothe branchial arches or ganglia (30). Furthermore, trans-planted rhombomeres maintain expression of Hox genes in-dicative of their original position even after being grafted to

Figure 3. A) A schematic diagram illustrating the operation of Dii labeling rhombomere 4 (R4) followed by 180#{176}rostrocaudal rotation

of r3/4 (R4 - R3). B) A confocal microscopic image of an embryo operated at the il-somite stage and fixed at the 20-somite stage.DiI-labeled cells have emerged from the injection site (large arrow) and have migrated caudally toward the endogenous otic vesicle (OT)

and into the second branchial arch. An ectopic otic vesicle (eOT) formed in this embryo at the r2/4 graft border, around which a small

stream (small arrow) of DiI-labeled cells migrated. C) A 20-ss embryo in which Dil-label was injected into r4 and an otic placode wasgrafted at the r2/3 border at the lO-ss. An ectopic otic vesicle (cOT) formed rostral to the endogenous otic vesicle (OT). Dil-labeled cellswere evident adjacent to the rostral portion of both the endogenous (OT) and grafted (cOT) otic vesicle (modified from Sechrist et al,

ref 29).

ARCH 1

ARCH2

Hox B4

Hox B2

HoxB3

Figure 4. Schematic diagram illustrating the rhombomeres, bran-chial arches and the expression patterns of known genes. Arrows in-dicate the directions of migrating neural crest cells arising from therhombomeres.

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an ectopic site (31, 32). Based on these expression studies, thecombination or code of gene expression has been proposedto specify both rhombomeres and theirneural crestderiva-tives at neural plate stages and thus underlie the patterningof the branchial arches (Fig. 4). Although selected homeo-box genes appear in a segment-specific pattern in the hind-brain and corresponding branchial arches, little is knownabout their expression in migrating neural crest cells thatpopulate the arches.

Another gene that may be involved in segmental pattern-ing, both in the hindbrain and neural crest,isthe zinc fingergene Krox-20. Krox-20 is expressed in alternating domains inthe neural plate that later correspond to r3 and r5, where itacts as a transcriptional regulator of HoxB2 expression (33).We have examined the expression and origin of the zincfinger gene Krox-20 in hindbrain neural crest cells in orderto gain some insightinto itsfunction in thispopulation. Inaddition to its expression in r3 and r5, Krox-20 is detectablein neural crest precursors in the dorsal midline of the neural

#{149}.

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Figure 5. Whole-mount view of an embryo after Dii injection intor5 followed by in situ hybridization with a Krox-20 probe. After fixa-tion, the Di! was photoconverted to a brown precipitate. The purplealkaline-phosphatase staining represents Krox-20 signal. Labeledneuralcrestcells(arrows)thatareKrox-20 positive arise from r5 andr6 and migrate caudal to the otic vesicle (01’).

tube extending from caudal r5 into r6. These Krox-20 ex-pressing cells leave the neural tube at the 13-somite stage,emerging near the r5/6 border and migrating caudally andventrally.

By combining Dii cell marking techniques with in situhybridization (34), we have been able to determine therhombomeric origin of some neural crest cells that carry theKrox-20 transcription factor (Nieto et al., unpublishedresults). Our results show that both r5 and r6 contribute tothe Krox-20 expressing neural crest cells that migrate cau-dally around the otic vesicle (Fig. 5). Krox-20 expressing cellsfirst migrate from mid-to-caudal r6 and shortly thereafterfrom rostral r6 and r5. Its expression is transient, becomingundetectable in the neural crest by the 23-somite stage. Simi-lar analyses should be possible with other transcription factors.

MIGRATION PATTERNS OF TRUNK NEURALCREST CELLS

In the trunk region, neural crest cells migrate along twomajor pathways: a dorsal pathway underneath the ectoderm,

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whose progeny become pigment cells, and a ventral pathwaythrough the somites whose progeny form sensory and sym-pathetic ganglia and adrenomedullary cells (2). Neural crestmigration along the dorsal pathway in unsegmented. In con-trast, cells following the ventral pathway migrate in a seg-mental fashion, moving selectively through the rostral, butnot caudal, portion of the somites (35). Similar to neuralcrest cells,motor axons emerging from the ventral neuraltube (36) navigate preferentiallythrough the rostralhalf ofeach somite. Perhaps molecular differences within the so-mites influence neural crest cell and motor axon movement.For example, there may be inhibitory cues in the caudal so-mite, attractive cues in the rostral somite, or both.

By rotating either the neural tube or the segmental plate(which gives riseto the somites) 180#{176}about its rostrocaudalaxis, Keynes and Stern (36) found that the orientation of thesomites, but not the neural tube, dictated the pattern of ax-onal movement. In support of the idea that the caudal somitemay inhibit axonal growth, Davis and colleagues (37) haveshown that a molecule recognized by peanut agglutinin,selectively found in the caudal somite, inhibits axon growth.

Analogous rotations experiments have been performed toanalyze what controls the pattern of trunk neural crestmigration (38). The results are similar to those observed formotor axons. After rostrocaudal mesodermal rotations, neu-ral crest cells emerge uniformly from the neural tube, then

Figure 6. The effects of inverting the rostrocaudal polarity of the

somites.The schematic diagram (left) illustrates the operation inwhich the segmental plate is rotated 180#{176}rostrocaudally, producing

somites with inverted polarity. A longitudinal section (left) stainedwith the HNK-l antibody through an embryo 24 h after such an

operation.The arrow indicatesa somite marking thegraftborder,above which the somiteshave been inverted.Rostralto the graft

border, neural crest cells migrate through the caudal (C) portion ofthe somite, which would have been rostral before rotation. Below

the graft in the unoperated region of the embryo, neural crest cellsmigrate through the rostral (R) but not caudal portion of the so-

mites. These results suggest that the somites are responsible for thesegmental pattern of neural crest migration (modified from

Bronner-Fraser and Stern, ref 38).

Figure 7. Top) Schematic diagram illustrating the way in which anectopic notochord (No) is implanted between the neural tube (NT)and somites (SOM). Bottom) A transverse section stained with the

HNK-l antibody through an embryo 24 h after such an operation.

Neural crest cells (arrow) do not approach the ectopic notochords

(eNo) (modified from Pettway et al., ref 45).

migrate through the rotated somites. However, their patternof migration is inverted such that neural crest cells are nowpresent in the caudal (original rostral) halves of the somitesafter rotation of the mesoderm (Fig. 6). Although their func-tion remains to be established, there are some possible candi-dates for inhibitory or attractive molecules within the so-mites. For example, molecules selectively distributed in thecaudal portion of the somite include T-cadherin (39),chon-

droitin sulfate proteoglycans (40), and molecules recognizedby peanut lectins (41). Other potentiallyattractivemoleculesare distributed selectively in the rostral portion of the somite;these include butyrlcholinesterase (42) and tenascin,although the latterappears as a consequence rather than acause of neural crestmigration (43).

Another tissuethat appears to be inhibitoryformigratingneural crestcellsisthe notochord. Ventrally migrating neu-ral crest cells in the trunk migrate throughout the rostral so-mitic sclerotome, with the exception of the region surround-ing the notochord. In tissue culture experiments, Newgreen

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(44) found that neural crest cells avoided the region sur-rounding notochords with which they were cocultured, sug-gesting that the notochord produces a substance that inhibitsneural crest migration. We have implanted a length of quailnotochord lateral to the neural tube of 2-day-old chicken em-bryos in order to examine the potential inhibitory effects ofthe notochord in vivo. Neural crest cells were observed toavoid the implanted notochord, consistent with the possibil-ity that itinhibitstheirmigration (Fig. 7).Furthermore, the

inhibition was sensitive to treatment with trypsin and chon-droitinase (45). A likely candidate for the inhibitorymolecule is a chondroitin sulfate proteoglycan that bears theHNK-1 epitope (46).

RECEFIORS AND EXTRACELLULAR MOLECULESON NEURAL CREST PATHWAYS

Along neural crest migratory pathways, there are abundantlevels of extracellular matrix molecules. The most prevalentglycoproteins are fibronectin (47), laminin (48), tenas-cin/cytotactin (49), and various collagens (50, 51). Fibronec-tin in particular has been suggested to play a major role inthe adhesion and motility of neural crest cells (47). In tissueculture, neural crest cells migrate avidly on both fibronectin(52) and laminin (53) substrates. Hyaluronic acid is presentin high concentrations during the early stages of migration(54); thus, initiation of neural crest cell movement occurs ina hyaluronate-rich region. Of the proteoglycans present wi-thin the embryo, heparan sulfate proteoglycans (51) appearon neural crest cell pathways, whereas chondroitin sulfateproteoglycans are generally present in regions from whichneural crest cells are absent (49, 51), such as the perinotochordalspace. Chondroitin sulfate proteoglycans tend to inhibit neu-ral crest cell migration in vitro, consistent with the idea thatthey may restrict or inhibit migration in the embryo.

Neural crest cells possess several integrin receptors thatrecognize a variety of extracellular matrix molecules. Invitro, antibodies against j3 integrins block neural crest at-tachment to fibronectin, laminin, and collagens, suggestingthat these are the primary mediators of neural crest cell at-tachment (55). However, little is known about the a subunitsassociated with fi integrins on neural crest cells. To charac-terize a subunits on neural crest cells, short anti-sense phos-phorothiol oligonucleotides (15- to 30-mers) have been usedto knock out mRNA for proteins in cultured cells. Selectedantisense oligonucleotides reduce the amounts of cell-surfacea1 and/or f3 integrin subunits by up to 95% and inhibit neu-ral crest cell attachment to laminin or fibronectin substrata.The operation of at least three distinct a integrin subunitsis indicated by substratum-selective inhibition of cell attach-ment (56). One of these is an a5 subunit (57). Although theidentity of the others remains unknown, antibody labelingexperiments suggest that they do not correspond to a5, a6,or a7 (M. Bronner-Fraser, unpublished results).

Microinjection of antibodies lateral to the cranial neuraltube in vivo can be used to functionally knock out selectedcell-matrix interactions along neural crest migratory path-ways. Such perturbation experiments suggest that some cx-tracellular matrix molecules play a functional role in cranialneural crest migration. Using this approach, it has beenshown that various molecules are necessary for properemigration of cranial neural crest cells. These include the ssubunit of integrin, fibronectin, laminin-heparan sulfateproteoglycan complex, tenascin, and galactosyltransferase

(58). The phenotypes arising after antibody injection include

an accumulation of neural crest cells within or adjacent tothe neural tube, together with various neural tube abnormal-ities. These observations suggest that numerous moleculesrather than a single molecule are necessary for properemigration of neural crest cells. This is consistent with theidea that cranial neural crest migration uses complex, andperhaps multivalent, interactions. Curiously, the above-described abnormalities are seen only after injection of anti-bodies into the cranial region; in contrast, similar injectionsinto trunk neural crest cell pathways have no detectableeffects, though these antibodies do affect migration of myob-last cells (59) in this region of the embryo. This observationsupports the idea that cranial and trunk neural crest popula-tions differ along the neural axis.

CONCLUSIONS

The ability to combine techniques in experimental embryol-ogy, cell, and molecular biology has clarified some of themechanisms underlying neural crest migration. The neuralcrest clearly is not a single population of cells, but rather aseries of overlapping populations that differ in their migra-tory pathways and derivatives. In the hindbrain, the segmen-tal migration of neural crest cells may be influenced both byinformation inherent to the rhombomeres coupled with en-vironmental signals from neighboring tissues, such as the

otic vesicle. In the trunk, cell-cell interactions may predom-inate, such that the mesodermal somites control the ros-trocaudal patterning of neural crest cells and the notochordprevents neural crest cells from crossing the midline. At amolecular level, interactions between integrin cell-surfacereceptors and extracellular matrix molecules in the periph-ery may influence neural crest cell emigration. Furthermore,changes in intercellular adhesion, manifested by cadherins,may be important for delamination of neural crest cells fromthe neural tube. The challenge for the future will be tocharacterize the system at the cellularand molecular level,clarifying how changes in gene expression control changes incell behavior that account for patterning of migration. jJ

Our work is supported by U.S. Public Health Service HD-l5527,HD-25138, DE10066, and by a grant from the Muscular DystrophyFoundation.

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