Review article

Role of the extracellular matrix during neural crest cell migration

Roberto Perrisa,b,*, Daniela Perissinottob

aDepartment of Functional and Evolutionary Biology, University of Parma, Viale delle Scienze, 43100 Parma, ItalybDivision for Experimental Oncology 2, The National Cancer Institute, Via Pedermonanta Occidentale 12, 33081 Aviano (PN), Italy

Received 1 March 2000; received in revised form 18 May 2000; accepted 18 May 2000

Abstract

Once speci®ed to become neural crest (NC), cells occupying the dorsal portion of the neural tube disrupt their cadherin-mediated cell-cell

contacts, acquire motile properties, and embark upon an extensive migration through the embryo to reach their ultimate phenotype-speci®c

sites. The understanding of how this movement is regulated is still rather fragmentary due to the complexity of the cellular and molecular

interactions involved. An additional intricate aspect of the regulation of NC cell movement is that the timings, modes and patterns of NC cell

migration are intimately associated with the concomitant phenotypic diversi®cation that cells undergo during their migratory phase and the

fact that these changes modulate the way that moving cells interact with their microenvironment. To date, two interplaying mechanisms

appear central for the guidance of the migrating NC cells through the embryo: one involves secreted signalling molecules acting through their

cognate protein kinase/phosphatase-type receptors and the other is contributed by the multivalent interactions of the cells with their

surrounding extracellular matrix (ECM). The latter ones seem fundamental in light of the central morphogenetic role played by the

intracellular signals transduced through the cytoscheleton upon integrin ligation, and the convergence of these signalling cascades with

those triggered by cadherins, survival/growth factor receptors, gap junctional communications, and stretch-activated calcium channels. The

elucidation of the importance of the ECM during NC cell movement is presently favoured by the augmenting knowledge about the

macromolecular structure of the speci®c ECM assembled during NC development and the functional assaying of its individual constituents

via molecular and genetic manipulations. Collectively, these data propose that NC cell migration may be governed by time- and space-

dependent alterations in the expression of inhibitory ECM components; the relative ratio of permissive versus non-permissive ECM

components; and the supramolecular assembly of permissive ECM components. Six multidomain ECM constituents encoded by a corre-

sponding number of genes appear to date the master ECM molecules in the control of NC cell movement. These are ®bronectin, laminin

isoforms 1 and 8, aggrecan, and PG-M/version isoforms V0 and V1. This review revisits a number of original observations in amphibian and

avian embryos and discusses them in light of more recent experimental data to explain how the interaction of moving NC cells with these

ECM components may be coordinated to guide cells toward their ®nal sites during the process of organogenesis. q 2000 Elsevier Science

Ireland Ltd. All rights reserved.

Keywords: Neural crest; Extracellular matrix; Cell migration; Morphogenesis; Pattern formation

1. Pre-migratory versus pro-motile NC cells

A primary unresolved question concerning the migration

of NC cells is how locomotion is initiated in the speci®ed

premigratory NC. That means how cells are converted from

a non-motile into a motile state. In higher vertebrates, this

transition obligatory requires a loss of N-cadherin/cadherin-

6-mediated cell-cell adhesion (Bronner-Fraser et al., 1992;

Monier-Gavelle and Duband, 1995; Inoue et al., 1997;

Nakagawa and Takeichi, 1998), which most likely triggers

a catenin-mediated signalling cascade capable of inducing

inside-out activation of integrins and possibly other cell

surface receptors. However, in certain amphibian embryos,

such as the Mexical axolotl where a contiguous basement

membrane separates the premigratory NC from the under-

lying neural tube, ultrastructural analysis reveals that cells

are not held together by specialized cell-cell contacts

(LoÈfberg et al., 1980, 1989). Thus, although an epithelial-

mesenchymal transition-speci®c loss of cadherin-mediated

adhesion seems to be of some importance for the formation

and dispersion of NC cells in Xenopusembryos (Kintner,

1992; Espeseth et al., 1998; Vallin et al., 1998), the delami-

nation process and initiation of NC cell movement may not

obey evolutionary conserved regulatory mechanisms.

At present there is no evidence that the acquisition of

Mechanisms of Development 95 (2000) 3±21

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* Corresponding author. Tel.: 139-0434-659-301; fax: 139-0434-659-

428.

locomotory capabilities by premigratory NC cells may be

controlled by an intrisic developmental clock. This is parti-

cularly obvious for the axolotl embryo where NC cells

remain `premigratory' for a signi®cant period of time,

despite of being fully competent to locomote (Perris and

LoÈfberg, 1986). This `stationary phase' could simply be

due to the `inactive state' of motility-associated GTPases

of the Rho/Rac/cdc42 family (Liu and Jessel, 1998), or

essential transcriptional elements encoded by genes such

as Slug, Twist, Wnts, Id2, Pax3, Sox 9 and 10, or to an

improper balance in the tyrosine phospatase/tyrosine kinase

signalling activity (Wehrle-Haller and Weston, 1996; Bren-

nan et al., 1999). Alternatively, it could be attributed to the

lack of an adequate focal adhesion mobility allowing for the

modulation of integrin af®nity in response to ECM cues

(Smilenov et al., 1999). How pro-motile genes could be

turned on is still unclear. However, the release of the signal-

ling molecules such as nogging, BMPs, or the recently

described noelin-1 (LaBonne and Bronner-Fraser, 1999;

Sela-Donenfeld and Kalcheim, 1999; Barembaum et al.,

2000) may afford a plausible RAS-dependent control

mechanism for inside-out integrin activation. Such an exter-

nal signalling could cooperate with that involving mobiliza-

tion of catenins, or could represent the inducing factor for

the rapture of the cadherin-mediated intercellular adhesion

in higher vertebrate embryos. Differential tethering of these

inducing molecules to various ECM components may be

decisive in creating threshold local concentrations, as well

as set up the proper morphogen gradients for evoking differ-

ential cell responses (Gurdon et al., 1999). Reciprocal inter-

actions of integrins with their cognate ECM ligands (Condic

and Letourneau, 1997) and the coaction of these adhesive

events and those involving cell-cell contacts may be suf®-

cient to `activate' presumptive NC cells residing within the

neural tube and convert them into pro-motile cells.

However, there is also experimental evidence that ECM

factors are directly implicated in the triggering of NC cell

movement in the axolotl embryo (LoÈfberg et al., 1985;

1988) and the possibility has to be considered that these

may be the same factors participating in the regulation of

the onset of cell migration in other embryos.

2. Functional relationship between positionalinformation and ECM interaction for the onset of NCcell migration

The next unresolved question concerns how NC cells are

directed along the dorsolateral or medioventral migratory

pathways at the onset of their dispersion through the embryo.

A closer light and electron microscopic analysis of the initial

phases of NC cell movement in the axolotl embryo discloses

the possible existence of prede®ned environmentally

controlled migratory patterns. Of the four cell-thick (on aver-

age) cord of premigratory cells, only the basal ones (i.e. those

apposing the neural tube basement membrane) migrate in a

ventromedial direction (Fig. 1). Conversely, the most apical

cells consistently migrate in a dorsolateral direction. This

putative positional information, apparently dictating the

migratory directionality, does not seem to be an intrinsic prop-

erty of cells at any given dorso-ventral position within the

premigratory NC because experimental dorsal-to-ventral rear-

rangement of single, or clusters of cells does not alter the cells'

migratory pattern (Epperlein et al., 1988, 1996; Perris, unpub-

lished). Similarly, the relative positional value of basal versus

apical cells is not intimately associated with the relative axial

position of a given NC cell along the neural axis. This could be

ruled out by reciprocal graftings of clusters of premigratory

NC cells at various positions along the dorsal neural tube,

which demonstrated that grafted cells preserved the `relative

position'-versus-`migratory route' relationship (Perris,

unpublished). Thus, the topographically de®ned migratory

pattern displayed by these cells seem to be mediated by envir-

onmental cues and less effectively by localized premigratory

cell-cell contacts.

The seemingly amphibian-speci®c paradigm depicted

above appears applicable to the avian NC where cells that

remain associated with the neural tube basement membrane,

soon after their appearance as single discernible cells,

migrate preferentially ventromedially (Fig. 1). In contrast,

NC cells replicating from the early delaminated ones and

remaining associated with each other, and hence not in

contact with the neural tube basement membrane, tend to

explore the dorsolateral migratory pathway (Fig. 1). In this

latter case, a speci®c directional signal could be generated

through the cadherin-mediated cell-cell interactions and

potential cross-talks of this cell adhesion system with that

represented by the integrins (Monier-Gavelle and Duband,

1997). It is presently unclear how early and to what extent

these pioneering NC cells actually proceed along the dorso-

lateral migratory pathway, as well as how tightly this migra-

tion could correlate with the differentiation state of the cells

(Erickson and Goins, 1995; Reedy et al., 1998; Wakamatsu

et al., 1998. Recent studies utilizing endothelin B receptor

expression as an early melanocytic marker indicate a rela-

tively early and metameric dorsolateral migration of these

NC derivatives (E. Dupin, personal communication). Thus,

it may be worth reconsidering the possibility that speci®c

responses to external cues may be more decisive than the

intrinsically regulated acquisition of pigment cell traits for

the choice of dorsolateral versus ventromedial migratory

directionality.

The implication of positional value in early migrating

cells, i.e. their cadherin-mediated contact or lack of contact

with the neural tube, capitalizes on the fact that cells that do

maintain neural tube contact appear tightly associated to its

basement membrane, and may even become polarized

following interaction with spatially oriented ECM structures

lining this membrane (LoÈfberg et al., 1980; Perris et al.,

1990; Perris, 1997; Fig. 1). Obviously, the positional

value/polarization concept is a not a static phenomenon,

since early migrating individual NC cells may constantly

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±214

reorganize in relation to their neighbours during movement.

In fact, high-resolution, real-time analysis of NC cell move-

ment in hind region of chick embryos reveals both extensive

cell-cell contacts and a certain degree of intermingling of

cells pertaining to different migratory streams (Kulesa and

Fraser, 2000).

A speci®c topographical organization of the early migrat-

ing NC cells would not be solely relevant for their migratory

behaviour, but could similarly be important for other vital

cellular functions (Chen et al., 1997). The front-rear polarity

established by early migrating NC cells is likely to be

dictated by `membrane raft microdomains' containing

higher densities of gangliosides and surface receptors for

signalling molecules (Manes et al., 1999, including the

Eph-receptors currently believed to participate in the

guidance of NC cell movement (Krull et al., 1997; Robinson

et al., 1997). In contrast to the overt basement membrane-

interaction engaged by NC cells apposing the neural tube,

NC cells overriding these basally localized neighbours are

conditioned by a homotypic cell-cell contact at their most

ventral side and by a cell-ECM interaction at their most

dorsal side. Hence, these latter cells have a reversed polar-

ization in comparison to the underlying ones with respect to

the linkages established with environmental cellular and

non-cellular components and the separate or convergent

sets of signals that they receive through these interactions.

Thus, depending upon the relative polarization, i.e. relative

distance to the neural tube basement membrane and whether

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±21 5

Fig. 1. Light and scanning electron microscopic images of initial NC cell dispersion in the Mexican axolotl and chick embryos, and ultrastructure of the ECM

deposited along the primary NC migratory pathways of the former embryo. (A) Scanning electron microscopic view of the initial phases of NC cell emigration

in the axolotl embryo highlighting the modes of dorsolateral (yellow arrows) versus medioventral (violet arrows) migration. (1, 6 and 7) Show NC cells that

have crawled over neighbouring cells and are progressing dorsolaterally. (2±5) Show NC cells that have moved away from the NC core population by migrating

in close contact with the neural tube basement membrane. These cells move preferentially on the medial side of the somites (S) or in between them (5). (B±C)

Illustrate the same phenomenon as in (A) in both axolotl (B) and chick (C) embryos, when observed by light microscopy in serial transverse sections. In (B),

note that cells indicated by green arrows are in close contact with the inner dermomyotomal basement membrane. (D) Shows the ®lopodium-ECM ®bril

interaction at the leading edge of axolotl NC cells proceeding medioventrally. Light blue arrows point to sites of cell-ECM contacts. (E,F) Are views of the

interstitial ECM of the dorsolateral (E) and medioventral (F) axolotl NC migratory pathways, following a speci®c ruthenium red-based ®xation procedure for

optimal preservation of the ECM architecture. Green arrows point to hyaluronidase-sensitive micro®laments. Courtesy Dr Jan LoÈfberg.

contacting other moving NC cells apically or basally, early

migrating NC cells may be biased to proceed either dorso-

laterally or ventromedially (Fig. 1).

3. `Maturation' of the dorsolateral interstial ECMtriggers the onset of NC cell movement

ECM isolated from the NC migratory pathways of axolotl

embryos at stages of advanced NC cell movement elicits a

precocious cell emigration when transplanted through

micromembrane carriers in younger embryos (Fig. 2;

LoÈfberg et al., 1985; 1988). It could experimentally be

ruled out that this strictly localized ECM effect was attrib-

uted to a shedding of long-range signalling molecules

sequestered by the implanted ECM, i.e. acting upon the

cells in a chemotactic and/or chemokinetic fashion such as

in the case of the FGF-2 and FGF-8 effects on mouse mesen-

cephalic NC cells (Kubota and Ito, 2000). These data high-

lighted at least two important points. First, they con®rmed

by a different, `in vivo' criterium that the stationary NC cells

residing on the dorsal aspect of the neural tube were fully

competent to locomote and simply required the proper

environmental signal to embark upon their dispersion

through the embryo. Secondly, they indicated that the

migration-triggering signal could be conveyed to the cells

by the interstitial ECM overlying the NC premigratory

population upon direct contact. ECM signalling, however,

seemed to be contingent upon the ECM's acquisition of

such signalling capacity. We have denoted this to be a

`maturation process' of the interstitial ECM, and this notion

is fully supported by the fact that ECM grafted isochroni-

cally (and orthotopically) cannot elicit precocious NC cell

movement (LoÈfberg et al., 1985, 1988).

Changes in the composition of the ECM responsible for

the stimulation of NC cell movement in the axolotl embryo

and possibly higher vertebrate embryos are likely to be

driven by a cell autonomously regulated developmental

program in the surrounding tissues. Once again, the most

clear evidence for such an intrinsic maturation derives from

unpublished experiments in the axolotl embryo showing

that, if dorsal ectodermal tissue pieces are explanted and

grown in vitro for a de®ned time-period and then back-

grafted into a host embryo at a premigratory NC stage,

they have retained the same capability to elicit precocious

cell movement as the directly transplanted ectodermal

tissue.

4. Disparate and coincident effects of spatially separatedECMs

Although studies in amphibians demonstrate that spatio-

temporally regulated alterations of the composition of the

interstitial ECM are implicated in the onset of NC cell

dispersion, it has yet not been possible to prove experimen-

tally that similar changes may occur in the pro-adhesive/

pro-migratory neural tube basement membrane. At early

phases of NC cell dispersion, the basement membrane

surrounding the epithelial somites may also be pro-adhe-

sive, as it is contacted by both NC cells approaching it

medially and laterally, as in the case of the few NC cells

moving between the somites. Immunohistochemical and

electron microscopical studies in chick embryos suggest

that structural and compositional properties of the dermo-

myotomal basement membrane renders it particularly pro-

adhesive, at somewhat later phases of NC cell migration,

such as to provide a preferred substrate for ventrally migrat-

ing NC cells (Tosney et al., 1994; Erickson and Perris,

1993). Conversely, there is presently no ultrastructural

evidence for a direct interaction of early migrating NC

cells with the ectodermal basement membrane, which is

penetrated by late migrating, fully differentiated melano-

cytes at later phases of development (Erickson et al.,

1992). Thus, at least three distinctly structured basement

membranes may differentially affect NC cell migration

during its different phases, i.e. that of the neural tube,

dermomyotome and ectoderm.

Compositional changes as those observed in the intersti-

tial ECM surrounding the premigratory NC are presumed to

also occur in the medioventral interstitial ECM lining the

neural tube-somite migratory pathway (Fig. 1). In fact,

heterochronic/heterotopic transplantations of this ECM

also induce precocious dorsolateral migration of NC cells

in the trunk region of the axolotl embryo (LoÈfberg et al.,

1988). Transplantions of both subectodermal and medioven-

tral ECMs in juxtaposition with the most dorsally located

premigratory NC cells, further sustain the importance of a

dorsal-to-ventral positional value for the directionality of

early migrating NC cells. Independent of the regional prove-

nience of the implanted ECM, the most dorsally localized

NC cells are consistently elicited to migrate dorsolaterally

(LoÈfberg et al., 1985, 1988).

5. Positional information and cell-cell association versusdifferential responsiveness to ECM signals may dictatethe pigment pattern of the axolotl larva

Establishment of pigment patterns in amphibia may

ideally be exploited as model systems for the study of the

inter-relationships of cell-cell and cell-ECM interactions in

the context of regulated cell motility. In the axolotl embryo,

some premigratory NC cells destined to become pigment

cells seem to deviate in their ECM responsiveness and initi-

ate a unique `sorting-out cascade'. While prospective mela-

nocytes migrate rapidly into the subectodermal space,

prospective xanthophores remain aggregated in irregularly

interspaced cell clusters on the dorsal neural tube (LoÈfberg

et al., 1989; Perris et al., 1990; Epperlein et al., 1996). A

number of experiments indicate that NC cells giving rise to

xanthophores are not predetermined to form this speci®c

pigment cell type, but, on the other hand, it is not clear

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±216

when and how premigratory NC cells become committed to

differentiate into xanthophores. Similarly, the cellular and

molecular changes occurring in these presumptive pigment

cells, and dictating their different migratory behaviour

(when compared to melanocytes), have not been elucidated.

An extended series of experiments suggests that increased

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±21 7

Fig. 2. Representation of the `microcarrier system', as originally devised for the axolotl embryo, and its experimental potential. Membrane carriers for ECM

transplantation are produced from uncharged polycarbonate membranes (t8 mm in thickness), or specially devised nitrocellulose-based micromembranes (t3

mm in thickness), which are modelled to a suitable size for implantation into the living embryo. Microcarriers are implanted into various regions of an embryo

at stages preceding the onset of NC cell migration and explanted after the desired time period for further use. The explanted cell-free ECMs can be analyzed

ultrastructurally, immunologically and biochemically, or transplanted with and without further modi®cation into a recipient embryo. Alternatively, they can be

used to assay the effect of native ECMs on cultured cells.

cell compaction, as a consequence of increased homotypic

cell-cell avidity, and a concomitant sorting out of prospec-

tive melanocytes and xanthophores by combined repulsive

effects and differential ECM responses, are critical factors

for the establishment of the vertical barred pigment pattern

of the axolotl larva (Epperlein et al., 1996).

Interestingly, ablation of the dorsolateral ectoderm retains

both the xanthophores and melanocytes within the same apical

cell clusters, normally exclusively composed of xanthophores

(Epperlein et al., 1988, 1996). First, this ®nding sustains the

notion that the ECM interaction of premigratory NC cells

committed towards the melanocytic lineage is essential for

their dorsolateral migration (see above). Secondly, it can be

concluded that failure to receive proper signalling from the

overlying interstitial ECM causes disturbances in the sorting

out of the two pigment cell phenotypes. In fact, implantation of

a micromembrane that physically separated the cells from the

overlying interstitial ECM, caused an aberrant co-clustering of

melanocytes and xanthophores similar to that observed after

ectoderm ablation (Epperlein et al., 1988, 1996). Further

evidence for the existence of a precise balance between cell-

cell and cell-ECM interaction in the control of early xantho-

phore clustering derives from transplantations of ECM

isolated from embryos at stages of xanthophore dispersion.

In these cases, premature movement of these pigment cells

and a failure to form premigratory clusters can be observed

at the site of the graft (LoÈfberg et al., 1988). Thus, a precise

relationship has to be maintained between the relative posi-

tional value of prospective pigment cells at their premigratory

location and their interaction with the motility-promoting

ECM to assure correct NC cell migration and pigment pattern

formation in this amphibian embryo.

6. Identi®cation of `permissive' and `non-permissive'ECM components supporting NC cell migration

There is substantial evidence that quantitative changes, as

well as changes in the supramolecular organization of both

basement membrane and interstitial ECMs may govern the

initial NC cell emigration from the neural tube. The immu-

nochemical and in situ hybridization studies compiled over

the years provide a rather extensive map of the ECM

components expressed at various phases of NC development

in various species (Table 1). The great majority of these

molecules have been tested in vitro, either singly or in

various combinations, in the attempt to establish their poten-

tial function as migration-promoting components. On the

basis of the outcome of these studies it is possible to classify

these components in three distinct categories: permissive,

i.e. those that support extensive NC cell attachment and

migration; non-permissive, i.e. those that promote a weak

cell adhesion, but do not sustain signi®cant locomotion; and

inhibitory, i.e. those that directly impede NC cell movement

(Table 1).

Indications about the in vivo role of the ECM molecules

are provided by gene deletion studies in mice (Table 1),

which show that ®bronectin, g1 chain-containing laminins,

PG-M/versions and hyaluronan (based upon data on the

HAS catalytic enzymes involved in its biosynthesis) may

be pivotal in the regulation of NC development. In contrast,

knock-out of genes encoding for several others ECM mole-

cules, such tenascins, vitronectin, thrombospondins, perle-

can, ®bulins and several collagen types, do not affect the

undifferentiated NC cells, or their derivatives, and appear

therefore of lesser importance (Table 1). Moreover,

although discordant results have been reported concerning

the ability of some of these molecules to in¯uence NC cell

migration, our cumulative in vitro data assign to them a

`neutral' role in this process, which is in accord with role

that can be extrapolated from gene manipulations. Recent

data gathered from abrogations of murine ECM genes also

corroborate other previous observations made in both avian

and amphibian embryos. First, in ovo injections of function-

blocking antibodies directed against either the integrin

receptors for ®bronectin and laminins (Bronner-Fraser,

1985, 1986), or antisense oligonucleotides interfering with

the function of these integrin receptors (Lallier and Bronner-

Fraser, 1993; Kil et al., 1996), perturb emigration of NC

cells from the neural tube. Similar results are obtained

when injecting an antibody against a laminin-8-agrin

complex (INO antibody; Bronner-Fraser and Lallier,

1988). Secondly, implantation of micromembranes coated

with either ®bronectin or laminin-1 onto the premigratory

NC of the axolotl embryo triggers massive premature

emigration (Figs. 2 and 3; Olsson et al., 1996a). Thus, it

seems that the laminin isoforms recently discovered to be

the prevailing ones during the course of NC cell dispersion,

i.e. laminin-1 and -8 (Perris, 1997; Bellina et al., 2000), and

®bronectin are fundamental components of the NC migra-

tory pathways.

7. De®ning the ECM constituents implicated in thetriggering of NC cell migration and its guidance

A primary role for laminin isoforms 1 and 8 in the regula-

tion of NC cell movement is supported by their superior

ability to promote NC cell migration in vitro, when

compared to other putative laminin isoforms of the NC

migratory pathways (Perris et al., 1996a; Bellina et al.,

2000). This bias also is sustained by the diverse effects

exerted by laminins on initial NC cell migration in vivo.

When implanted ectopically into the chick embryo lami-

nin-1 and -8 redirect ventrally migrating NC cells into the

dorsolateral pathway, whereas laminin-4 and -9 seem to

cause clustering of the dispersing cells in the vicinity of

the neural tube (Bellina et al., 2000). Furthermore, similar

to ®bronectin, the macromolecular organization of both

laminin-1 and -8 may diversely affect NC cell migration,

consistent with the observations that cells may utilize differ-

ent integrin receptors and recognition mechanisms for their

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±218

adhesive events (Lallier and Bronner-Fraser, 1991, 1992;

Lallier et al., 1994; Perris et al., 1996a). Whereas ®brils of

polymeric ®bronectin may provide guiding cues (Hocking

et al., 2000), distinctly assembled laminins affect the rate

and extent of NC cell movement. Accordingly, we ®nd that

laminin-1 and -8, but not laminin-2, -4 and -9, optimally

promote NC cell motility at submaximal substrate concen-

trations. This effect is likely to be associated with the dispa-

rate oligomerization degree of these laminins at the different

concentrations and is not due to a diverse avidity displayed

by the NC cells at these different substrate concentrations

(Bellina et al., 2000). In addition, since laminin-2/4 has been

proposed to be speci®cally involved in the migration of

Schwann cells (Anton et al., 1994), a scenario may be

portrayed in which different laminin isoforms act diversely

upon the moving NC cells at different phases of their conco-

mitant phenotypic diversi®cation.

Our recent data indicate that laminin-1 has a widespread

distribution in the early embryo, as shown by a distribu-

tional analysis of the chick a1 chain transcript, whereas

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±21 9

Table 1

Proposed functional classi®cation of ECM components present during NC cell migration and following gangliogenesis

Permissive componentsa Non-permissive componentsa Inhibitory componentsa Importance in vivob

Initial NC cell migration Relevant Not relevant

Fibronectinc Tenascin-Cd Aggrecan Fibronectin Tenascin-C

Vitronectin Collagen type II Aggrecan ?e Collagen type II

Laminin-1 Collagen type IX Laminin-1 Vitronectin

Laminin-8 Nidogen-1 Laminin-8f Collagen type IX

Collagen type I Perlecan Hyaluronan ?g Nidogen-1

Collagen type III Fibulin-1 PG-M/versicans Collagen type I

Collagen type IV Agrinh, i Perlecan

Collagen type VI Hyaluronan Collagen type III

PG-M/versican V0d Link protein Fibulin-1

PG-M/versican V1d Collagen type VI

Thrombospondin-1 Agrin

Fibromodulin

Advanced migration Link protein

Laminin-2 Collagen type V Laminin-2

Laminin-4 Collagen type VIII Collagen type V

Laminin-9 Collagen type XVIIIh, j Laminin-4

PG-M/versican V2 Fibulin-2 Osteopontin

Thrombospondin-2 Thrombospondin-1

Tenascin-Yh, k Thrombospondin-2

Osteopontin Osteopontin

Laminin-5

Laminin-10

Postgangliogenesis Laminin-11

Laminin-5 Collagen type XI Fibrillin-1

Laminin-10 Collagen type XII Decorin

Laminin-11 Collagen type XIV Biglycan

PG-M/versican V3e Fibrillin-1 Matrilin-1

Nidogen-2e

Decorin

Biglycan

Matrilin-1

Fibromodulin

a Based upon combined in vitro and in vivo data, except gene deletions in mice.b Proposed importance based upon the outcome of the published gene deletion studies in mice showing no phenotype, postnatal phenotypes not affecting the

NC derivatives, or prenatal death not affecting the NC derivatives.c Includes ®bronectins containing, or not, the EIIIA, EIIIB and V spliced axons (Peters and Hynes, 1996).d Not formally tested in functional assays in vitro.e Not formally established, since mutations in the chick and mouse genes give partial deletions of the aggrecan gene in cartilage with subsequent

chondrodyplastic phenotypes.f Predicted on the basis of knock-out of the g1 chain.g Based upon the severe phenotypes obtained after knock-outs of HAS1 and HAS3 catalytic enzymes.h Agrin may be recognized by avb1 integrins of the NC cells (Martin and Sanes, 1997), and hence, the inhibitory effect of the INO antibody (Bronner-Fraser

and Lallier, 1988) may be twofold, perturbing both the laminin-8- and agrin-NC cell interaction.i Differ from other `permissive' ECM components in that they exert an indirect effect on NC cell migration.j Halfter et al. (1998).k Tucker et al. (1994); Hagios et al. (1996).

laminin-8 (a4 chain mRNA) is more tissue restricted, being

particularly concentrated in the neural tube (Bellina et al.,

2000). The working hypothesis that we have formulated

accordingly is that the relative ratio of laminin-1 versus

laminin-8, as well as the macromolecular con®guration of

these two laminins, may be critical factors for the retain-

ment of early migrating NC cells in close apposition to the

neural tube surface. As discussed above, such polarization

would favour a ventromedial migration and the same quan-

titative and qualitative parameters may dictate the preferen-

tial NC cell migration in close contact with the inner

dermomyotomal basement membrane. This phenomenon

can be observed both in the chick and axolotl embryo

(Fig. 1; Erickson and Perris, 1993) and suggests that

combined laminin isoform-speci®c and conformation-

related responses of early migrating NC cells may be critical

for their directional choices.

The experimental paradigm involving stimulation of

premature NC cell migration by implantation of membrane-

bound ECMs, or single ECM constituents (Fig. 2), can ef®-

ciently be exploited to identify critical factors in the motility-

promoting interstitial ECM. Particularly useful in this endea-

vour is the availability of a naturally occurring mutant, the

white axolotl mutant (dd), which manifests an early ECM-

dictated NC migratory de®cit. In contrast, less useful are the

analogous mouse mutants steel and splotch in which the muta-

tions affect NC development at different phases of the migra-

tory process, acting simultaneously on both cell movement

and other cellular processes including survival and phenotypic

speci®cation. Implantation of micromembranes coated with

various puri®ed ECM components clearly support a prevailing

role for ®bronectin and laminin-1 as candidate molecules

directly responsible for the triggering and support of initial

NC cell dispersion. Both ECM components effectively stimu-

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±2110

Fig. 3. Transplantation of native ECM bound to micromembranes (boxed area in upper light microscopy micrograph) from older (i.e. at the time of the onset of

NC cell migration) to younger axolotl embryos elicits precocious NC cell movement at the site of the graft. Prior treatment of the ECM to be transplanted with

GAG-degrading enzymes differentially (`2' ± `1111') abrogates its migration-promoting ability. Out of .20 puri®ed ECM molecules tested, only the six

represented possess the ability to mimic the migration-inducing effect of native ECMs to various degrees (`1' ± `1111').

late precocious NC cell movement in both wild type and

migration-defective white mutant axolotl embryos (Olsson

et al., 1996a). Conversely, several of the collagen types

known to be abundantly expressed in the ECM surrounding

the premigratory NC are less effective and are unlikely to be of

signi®cant importance at these early phases of NC cell move-

ment (Fig. 3).

8. Aggrecan ± the `repulsive' hyaluronan-bindingproteoglycan

The ECMs of the NC migratory pathways are exuberantly

rich in proteoglycans, the primary and earliest expressed

ones being perlecan, aggrecan and PG-M/versicans.

Whereas perlecan is ubiquitously expressed in basement

membranes throughout NC development (Perris et al.,

1991; Perris, 1997), aggrecan and PG-M/versicans (Fig. 4)

exhibit virtually mutually exclusive tissue distribution (Peri-

ssinotto et al., 2000; Fig. 5). Aggrecan is thought to be the

primary proteoglycan produced by the notochord and,

accordingly, it is largely restricted to the perinotochordal

ECM. Its deposition in the perinotochordal ECM is spatio-

temporally regulated in a metameric pattern that alternates

the segmental distribution of the peripheral nervous ganglia

and accompanies chondrogenesis (Bundy et al., 1998; Peri-

ssinotto et al., 2000). The precise molecular composition of

the aggrecan gene product of the early developing avian

embryo remains ambiguous. A number of studies indicate

an abundant presence of keratan sulfates in the perinoto-

chordal region, which is spatiotemporally coincident with

that of aggrecan (Perris et al., 1990, 1991; Olsson et al.,

1996b; Perris, 1997; Perissinotto et al., 2000), whereas

others propose the occurrence of a unique `notochordal

aggrecan' lacking such side chains (Domowicz et al.,

1995; Kerr and Newgreen, 1997. This discrepancy, as

well as combined immunochemical and biochemical analy-

sis of the perinotochordal/ventromedial ECM composition

(Stigson and Kjellen, 1991, 1996; Perissinotto et al., 2000),

has led us to hypothesize that novel keratan sulfate-bearing

proteoglycans could be expressed by the notochord at initial

phases of NC cell migration in both axolotl and chick

embryos.

When tested in functional assays in vitro, aggrecan

strongly interferes with NC cell motility. In fact, this proteo-

glycan emerges as the sole ECM molecule capable of block-

ing NC movement on a number of motility-promoting

substrates. This effect is thought to be exerted through a

mechanism involving a hyaluronan-mediated association

with the NC cell surface and a consequent side chain-

mediated inhibitory effect on integrin recognition of their

cognate ligands (Perris and Johansson, 1987, 1989; Perris et

al., 1996b; Fig. 6). The implication of these ®ndings is rather

indisputable when considering a number of original in vitro

and in vivo observations indicating that glycosaminogly-

cans associated with the notochord negatively in¯uence

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±21 11

Fig. 4. Schematic representation (A) of the overall structure and molecular interactions of the two hyaluronan-binding proteoglycans expressed during NC cell

movement in the trunk region of the embryo. (B±C) Show hyaluronan-aggrecan-link protein (B) and hyaluronan-PG-M/versican V2-link protein ternary

complexes (C) viewed by transmission electron microscopy following rotary shadowing. Courtesy Dr Matthias MoÈrgelin.

both NC cell migration and motor axon outgrowth (Pettway

et al., 1990; Oakley et al., 1994; Erickson and Perris, 1993;

Newgreen et al., 1996; Nakamoto and Shiga, 1998). They

are also in accordance with the ®nding that an anti-aggrecan

antibody alleviates the negative effect of ectopically intro-

duced notochordal tissues on avian NC cell movement in

vivo (Pettway et al., 1996). In concert, these latter observa-

tions, the tissue-restricted distribution of aggrecan during

the course of NC cell dispersion, and the spatiotemporal

distributional changes that the proteoglycan seems to

undergo with progressive chondrogenesis ®rmly sustain an

indirect guiding function for this macromolecule during the

course of NC cell migration. In further support of this

conclusion are data derived from experimental ablation of

the notochord showing a markedly altered peripheral gang-

liogenesis (Teillet and Le Douarin, 1983; Stern et al., 1991)

and implantations of micromembranes coated with puri®ed

aggrecan (or other ECM molecules) into various regions of

the chick embryo causing halted migration at the site of the

implant (Fig. 7; Perissinotto et al., 2000).

9. PG-M/versicans ± the `haptotactic' hyaluronan-binding proteoglycans

The dominating proteoglycans of NC migratory pathways

are products of the PG-M/versican/CSPG2 gene (William-

son et al., 1991; Landolt et al., 1995; Stigson et al., 1997;

Perissinotto et al., 2000). In analogy with aggrecan, these

proteoglycans do not directly support NC cell migration

(Perris et al., 1996b). This ®nding has mistakenly led several

authors to propose that they also could act as `inhibitory'

macromolecules. In particular, several studies have put

forward the idea the PG-M/versicans (or analogous proteo-

glycans) organize in a metameric fashion within the caudal

portion the somites and thereby participate in the segmental

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±2112

Fig. 5. Reconstructed map of the proteoglycan distribution during the course of NC cell migration and peripheral neurogenesis in the chick embryo. Data derive

from combined qualitative and quantitative light and confocal laser microscopic evaluations of immunohistohemical stainings obtained with .20 monoclonal

antibodies directed against the PG-M/versican and aggrecan core proteins and their glycosaminoglycan/oligosaccharide-speci®c moieties. In each panel, a light

microscopic image of a representative immunohistochemical staining at the given developmental stage was used as a framework for the graphic representation.

migration by excluding NC cells from the rostral portion

(Tan et al., 1987; Newgreen et al., 1990; Landolt et al.,

1995). However, an assessment of the precise distribution

of PG-M/versicans within the epithelial somites, and the

developing sclerotome, during the course NC cell migration

in intact and NC-deprived embryos rejects this idea. In fact,

in situ analysis of the core protein mRNA, the translated

protein and the characteristic glycosaminoglycan moieties

of PG-M/versicans reveal that these proteoglycans undergo

a distributional rearrangement within the sclerotome that is

strictly correlated with the movement of the cells (Perris et

al., 1991; Perissinotto et al., 2000). An important function in

the regulation of NC cell migration through the somites may

still be imputable to PG-M/versicans, based upon the distur-

bances in NC cell movement noted in this region after injec-

tion of soluble lectins (Krull et al., 1996 and synthetic

metabolic inhibitors of sulfation and proteoglycan secretion

(Kubota et al., 1999. Whether this could be due to an

impaired NC cell haptotaxis in response to dorsal-to-ventral

PG-M/version gradients within the sclerotome (see below),

or whether it is due to an as yet unidenti®ed rostro-caudal

concentration difference of these, or other guidance mole-

cules, remains to be determined.

The notion of guidance by local accumulations of `inhibi-

tory' PG-M/versions has also been extended by some inves-

tigators to other apparently `non-permissive' areas of the

embryo, such as the subectodermal space of the avian embryo

(Oakley et al., 1994). However, even in this case there

appears to be a misconception when it comes to the interpre-

tation of the actual role of PG-M/versicans. Some of our

previous studies indicated that, opposite to aggrecan, PG-

M/versican isoform V0 was not capable of arresting NC

cell movement in vitro (Perris et al., 1996b), although

being fully capable of binding to cell surface hyaluronan

(Fig. 4). A three-dimensional reconstruction of the PG-M/

versican expression within the sclerotome at the time of NC

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±21 13

Fig. 6. Proposed model for the mechanisms by which aggrecans (but not PG-M/versicans) may participate in the guidance of NC cell movement through the

embryo. Panels to the left show the in situ distribution of hyaluronan, highlighted through ¯uorescent detection with a biotinylated G1-containing (Fig. 4)

fragment of bovine cartilage aggrecan, in a chick embryo at stages of advanced NC cell migration (upper panel), and immunolocalization of aggrecan at a

somewhat later developmental stage (lower panel). Arrowheads in the upper panel point to early migrating NC cells covered by hyaluronan. Panels to the right

show schematically the modes of aggrecan interaction with migrating NC cells and the subsequent effect on motility. Synthesis of NC hyaluronan is proposed

to be catalyzed by HAS2, but the concomitant expression of other hyaluronan syntheses, e.g. HAS3, is likely. Experimental evidence suggests that, following

cell surface-association via linkage to hyaluronan, the glycosaminoglycan side chains of aggrecan (in particular the keratan sulfate ones) interfere with integrin

function. The basic mechanisms of this interference are not known, but they may involve both a direct impediment of integrin binding to the cognate ligands

and an alteration in the prerequired cell surface mobility of the receptors. According to the model, modulation of the hyaluronan synthesis by migrating NC

cells may determine the extent of aggrecan engagement and thereby in¯uence both speed and directionality of NC cell locomotion. On the other hand,

proteolytic processing of the glycosaminoglycan chains of the aggrecan may represent a potential way to modulate the inhibitory effect exerted by the

immobilized proteoglycan, as indicated experimentally, and may thereby have a direct repercussion on NC cell motility.

cell migration shows two apparent distributional gradients:

one established along the neural axis by higher densities of

PG-M/versicans in more anterior regions when compared to

the more posterior ones and one established by the relative

higher expression of PG-M/versicans in the dorsal portion of

the embryo when compared to the more ventral areas. With

the exception of the hyaluronan produced by the migrating

NC cells themselves, the distribution of this glycosaminogly-

can largely coincides with that of PG-M/versicans (Fig. 5),

and the possibility remains that other ECM components

(linking or not to PG-M/versicans) may organize in a similar

fashion. Thus, ventrally migrating NC cells move from a

region relative scarce in PG-M/versicans to a region with

denser distribution of these macromolecules (Fig. 5) and

may do this in a haptotactic manner (Perissinotto et al.,

2000). Alternatively, by virtue of their well-know capacity

to bind diffusible molecules, the apparent gradients of PG-M/

versicans could actually predispose gradients of signalling

molecules essential for the regulation of NC cell motility.

Based upon its tissue distribution, a candidate such molecule

could be F-spondin (Debby-Brafman et al., 1999).

If NC cells consistently migrate toward areas richer in

PG-M/versicans, why would they not migrate extensively

in the subectodermal and perinotochordal areas where these

proteoglycans are particularly concentrated? A number of

previous and recent observations may provide a rational

explanation for this apparent discrepancy. First, studies in

vitro in which moving avian NC cells are confronted with

variable concentrations of PG-M/versicans, i.e. alternating

low/high concentrations, in a number of `haptotaxis assays'

demonstrate that NC cells migrate more pronouncedly if

encountering progressively higher concentrations of proteo-

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±2114

Fig. 7. Effect of aggrecan on NC cell migration in vitro (lower panels) and in vivo (upper panels). Lanes containing laminin-1 (left) or ®bronectin (right),

alternated by the chick aggrecan and viewed in phase contrast (left), or by indirect immuno¯uorescence (right) following ¯uorescent tagging of the coated

®bronectin, and DAPI staining of the migrated NC cells. Implantation of micromembranes coated with the chick aggrecan in the dorsolateral migratory

pathway, i.e. between neural tube and somites (red arrow), retains the migrating NC cells in contact with the ectopic aggrecan substrate (white arrow).

glycans; cells proceed until they reach the densest concen-

tration which apparently stops their movement. Conversely,

NC cells are largely impeded in their movement if

confronted with a reversed gradient, i.e. highest density

®rst and then progressively lower concentrations. NC cells

are similarly restricted in their movement if faced with a

step-like gradient with a large difference between the ®rst,

second and third concentration (Perissinotto et al., 2000;

Dillon and Perris, unpublished). Thus, it appears that NC

cells must sense a precise concentration ratio for optimal

motility and are clearly interfered in their migration when

encountering excessive concentrations of PG-M/versicans.

In this case, a corollary critical factor could be local differ-

ences in the glycanation/glycosylation patterns of the

proteoglycans. A clear bias of avian NC cells to move

toward embryonic areas richer in PG-M/versican was

recently demonstrated through ectopic implantations of

these proteoglycans in selected regions of the chick embryo

(Perissinotto et al., 2000). Furthermore, orthotopic implan-

tation of PG-M/versicans in the axolotl embryo elicits

precocious NC cell migration at the site of graft (Fig. 3).

Further support for the necessity of establishing precise

proteoglycan concentration gradients for proper NC cell

movement are afforded by experiments involving dermo-

myotomal ablations and injection of target complexes of

glycosaminoglycan-degrading enzymes into the subectoder-

mal region (Fig. 8). In both cases, the outcome of the experi-

mental manipulation is predicted to be an altered local

distribution of PG-M/versicans in the dorsolateral migratory

pathway, or as could be better stated, the creation of a more

gradient-like distribution of these proteoglycans along this

path. Indeed, both experimental manipulations increase the

propensity of the NC cells to prematurely enter the subec-

todermal space or to invade it more extensively (Oakley et

al., 1994; Perris et al., unpublished). In addition, these in

vivo treatments also seem to interfere with a normal

entrance of NC cells into the somites, causing their cluster-

ing in the vicinity of the neural tube (Fig. 8). Since these

induced local alterations of the proteoglycan composition

affect the glycosaminoglycan side chains of these macro-

molecules (and not their core proteins), a further emphasis

may be given to possibility that these moieties are the ones

responsible for the sequestering of appropriate signalling

molecules in the relevant extracellular spaces.

10. Studies on mutant embryos sustain a `permissive'role for PG-M/versicans

The mutation of the white Mexican axolotl differs from all

other NC-related mutations and many other naturally occur-

ring mutations in that it is a locally restricted and early mani-

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±21 15

Fig. 8. Schematic illustration of the experimental paradigm conventionally denoted `antibody-targeted enzymatic treatment' and its potential in probing the

function of the ECM during NC cell migration in vivo. (A) Enzymes are covalently linked to high molecular weight, ¯uorescently tagged dextran (that

functions as a carrier molecule), or alternatively ¯uorescent microbeads, to which an antibody directed against an antigen localizing in the vicinity of the

speci®c enzyme substrate is similarly coupled. This ¯uorescent enzyme-antibody complex can be microinjected into selected regions of the embryo to cause

locally restricted enzyme degradations. (B) Shows an HNK-1-stained section derived from a chick embryo that had received a subectodermal chondroitinase

ABC-complex (Chase ABC) injection. Note the relatively advanced dorsolateral NC cell movement when compared to the controlateral uninjected side of the

embryo. (C) Shows a similar section from an embryo that had received an intrasomitic midtrunk injection of heparitinase III-complexes (Hep III). This

manipulation was not found to signi®cantly alter NC cell movement, but, in this case, evoked an abnormal dermomyotome formation (arrow). (C) Shows

disturbed NC cell migration following extensive intrasomitic injection of chondroitinase ABC-complexes, with clustering of NC cells along the neural tube

(NT) surface and consequent reduced amount of NC cells entering the somites (S). The section was double-stained with HNK-1 (Texas Red) and the anti-

chondroitin sulfate antibody CS56 (Bodipy) to ascertain the reduction in chondroitin sulfate in the injected region (compare with Figs. 4 and 7).

fested heterochronic one. It affects the early ectoderm such

that it cannot exert its normal function during the time period

that NC cells are competent to respond to its signals. This

phenomenon could be demonstrated by reciprocal graftings

of subectodermal and medioventral ECMs from embryos at

different phases of NC cell migration (LoÈfberg et al., 1988).

The original hypothesis that proteoglycans could be the

defective components of this ECM (Perris et al., 1990) was

later con®rmed by accurate analyses of the proteoglycan

content in wild type and white mutant embryos at times of

initial and advanced NC cell movement (Stigson et al., 1991,

1997). Parallel studies identi®ed the defective proteoglycan

of the white mutant axolotl ectoderm as being a homologue

of the PG-M/versican (Stigson et al., 1996). They also

demonstrated that this proteoglycan was temporally under-

expressed in the ectodermal tissues and thereby corroborated

a direct role for PG-M/versicans in the regulation of NC cell

movement in the amphibian embryo as well. Functional

assertion of the involvement of PG-M/versicans, as well as

other proteoglycans, in NC cell migration has similarly been

provided by implantation of membranes coated with these

macromolecules, which demonstrated that ectopic proteo-

glycans could stimulate precocious NC cell emigration

(Olsson et al., 1996a; Perris et al., unpublished).

Genetic linkage analyses indicate that the dd mutation of the

white axolotl is not directly associated with the AxPG PG-M/

versican gene (Pahiery et al., 1999), suggesting that the inher-

ited defect of this amphibian only acts secondarily on the

transcription of this gene. A strikingly similar situation is

found in the mouse splotch mutant. In this case, the mutation

is known to affect the Pax3 gene and is associated with a

pronounced alteration of the transcription of PG-M/versicans

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±2116

Fig. 9. Diagrammatic representation of the currently identi®ed and putative integrin receptors of early migrating NC cells and their cognate ligands. Arrows

indicate the approximate location of the integrin binding sites, as determined by utilizing function-blocking antibodies, antisense oligonucleotides and

proteolytic/recombinant fragments. Both cation-dependent and -independent cell binding sites on laminin-1 and -8 (as well as other laminin isoforms

recognized by NC cells in vitro) are conformation-dependent and require integrity of the heterotrimeric structure of the laminins. Integrin a1b1, or possibly

a11b1, is thought to carry the carbohydrate moiety recognized by the HNK-1 antibody and may act in conjunction with a3b1 and a8b1 to promote full

interaction of NC cells with laminins. The relative involvement of these integrins and the modes by which they operate is dependent upon the supramolecular

organization of the laminins, which is dictated by both oligomerization of the proteins and their interaction with other basement membrane components.

Integrin a8b1 is also the candidate integrin for tenascin-C, a ECM component to which NC cells bind, but fail to migrate on in vitro. Comparisons of studies on

NC and tumor cells strongly suggest that the NC a4b1 integrin recognizes multiple binding sites within the ®bronectin molecule, including a synergistic one

residing within the 3Fn8 type III module. The NC cell-binding site residing within the NC1 globular domain of collagen type IV is cryptic. CNBr3 denotes

cyanogen bromide-derived fragment 3 of the collagen type I b1(I) chain.

during early NC development (Henderson et al., 1997). The

down-stream consequences of this transcriptional alteration,

with regard to the translated protein products (i.e. PG-M/versi-

can isoform expression) and their glycanation/glycosylation

patterns, have not yet been de®ned. However, according to the

above discussion, misexpression of PG-M/versicans during

the course of NC cell migration is presumed to perturb the

well-balanced spatiotemporal gradients of PG-M/versican

distribution along the NC migratory pathways and thereby

cause the overt disturbances of NC cell movement in this

mutant embryo.

11. NC cell integrins and their modes of action

The precise integrin pro®le of early migrating NC cells is

not presently known in any animal species. Yet, for the ones

identi®ed on chick and/or mouse NC cells (Perris, 1997;

Testaz et al., 1999), gene deletion studies currently provide

some indications about their relative importance during NC

cell movement. For instance, the a4b1 integrin discovered

to be the primary ®bronectin-binding integrin (Fig. 9) of

early migrating NC cells (Stepp et al., 1994; Perris, 1997;

Kil et al., 1999), seems to be essential for early mouse

development, including the development of the NC (Yang

et al., 1999). Given the analogous cardinal role played by

®bronectin during mouse development, and as a migratory

substrate for dispersing NC cells in vitro, it may not be

surprising that this receptor-ligand pair appears central for

NC development. However, the modes of binding the NC

cell a4b1 integrin to ®bronectin are not completely under-

stood, nor has it been de®ned whether a a4b1-VCAM-1

interaction is of any signi®cance for the homing of NC

cells. Although avian NC cells have been proposed to recog-

nize the CS1 site of the alternatively spliced IIICS domain

of ®bronectin (Dufour et al., 1988; Testaz et al., 1999), the

recently reported sequence of the mouse a4 subunit shows

that it has not retained full conservation of the amino acids

found to be critical for CS1-binding in the human homolo-

gue (Irie et al., 1995). On the other hand, recent studies on

human tumour cells provide an explanation of the RGD

sensitivity of the a5b1-independent (since this integrin is

not present on early migrating NC cells) NC cell interaction

with ®bronectin, which is fully supported by the central cell-

binding domain of the molecule (Perris et al., 1989). These

studies demonstrate that the a4b1 reacts with both the CS1

and central cell-binding domain of ®bronectin and does so in

an RGD-dependent manner (Yin et al., 1999).

The precise function of av integrins on early migrating

NC cells (Delannet et al., 1994; Testaz et al., 1999; Fig. 9)

remains even more controversial, as their primary ligand,

vitronectin, has thus far not been localized within the NC

migratory pathways. Thus, the possibility remains that av

integrins may cooperate with other NC cell integrins in their

interactions with ®bronectin and other ECM/cell surface

ligands (Testaz et al., 1999). Furthermore, these cell surface

receptors may not be fully relevant for NC cell development

as their abrogation by gene manipulation affects only vascu-

lar tissues in older embryos.

A number of previous investigations from our group have

suggested that the a1b1 integrin may be critically involved

in the process of NC cell adhesion and migration on lami-

nins (Lallier and Bronner-Fraser, 1991, 1992, 1993; Lallier

et al., 1994; Perris et al., 1996a; Fig. 9), whereas this integ-

rin has not been possible to detect on migrating NC cells in

situ (Duband et al., 1992). A number of indirect evidences

now suggest that this integrin may actually correspond to

the avian homologue of the recently discovered a11b1

integrin. Furthermore, previous studies from our group

have indicated that NC cell interaction with laminins is

mediated by at least two b1 integrins (Lallier and Bron-

ner-Fraser, 1991, 1992; Lallier et al., 1994; Perris et al.,

1996a). Since early migrating avian NC cells, lack a6 integ-

rins, primary candidates for the cooperating laminin-bind-

ing integrins of NC cells are the a3b1 and a8b1. The

former integrin is also likely to be responsible for the inter-

action of thrombospondin-1 with NC cells (Tucker et al.,

1999), whereas the latter may represent a putative tenascin-

binding NC integrin (Fig. 9).

12. Other putative ECM receptors and cooperatingmechanisms of ECM interaction

Apart from the above described mechanism whereby cell

surface-associated hyaluronan may act as a linking mole-

cule for the modulation of the NC cells' interaction with

aggrecan and PG-M/versicans, a synergistic NC-proteogly-

can interaction seems to be mediated by HNK-1-reactive

sulfoglucuronyl glycolipids (Jungalwala et al., 1992; Peri-

ssinotto et al., 2000). These bind speci®cally to the lectin-C

type modules (Miura et al., 1999) characterizing both aggre-

can and PG-M/versicans and thereby may alternatively rein-

force the cell surface retention of aggrecans (Perris et al.,

1996b), and/or mediate the haptotactic response of NC cells

to PG-M/versicans (Perissinotto et al., 2000). This interac-

tion can be competed out by exogenous lectins and this fact

highlights a potential mechanism underlying the altered

segmental NC cell migration through the sclerotome follow-

ing in vivo lectin injection (Krull et al., 1995). Another

important role in the interaction of NC cells with proteogly-

cans may be played by tenascins. At least one tenascin type

is produced by the migrating NC cells (Erickson and Perris,

1993; Perris, 1997) and the importance of the production

and secretion of this ECM molecule by dispersing NC cells

remains unknown. However, the complementary distribu-

tion patterns displayed by tenascin-C and PG-M/versicans

during NC cell movement (Tan et al., 1987), especially

evident within the sclerotome, and the interacting capability

of these molecules strongly suggest that secreted NC tenas-

cin may in¯uence the cells' local response to different

concentrations of PG-M/versicans. The potential release of

R. Perris, D. Perissinotto / Mechanisms of Development 95 (2000) 3±21 17

tenascin-C by dispersing NC cells may also explain the

inhibitory effect on cell movement in vivo observed after

in ovo injection of an anti-tenascin antibody (Bronner-

Fraser, 1988), known to react with an epitope residing

within a region of the molecule distinct from that implicated

in the integrin-mediated cell interactions.

Additional cooperative ECM receptor activities may be

exerted in late migrating NC cells by b-dystroglycan, CD44

and an unidenti®ed member of the syndecan/glypican family

of integral proteoglycans (Perris, 1997). The ®rst two trans-

membrane molecules seems to exert its well-established brid-

ging function in the recognition of laminins by NC-derived

Schwann cells (Ikeda et al., 1996) and neuronal and non-

neuronal NC precursors invading the gut and destined to

form gastric smooth muscle and enteric neurones (Chalazoni-

tis et al., 1997). The putative function of the second transmem-

brane proteoglycan molecule(s) is still obscure.

In contrast to the aforementioned cell surface components

proposed to cooperate with integrins in the NC cell interac-

tion with the ECM, other recently discovered signalling

mechanisms may assist integrin function through engage-

ment of cell surface tyrosin kinase receptors. For instance,

integrin binding activity may readily be regulated by ephrin-

B1 and its Eph-B1-3 receptors, which may modulate both

b1- and b3-integrin functions through a R-RAS-dependent

mechanisms (Huynh-Do et al., 1999; Zou et al., 1999).

Interestingly, engagements of different EphB receptors

seems to have opposite effects on integrin mediated cell

adhesive functions (Huynh-Do et al., 1999; Zou et al.,

1999) and may therefore effectively control the process of

directional cell locomotion requiring dynamic modulations

of substrate adhesivness. Such a complementary mechanism

of integrin modulation may be particularly relevant for NC

cell migration through the rostral portion of the sclerotome,

which is thought to be dictated by signalling through Eph-

B3-ephrin-B1 interactions (Krull et al., 1997). Thus, rather

than acting as a pure `repellent' phenomenon, concentra-

tion-dependent ephrin signalling may govern the rostrocau-

dal pattern of NC cell migration through the somites by

controlling the NC cell interaction with the putative migra-

tory substrate molecules, via modulation of the relative

integrin af®nity for different ECM ligands. Subtle concen-

tration differences of ECM molecules in the rostral versus

caudal somite halves, sensed by the ephrin-Eph receptor

system, would be undetectable by the currently available

technical means, but would be instrumental in creating

speci®c substrate conditions favouring directional move-

ments. Furthermore, the importance of complementary

signalling events exerted through semaphorin IIIa has also

to be taken into consideration (Eickholt et al., 1999)

13. Conclusive remarks

Our present knowledge about the composition of the

ECM expressed during NC development, as well as the

modes by which migrating NC cells interact with this

ECM, suggest that ®bronectin may provide a ground

substrate for the movement of the cells. Depending upon

its degree of polymerization (Pasqualini et al., 1996), it is

additionally thought to provide guiding cues in the form of

organized ®brillar structures associated with the interstitial

collagen network. Two laminin isoforms, laminins 1 and 8,

seem to be critically involved in the regulation of initial NC

cell migration by in¯uencing the extent of basement

membrane association of the migrating NC cells. Another

organizational level of NC cell guidance by these laminins

is afforded by their supramolecular arrangement within

basement membranes, which may render them permissive

or non-permissive for NC cell movement. Interaction of NC

cells with both ®bronectin and laminins through their integ-

rin receptors is expected to provide the necessary survival

signals, may contribute to the regulation of gene expression,

and may be controlled by concurrent signalling through

tyrosine kinase-type receptors and their ligands.

A primary function in the guidance of NC cell migration

can be attributed to the proteoglycans aggrecan and PG-M/

versicans, which based upon gene deletions and studies of

mutant murine and amphibian embryos appear indispensa-

ble for proper NC development. These proteoglycans seem

to exert opposite functions during NC cell migration: aggre-

can direct movement by demarcating the ventral migratory

route, whereas PG-M/versicans may be responsible for the

de®nition of the spatiotemporal progression of NC cell

dispersion through various regions of the embryo. Modula-

tion of the synthesis of hyaluronan by the moving NC cells

is likely to be a critical factor in the way NC cells respond to

different local concentrations of these proteoglycans. An

ancillary role in the modulation of this response may be

played by tenascins secreted by the migrating NC cells.

Finally, the control of the aggrecans' and PG-M/versicans'

effect on NC cell movement is presumed to be governed by

the spatiotemporal regulation of the glycanation/glycosyla-

tion of these macromolecules.

Acknowledgements

The authors are particularly thankful to a number of colla-

borators, who over the years have made substantial contri-

butions to the published and unpublished data discussed in

this article. The authors also wish to thank Marianne Bron-

ner-Fraser, Sally Moody and Chaya Kalcheim for helpful

comments on the manuscript. The invaluable ®nancial

support from Fondo Sanitario Nazionale (FSN-RF96) is

gratefully acknowledged.

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