Avian neural crest cell migration on laminin: interaction ... · promote migration of neural crest...

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INTRODUCTION During embryonic development, certain groups of cells can transiently exhibit locomotory capacities that allow them to migrate long distances from their sites of origin and populate other areas of the embryo where they undergo terminal differ- entiation. Such is the case for the neural crest population, which originates from the dorsal part of the neural tube and gives rise after migration to a large variety of tissues, including peripheral ganglia, melanocytes and craniofacial structures (Le Douarin, 1982; Levi et al., 1990; Erickson and Perris, 1993; Le Douarin et al., 1993; Stemple and Anderson, 1993; Bronner- Fraser, 1995). During migration, neural crest cells penetrate a fibrillar network of extracellular matrix molecules, which serves as a scaffold onto which cells migrate in an integrin- dependent manner (Bronner-Fraser, 1993; Erickson and Perris, 1993; Duband et al., 1995b). Beside this fibrillar matrix, neural crest cells come in contact with the basal laminae of the epi- thelial tissues that line the migratory pathways, e.g. the ectoderm, neural tube, somites and myotomes in the trunk region (Krotoski et al., 1986; Sternberg and Kimber, 1986; Duband and Thiery, 1987; Perris et al., 1991a). As neural crest cells cannot penetrate through them (Erickson, 1987), basal laminae may act as physical barriers to cell dispersion, thereby imposing to some extent the plan of the migratory routes. In addition, a number of constituents of basal laminae are able to promote migration of neural crest cells in vitro (Perris et al., 1989, 1993b) and, in vivo, antibodies to these constituents can perturb cranial neural crest cell migration (Bronner-Fraser and Lallier, 1988). Moreover, in some instances, neural crest cells have been found to migrate preferably on basal laminae over fibrillar matrices (Tosney et al., 1994). Thus, basal laminae 2729 Journal of Cell Science 110, 2729-2744 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JCS4418 In the present study, to further elucidate the molecular events that control neural crest cell migration, we have analyzed in vitro the adhesive and locomotory response of avian trunk neural crest cells to laminin-1 and searched for the integrin receptors involved in this process. Adhesion of crest cells on laminin-1 was comparable to that found on fibronectin or vitronectin. By contrast, migration was sig- nificantly greater on laminin-1 than on the other substrate molecules. Interaction of crest cells with laminin-1 involved two major cell-binding domains situated in different portions of the molecule, namely the E1and E8 fragments, which elicited different cellular responses. Cells were poorly spread on the E1fragment whereas, on E8, they were extremely flattened and cohesive. Either fragment supported cell locomotion, albeit not as efficiently as laminin-1. Immunoprecipitation and immunocytochem- istry analyses revealed that crest cells expressed the α1β1, α3β1, α6β1 and αvβ3 integrins, as well as β8 integrins, as presumptive laminin-1 receptors, but not α6β4 and α2β1. Immunofluorescence labeling of cultured cells showed that the α1, αv, β1 and β3 subunits were diffuse on the cell surface and in focal contacts. In contrast, α3 and β8 were diffuse, while α6 was mostly intracytoplasmic and, secon- darily, in focal contacts. Inhibition assays of cell adhesion and migration with function-perturbing antibodies demon- strated that α1β1 played a predominant role in both adhesion and migration on laminin-1 and interacted with either binding sites in the E1and E8 fragments. αvβ3 was also implicated in neural crest cell migration. In contrast, α3β1, α6β1 and the β8 integrins appeared to play only sub- sidiary roles in cell adhesion and migration. Finally, the ability of neural crest cells to interact with laminin-1 was found to increase with time in culture, possibly in correla- tion with changes in α3 distribution on the cell surface. In conclusion, our study indicates that (1) the preferential migration of neural crest cells along basal laminae can be accounted for by the ability of laminin-1 to promote migration with great efficiency; (2) interaction with laminin-1 involves two major cell binding domains that are both recognized by the α1β1 integrin; (3) α1β1 integrin can elicit different cellular responses depending on the laminin- 1 domains with which it interacts; and (4) changes in the repertoire of integrins expressed by neural crest cells are consistent with the modulations of cell-substratum adhesion occurring throughout migration. Key words: Avian embryo, Neural crest, Cell migration, Laminin, Integrins SUMMARY Avian neural crest cell migration on laminin: interaction of the α1β1 integrin with distinct laminin-1 domains mediates different adhesive responses Nathalie Desban and Jean-Loup Duband* Institut Jacques Monod, CNRS et Université Denis Diderot, and Laboratoire de Biologie Moléculaire et Cellulaire du Développement, CNRS URA 1135 et Université Pierre et Marie Curie, Paris, France *Author for correspondence at Laboratoire de Biologie Moléculaire et Cellulaire du Développement, CNRS URA 1135 et Université Pierre et Marie Curie, 9 quai St Bernard, 75005 Paris, France (e-mail: [email protected])

Transcript of Avian neural crest cell migration on laminin: interaction ... · promote migration of neural crest...

Page 1: Avian neural crest cell migration on laminin: interaction ... · promote migration of neural crest cells in vitro (Perris et al., 1989, 1993b) and, in vivo, antibodies to these constituents

2729Journal of Cell Science 110, 2729-2744 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS4418

Avian neural crest cell migration on laminin: interaction of the α1β1 integrin

with distinct laminin-1 domains mediates different adhesive responses

Nathalie Desban and Jean-Loup Duband*

Institut Jacques Monod, CNRS et Université Denis Diderot, and Laboratoire de Biologie Moléculaire et Cellulaire duDéveloppement, CNRS URA 1135 et Université Pierre et Marie Curie, Paris, France*Author for correspondence at Laboratoire de Biologie Moléculaire et Cellulaire du Développement, CNRS URA 1135 et Université Pierre et Marie Curie, 9 quai StBernard, 75005 Paris, France (e-mail: [email protected])

In the present study, to further elucidate the molecularevents that control neural crest cell migration, we haveanalyzed in vitro the adhesive and locomotory response ofavian trunk neural crest cells to laminin-1 and searched forthe integrin receptors involved in this process. Adhesion ofcrest cells on laminin-1 was comparable to that found onfibronectin or vitronectin. By contrast, migration was sig-nificantly greater on laminin-1 than on the other substratemolecules. Interaction of crest cells with laminin-1 involvedtwo major cell-binding domains situated in differentportions of the molecule, namely the E1′ and E8 fragments,which elicited different cellular responses. Cells werepoorly spread on the E1′ fragment whereas, on E8, theywere extremely flattened and cohesive. Either fragmentsupported cell locomotion, albeit not as efficiently aslaminin-1. Immunoprecipitation and immunocytochem-istry analyses revealed that crest cells expressed the α1β1,α3β1, α6β1 and αvβ3 integrins, as well as β8 integrins, aspresumptive laminin-1 receptors, but not α6β4 and α2β1.Immunofluorescence labeling of cultured cells showed thatthe α1, αv, β1 and β3 subunits were diffuse on the cellsurface and in focal contacts. In contrast, α3 and β8 werediffuse, while α6 was mostly intracytoplasmic and, secon-darily, in focal contacts. Inhibition assays of cell adhesion

and migration with function-perturbing antibodies demon-strated that α1β1 played a predominant role in bothadhesion and migration on laminin-1 and interacted witheither binding sites in the E1′ and E8 fragments. αvβ3 wasalso implicated in neural crest cell migration. In contrast,α3β1, α6β1 and the β8 integrins appeared to play only sub-sidiary roles in cell adhesion and migration. Finally, theability of neural crest cells to interact with laminin-1 wasfound to increase with time in culture, possibly in correla-tion with changes in α3 distribution on the cell surface. Inconclusion, our study indicates that (1) the preferentialmigration of neural crest cells along basal laminae can beaccounted for by the ability of laminin-1 to promotemigration with great efficiency; (2) interaction withlaminin-1 involves two major cell binding domains that areboth recognized by the α1β1 integrin; (3) α1β1 integrin canelicit different cellular responses depending on the laminin-1 domains with which it interacts; and (4) changes in therepertoire of integrins expressed by neural crest cells areconsistent with the modulations of cell-substratumadhesion occurring throughout migration.

Key words: Avian embryo, Neural crest, Cell migration, Laminin,Integrins

SUMMARY

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INTRODUCTION

During embryonic development, certain groups of cells ctransiently exhibit locomotory capacities that allow them migrate long distances from their sites of origin and populother areas of the embryo where they undergo terminal difentiation. Such is the case for the neural crest populatwhich originates from the dorsal part of the neural tube agives rise after migration to a large variety of tissues, includperipheral ganglia, melanocytes and craniofacial structuresDouarin, 1982; Levi et al., 1990; Erickson and Perris, 1993;Douarin et al., 1993; Stemple and Anderson, 1993; BronnFraser, 1995). During migration, neural crest cells penetrafibrillar network of extracellular matrix molecules, whicserves as a scaffold onto which cells migrate in an integdependent manner (Bronner-Fraser, 1993; Erickson and Pe

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ing (Le Leer-

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1993; Duband et al., 1995b). Beside this fibrillar matrix, neurcrest cells come in contact with the basal laminae of the ethelial tissues that line the migratory pathways, e.g. thectoderm, neural tube, somites and myotomes in the truregion (Krotoski et al., 1986; Sternberg and Kimber, 1986Duband and Thiery, 1987; Perris et al., 1991a). As neural crcells cannot penetrate through them (Erickson, 1987), balaminae may act as physical barriers to cell dispersion, thereimposing to some extent the plan of the migratory routes. addition, a number of constituents of basal laminae are ablepromote migration of neural crest cells in vitro (Perris et al1989, 1993b) and, in vivo, antibodies to these constituents cperturb cranial neural crest cell migration (Bronner-Fraser aLallier, 1988). Moreover, in some instances, neural crest cehave been found to migrate preferably on basal laminae ovfibrillar matrices (Tosney et al., 1994). Thus, basal lamina

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may play a critical role in neural crest cell migration, providinboth a suitable substratum for migration and essential guidacues for the cells.

Laminin is a major structural component of basal laminwith numerous biological functions in cell adhesion, migratioand differentiation (Engel, 1992; Tryggvason, 199Yurchenco and O’Rear, 1994; Mercurio, 1995; Ekblom aTimpl, 1996; Timpl, 1996). Laminin is a large multidomaiglycoprotein composed of three chains, termed α, β and γ, thatare assembled into a cross-shaped structure. During the years, because of the diversity of the chains that can assointo different combinations, at least 11 laminin isoforms, deignated laminin-1 to laminin-11, have been listed. Receptfor laminin include at least nine members of the integrfamily: α1β1, α2β1, α3β1, α6β1, α7β1, α9β1, αvβ3, α6β4and β8 integrins (Kühn and Eble, 1994; Mercurio, 1995Laminin is the sole identified ligand among extracellulmatrix molecules for α6β1, α7β1 and α6β4, while other matrixcomponents such as fibronectin, collagens and tenascinpotential ligands for the other laminin-binding integrins. Iepithelial cells, laminin interacts with the cells’ surfacprimarily through α6β4 and α3β1 (Sonnenberg et al., 1990aLee et al., 1992; Giancotti, 1996), whereas in other cell typincluding neurons, interaction occurs essentially via α1β1,α3β1, α6β1 and α7β1 (Sonnenberg et al., 1988, 1990aWayner et al., 1988; Tomaselli et al., 1990; Bronner-Fraseal., 1992; Duband et al., 1992; de Curtis and Reichardt, 19George-Weinstein et al., 1993). On the other hand, α1β1 andα3β1 may not bind all laminin isoforms while others, e.gα6β1, are supposedly able to interact with all the laminilisted so far. The main integrin-recognition domains within thlaminin-1 molecule are now being mapped. Thus, binding sifor α6β1 and α1β1 are located at opposite regions of the croat both ends of the α1 chain (Sung et al., 1993; ColognatoPyke et al., 1995).

The molecular basis for neural crest cell interaction wilaminin has been explored in recent years (Perris et al., 19Lallier and Bronner-Fraser, 1991, 1992; Lallier et al., 1994).was found that crest cell migration on laminin-1 is mediatexclusively by the E8 fragment and that recognition of lamini1 involves different integrins of the β1 family, among whichα1β1 plays a major role (Lallier and Bronner-Fraser, 1991992; Lallier et al., 1994). These studies then revealed rather unexpected and surprising feature that, in neural ccells, the α1β1 integrin binds the E8 domain and not the E′one, contrasting with the other cell types examined so where α1β1 binds preferentially the E1′ domain (Forsberg etal., 1990; Hall et al., 1990; Sonnenberg et al., 1990b; Tomaset al., 1990; Goodman et al., 1991; Colognato-Pyke et 1995). The nature of the other integrins that participate laminin-1 recognition by neural crest cells has not, howevbeen elucidated. In the present study, we have therefore ramined in detail the mechanism of neural crest cell interactwith laminin-1; we have tentatively established the complerepertoire of the laminin-1-binding integrins in these cells ainvestigated their possible implication in cell migration. Wdemonstrate that interaction with laminin-1 does not involsolely the E8 fragment but the E1′ fragment as well, and thatthese domains may be implicated in separate functions. also provide evidence that neural crest cells express mlaminin-1-binding integrins than was previously thought an

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that, among them, α1β1 and, to a lesser extent, αvβ3 play apredominant role in cell migration, while α3β1, α6β1 and theβ8 integrins play only secondary roles, if any. We confirm thain neural crest cells, α1β1 binds the E8 fragment but we showthat, as in other cells, it also binds the E1′ domain, thus raisingthe interesting feature that α1β1 interaction with distinctlaminin-1 binding sites may mediate different adhesivresponses of cells. Finally, we demonstrate that laminin-binding properties of neural crest cells and surface distributiof integrins change over time in culture and this may be relato the natural history of these cells in the embryo.

MATERIALS AND METHODS

Adhesive proteins, antibodies and cell culturesMouse laminin-1 was purchased from Sigma or Life Technologieswas purified according to the original protocols (Timpl et al., 1979Bovine fibronectin was purchased from Sigma and vitronectin wpurified from bovine serum by affinity chromatography on a hepariSepharose column as described (Yatohgo et al., 1988). Laminielastase fragments E8 and E1′ were provided by Dr P. Yurchenco(University of New Jersey, Piscataway, USA) and Dr J.-C. Lissitzk(INSERM, Marseille, France). Rabbit polyclonal antibodies tlaminin-1 have been described previously (Duband and Thiery, 198and rabbit polyclonal antibodies to the E8 and E1′ fragments wereprovided by Dr P. Yurchenco. Polyclonal antibodies (2992) to thchicken β1-integrin subunit were provided by Dr K. Yamada (NIHBesthesda, USA; Chen et al., 1985). The CSAT hybridoma (anchicken β1 subunit) was donated by Dr C. Buck (Wistar InstitutePhiladelphia, USA; Horwitz et al., 1985). The mouse monoclonantibody (mAb) TASC anti-chicken β1 subunit was provided by DrL. Reichardt (Howard Hughes Medical Institute, UCSF, SaFrancisco, USA; Neugebauer and Reichardt, 1991). Rabbit antiserthe chicken α1β1 integrin (serum 178) and to the α1 subunit (serum233) were provided by Drs M. Paulsson and J. Syfrig (University Bern, Switzerland; Syfrig et al., 1991). The mouse mAb LM609 the human αvβ3 integrin was from Dr D. Cheresh (Scripps ResearcInstitute, La Jolla, CA; Cheresh and Spiro, 1987). The rabbit antisraised against synthetic peptides corresponding to the cytoplasdomains of the chicken αv, α6A and α6B subunits and of the humanα2, α3 and β3 chains, and the rabbit antiserum β8-EX against theamino-terminal portion of the chicken β8 subunit, were provided byDr L. Reichardt (Bossy and Reichardt, 1990; de Curtis and Reicha1993; Delannet et al., 1994; Venstrom and Reichardt, 1995). Trabbit antiserum α6-EX against the amino-terminal portion of thechicken α6 subunit was from Dr I. de Curtis (DIBIT, Milan, Italy; deCurtis and Reichardt, 1993; Cattelino et al., 1995). Other polyclonantibodies to the cytoplasmic domain of the human α2 and α3subunits were from Dr G. Tarone (University of Torino, Italy) or werpurchased from Chemicon. The mAb P1B5 to the human α3 chainand recognizing the avian α3 subunit was from Becton-Dickinson.The mAb MEP17 to the chicken α2 integrin subunit was provided byDr K. McNagny (EMBL, Heidelberg, Germany; Bradshaw et al1995). Trunk neural crest cell cultures were generated from quembryos as described (Duband et al., 1995a). Cells were cultureDMEM with calf serum depleted in fibronectin or in serum-fremedium containing 0.1% ovalbumin, transferrin and insulin, each10 µg/ml.

Assays for cellular adhesion and migrationCellular adhesion assays were performed in bacteriological plasdishes. Small areas of the dishes were incubated at 37°C for 1 hwith 50 µl of the adhesive proteins to be tested in phosphate-buffesaline (PBS), followed by incubation with bovine serum albumi

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2731Neural crest cell migration on laminin

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(BSA) in PBS at 10 mg/ml for 30 minutes. Neural crest cells wegenerally obtained from at least 50 neural tube explants cultured18 hours. Cells were harvested using treatment for 3 minutes at 3with an enzyme-free cell dissociation buffer (Life TechnologiesAfter addition of DMEM, cells were collected, sedimented at 10rpm for 10 minutes, resuspended in serum-free DMEM, and counA 50 µl sample of cell suspension containing about 3000 cells wdeposited on the substratum. The dishes were then incubated at for 1 hour, rinsed in PBS to remove the non-adherent cells, and fiin a 3.7% formaldehyde solution in PBS. Attached and spread cwere counted under a Nikon inverted phase contrast microscoResults were expressed as the percentage of attached and spreawith relation to the total number of cells deposited on the substratand values were obtained from at least six different measuremenat least three independent experiments.

Migration assays were performed in bacteriological or cell cultuplastic dishes. The extent of migration of neural crest cells westimated by measuring the linear distance between the neural and the front of migration of neural crest cells (Duband et al., 199Time-lapse videomicroscopy analyses were performed in Terasplates (Dufour et al., 1988). Values were obtained from at leastdifferent measurements in at least three independent experiment

Immunofluorescent stainingsFor immunofluorescent labeling of cultures, neural tubes weexplanted onto laminin-1-coated glass coverslips and culturedDMEM with 5% serum. After washes in DMEM, cultures were fixeusing different procedures to visualize integrins that are diffusibly dtributed over the cell surface or concentrated in focal adhesions:cold methanol for 5 minutes followed by cold acetone for 1 minu(2) 3.7% formaldehyde-5% sucrose in PBS for 1 hour at 20followed or not by permeabilization with 0.2% Triton X-100 in PBfor 3 minutes, (3) 3.7% formaldehyde-0.2% Triton X-100-5% sucroin PBS for 5 minutes followed by a 1-hour incubation in 3.7formaldehyde in PBS. Cultures were subjected to immunofluorescstaining using biotinylated secondary antibodies and fluorescein-cjugated streptavidin (Amersham). Preparations were observed wiLeica epifluorescence microscope and photographed using TMX-Kodak film.

ImmunoprecipitationsFor immunoprecipitation experiments, cells were metabolicalabelled with [35S]-methionine (Amersham) at 250 µCi/ml for at least8 hours. The viability of cells during labeling was regularly confirmewith an inverted microscope. After labeling, cells were washed in Psupplemented with 1 mM CaCl2 and MgCl2 and extracted for 20

Coating concentration (µg/ml)

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Fig. 1.Comparative attachment (A), spreading (B) and migration (open circles) and vitronectin (VN; open triangles) adsorbed onto bwith 3% serum, and results are expressed as the linear distance ineural tube explant after 18 hours of culture.

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RESULTS

Adhesion and migration of neural crest cells onlaminin-1The ability of neural crest cells to adhere to and migrate laminin-1 was analyzed in in vitro assays and was compawith that on fibronectin and vitronectin (Fig. 1A-C). Theprofiles of the dose-response curve of neural crest cell attament, spreading and migration on laminin-1 were very similto those obtained with fibronectin or vitronectin. Attachmeand spreading were dose-dependent at concentrations belowµg/ml and were maximal at higher concentrations. Maximvalues for attachment and spreading on laminin-1 were coparable to those obtained with fibronectin or vitronectiOutward migration of neural crest cells on laminin-1 was dosdependent within the range of 0.1-10 µg/ml and was generallylower than on fibronectin. In contrast, at concentrations laminin-1 above 10 µg/ml, the expansion of the cell populationwas systematically higher than on fibronectin and vitronectMaximal mean values for migration reached on vitronectifibronectin and laminin-1 were about 480 µm, 590 µm and 700µm, respectively. The number of migrating neural crest ceon laminin-1 was found to be comparable to that on fibronecor vitronectin for coating concentrations promoting equal ratof migration (Fig. 2A; for comparison, Duband et al., 1991Delannet et al., 1994). However, cells on laminin-1 differed their morphology and density. Individual cells exhibited a reatively less spread morphology with fewer processes a

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Fig. 2. (A,B) Morphology and migration of neural crest cells cultured for 18 hours on laminin-1 at 25 µg/ml in the presence of 5% serum. (A) isan overall view of the neural crest cell outgrowth and (B) shows the morphological aspect of individual cells. (C) Neural crest cell outgrowth inthe presence of polyclonal antibodies to laminin-1 at 1 mg/ml. Cells were allowed to emigrate from the neural tube for 5 hours and antibodieswere then applied to the culture for 15 hours. nt, neural tube. Bars, 100 µm (A); 50 µm (B,C).

reduced intercellular contacts (Fig. 2B). Migration was alevaluated in the presence of varying concentrations of se(1-10%) and in serum-free medium. Values for migration wenot substantially affected by the serum, except at coating ccentrations higher than 10 µg/ml, on which cells migrated sig-nificantly farther by about 10-20% in low serum or in serumfree medium (e.g. about 800 µm in the absence of serum an640 µm with 10% serum; data not shown). In the subsequexperiments, migration assays were performed on laminin-25 µg/ml in the presence of 3-5% serum or in serum-frmedium.

To further demonstrate the direct interaction between crcells and laminin-1, migration assays were performed in presence of antibodies to laminin-1 at varying concentratio(Table 1). At 1 mg/ml, the antibodies severely perturbed crcell migration. Cells rounded up almost immediately, followeby extensive aggregation (Fig. 2C), and they covered less

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Table 1. Effects of antibodies to laminin-1 and laminin-1fragments on neural crest cell migration

MigrationAntibodies Substratum (% of control)

Anti-LN (1 mg/ml) LN 37±14E8 75±14E1′ 0±0FN 97±4

Anti-LN (0.1 mg/ml) LN 65±13E8 96±4E1′ 46±21FN 97±1

Anti-E8 (0.1 mg/ml) E8 0±0LN 20±11FN 95±12

Anti-E1′ (0.1 mg/ml) E1′ 89±10E8 104±16LN 87±7

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Migration of the neural crest population over laminin-1 waalso monitored using videomicroscopy (Fig. 3A). Crest cewere seen to adhere onto the substratum only 3-4 hours athe neural tube has been explanted in culture. On fibronecin contrast, cells emigrated from the neural tube almost immdiately and, by 3 hours, they formed an outgrowth sevelayers wide along the entire border of the neural tube (Delanand Duband, 1992). In addition, during the first houfollowing their separation from the neural tube, crest ceremained poorly spread on laminin-1 and formed small clusteof several individuals. Although cells were very motile, thedid not show any preferential direction of movement and, cosequently, the expansion of the entire cell population was sloOn fibronectin, in contrast, cells were usually spread winumerous protrusions, and they migrated with a preferentdirection of movement but slightly less rapidly than olaminin-1. After about 12 hours of culture, expansion of thcell population became extremely rapid on laminin-1, and cebecame spread.

These observations therefore suggested that the lamininadhesion properties of neural crest cells may vary with timeculture. We then measured the ability of crest cells cultured varying periods of time to attach to and spread on laminin-1fibronectin (Fig. 3B). Attachments to laminin-1 and fibronectiwere comparable and constant over time. In contrast, while cspreading was also relatively constant on fibronectin during tfirst 24 hours of culture, it was quite low on laminin-1 durinthe first 12 hours. Thus, consistent with the videomicroscoanalyses, neural crest cells gradually acquired a greacapacity to spread on laminin-1 in culture. To determinwhether this increase in cell spreading might result frochanges of the surface properties of cells or from modificatioin the matrix organization or composition, we evaluatelaminin-1 adhesion over time for cells that have been cultur

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2733Neural crest cell migration on laminin

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Fig. 3. (A) Migration tracks and morphologies of neural crest cells on fibronectin at 10 µg/ml and laminin-1 at 25 µg/ml. Neural tubes weredeposited in Terasaki wells in serum-free medium and, once neural crest cells were seen to be separated from the neural tube and adherent tothe substratum (defined as t0hr), their migration was recorded using time lapse videomicroscopy. Migration tracks of several cells as well astheir positions and shapes every hour were plotted, and the total distance of migration was measured. A typical track and values of cell velocity(v) and persistence of movement (p), defined as the ratio between the linear distance and the total distance covered by the cells, are indicated.(B) Comparative attachment (closed symbols) and spreading (open symbols) of neural crest cells on laminin-1 (LN; squares) and fibronectin(FN; circles) over time in culture. Crest cells were cultured for varying periods of time (5-24 hours) on laminin-1 or fibronectin, before beingcollected and subjected to adhesion assays on laminin-1 at 25 µg/ml and fibronectin at 10 µg/ml.

first on fibronectin and vice versa. It was found that the tyof substratum onto which neural crest cells were initiacultured had no influence on their adhesion propertisuggesting that changes in laminin-1-adhesion propertiesnot depend on a possible modification of the substratum owhich cells were deposited.

Laminin-1 domains involved in neural crest celladhesion and migrationThe adhesive properties of laminin-1 result primarily from tpresence of two major, separable cell-binding domains locain the E1′ and E8 fragments, respectively (Fig. 4A; Yurchenand O’Rear, 1994; Mercurio, 1995). We tested the abilitythese fragments to support attachment, spreading migration of neural crest cells. In adhesion assays, thefragment was as efficient as intact laminin-1 in supporting cattachment and spreading and the profiles of the dose-respcurves and values for maximal attachment and spreading walmost identical for both molecules (Fig. 4B,C). In contrathe E1′ fragment permitted cell attachment only partially andid not allow cell spreading at all (Fig. 4B,C).

In migration assays, both the E8 and E1′ fragments werefound to promote efficient neural crest cell migration. On tE1′ fragment, outward migration was dose-dependent witthe range 0.1-10 µg/ml and was maximal at higher concentrtions. However, values never reached those attained laminin-1 (Fig. 4D), and cells were poorly spread on the sstratum (Fig. 5A). On the E8 fragment, the shape of the doresponse curve differed from those on the E1′ fragment orlaminin-1 (Fig. 4D). Migration was systematically greater thon laminin-1 at concentrations below 5 µg/ml and becamemaximal at 10 µg/ml. However, the curve declined at concetrations above 10 µg/ml and values for migration became significantly lower than on laminin-1. In addition, cells wergenerally more flattened and more cohesive on the E8 fragmthan on laminin-1 (Fig. 5B,C). We then evaluated wheth

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combination of the E1′ and E8 fragments at varying concentrations could modify the migratory properties of neural crecells. As shown in Fig. 4E, addition of the E1′ fragment to E8produced a slight increase in the extent of cell migration whthe E1′ fragment was in large excess compared to E8 and tE8 was coated at low concentrations. Under these conditiocells exhibited the same elongated shape as on intact lami1. In contrast, when the E8 fragment was at concentratiohigher than 5 µg/ml, addition of E1′ even at high concentra-tions did not modify the locomotory response and cellulmorphology of neural crest cells as compared to the fragment alone.

To further characterize the binding domains of laminin-1 thare involved in neural crest cell migration, we measured effect of antibodies specifically directed to the laminin-fragments on cell migration (Table 1). Antibodies directeagainst E8 totally abrogated crest cell migration, not only on E8 fragment, but on intact laminin-1 as well. At 0.1 mg/ml, thewas at least a fivefold decrease in the migratory potential of cells. Cells confronted with the anti-E8 antibodies exhibitedround morphology and formed multicellular aggregates, as in presence of anti-laminin-1 antibodies (not shown). In contraantibodies to E1′ showed virtually no inhibitory activity on cellmigration on both laminin-1 and the E1′ fragment. The reasonfor this result is that these antibodies are intrinsically poinhibitors of E1′ binding (P. Yurchenco, personal communication). However, cell migration on the E1′ fragment was totallyblocked by antibodies to laminin-1, thereby confirming that ceactually migrated over the fragment.

Finally, the ability of neural crest cells to attach and spreon the E1′ and E8 fragments was evaluated as a function of duration of cell culture (Fig. 4F). Attachment of neural crecells to either fragment was not found to vary significantduring the first 24 hours following their migration from thneural tube. By contrast, cell spreading on E8 was minimduring the first 10-12 hours and gradually increased to valu

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Fig. 4. (A) Diagram showing thestructure of laminin-1 with itsthree α1, β1 and γ1subunits, thelocation within the molecule of theE1′ and E8 fragments, and themajor integrin-recognition sites.Note that the αvβ3 integrin-binding site is only indicated forthe E1′ fragment as it is notfunctional in the native molecule.(B, C) Comparative attachment (B)and spreading (C) of neural crestcells on laminin-1 (LN; squares)and on the E8 (triangles) and E1′(circles) fragments.(D) Comparative migration ofneural crest cells after 18 hours ofculture in serum-free medium onlaminin-1 (squares) and on the E8(triangles) and E1′ (circles)fragments adsorbed on cell cultureplastic dishes. Values for BSA areindicated by two inverted triangles.(E) Migration of neural crest cellsin serum-free medium on the E8fragment at 0.5, 5 and 25 µg/mlcombined with E1′ at varyingconcentrations. (F) Comparativeattachment (closed symbols) andspreading (open symbols) of neuralcrest cells on laminin-1 (squares)and on the E8 (triangles) and E1′(circles) fragments over time inculture. Cells were cultured forvarying periods of time (5-24hours) on laminin-1 before beingcollected and subjected to adhesionassays on laminin-1 and on thefragments, each at 25 µg/ml.

that were usually obtained by 18 hours in culture. Interestingthe curve for the E8 fragment exactly paralleled that laminin-1.

Integrin receptors for laminin-1 on neural crest cellsTo characterize the laminin-1-binding integrins expressedneural crest cells, extracts of metabolically labelled cells wsubjected to immunoprecipitation with antibodies raisagainst the chicken β1, α1 and α2 integrin subunits, thechicken α1β1 heterodimer, the two variants of the chicken α6subunit (α6A and α6B), the cytoplasmic domain of the chickeαv subunit, and the cytoplasmic domains of the human α2, α3and β3 integrin subunits. Anti-β1 antibodies precipitated aseries of proteins of Mr (×10−3) 180, 160-140, 110 and 100corresponding to various α chains, the β1 subunit and itsbiosynthetic precursor, respectively (Fig. 6, lane 1; Akiyamaal., 1986; Delannet et al., 1994). Antibodies to α1β1 precipi-tated two proteins of 180 and 110 ×10−3 Mr, corresponding to

ly,for

byereed

n

,

et

the α1 and β1 chains (Fig. 6, lane 2; Duband et al., 1992). Nband could be visualized in immune precipitates of cell extracwith antibodies to the α2 subunit, either monoclonals or poly-clonals to the cytoplasmic domain (Fig. 6, lane 3). In contratwo intense bands of 140 and 110 ×10−3 Mr, corresponding tothe α3 and β1 subunits, could be detected with anti-α3 anti-bodies (Fig. 6, lane 4; Wayner et al., 1988). Antibodies to thA variant of α6 precipitated two bands of Mr 140 and 110 ×10−3, corresponding to α6 and β1, but no detectable band wasobtained with antibodies to the B variant (Fig. 6, lanes 5 a6; de Curtis and Reichardt, 1993). No band correspondingthe β4 chain could be obtained in association with α6. Theantibody to αv precipitated a set of proteins of Mr of about 150-140, 110, 95 and 85 ×10−3, corresponding to the αv subunitand its biosynthetic precursor, the β1, β3 and β5 chains, respec-tively (Fig. 6, lane 7; Bossy and Reichardt, 1990; Delannet al., 1994). The antibody to the β3 subunit precipitated twoproteins of Mr 140 and 95 ×10−3, corresponding to αv and β3,

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2735Neural crest cell migration on laminin

st.he

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Fig. 5.Morphology and migration of neural crest cells cultured for 18 hours in serum-free medium on the fragments E1′ (A) and E8 (B), and onintact laminin-1 (C), each adsorbed at 25 µg/ml on cell culture plastic dishes. In each picture, the neural tube is to the left. Bar, 50 µm.

and occasionally another non-identified protein of Mr of about80×10−3 (Fig. 6, lane 8; Bossy and Reichardt, 1990; Delannet al., 1994).

The surface distribution of integrins in neural crest cells wexamined by immunofluorescence using antibodies to theβ1,β3, β8, α1, α3, α6 and αv subunits. At first, integrin distribu-tion was studied in cells that had been cultured for 18-24 hoThe β1 subunit could be visualized in focal contacts, but onwhen formaldehyde fixation was employed in association w

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Fig. 6. Integrin receptors for laminin-1 on neural crest cells. Crestcells cultured on laminin-1 were metabolically labeled, extracted indetergent and the lysates subjected to immunoprecipitation using2992 polyclonal antibody to the chicken β1 subunit (lane 1), thepolyclonal antibody 178 to the avian α1β1 integrin (lane 2),polyclonal antibodies to the cytoplasmic domains of the human α2and α3 subunits (lanes 3 and 4), to the cytoplasmic domains of thechicken α6A (lane 5), α6B (lane 6) and αv (lane 7) subunits, and tothe cytoplasmic domain of the human β3 chain (lane 8). Sampleswere resolved by SDS-PAGE on 7% acrylamide gels undernonreducing conditions and radiolabeled bands were visualized bfluorography. Relative molecular mass markers are indicated on thleft and relative positions of the integrin subunits on the right.

et

as

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Triton X-100 (Fig. 7A). It should be noted that neural crecells on laminin-1 exhibited relatively few focal contactsWhen permeabilization treatment was performed after tfixation step, staining for β1 was exclusively uniform anddiffuse on the cell surface (not shown, but see Duband et 1986). The α1 subunit showed a distribution pattern virtuallidentical to that of β1. Staining was strong and uniform ovethe entire cell surface (Fig. 7B) and, when the cell membrawas extracted with detergents, staining of focal contabecame apparent. The α3 subunit was never found in focacontacts, whatever fixation procedure was used (Fig. 7C).contrast, α6 could be found in association with focal contasites, but the intensity of the labeling was markedly weakthan for α1 and β1 (Fig. 7D). In addition, strong intracellularstaining could be seen around the nucleus. Staining for αv wasprominent in focal contacts (Fig. 7E), while staining for β3 wasessentially diffuse over the cell surface but colocalization wfocal contacts was also observed (Fig. 7F). Finally, the β8subunit was as a weak diffuse staining on the cell surface (shown).

The surface distribution of integrins was also examinedneural crest cells that had been cultured for brief periodstime on laminin-1 (5-10 hours). In those cells, the β1 chain wasfound in tiny focal contacts in the cell periphery (Fig. 8A) buin contrast to cells cultured for 18 hours, it was not associawith αv, which instead showed a weak and diffuse staining the cell surface (not shown). In contrast to α1, which wasabundant on the entire surface of neural crest cells (Fig. 8the α3 and α6 subunits presented cellular distributions strikingly different from those seen in cells cultured for 18-2hours. α3 staining was particularly enriched in the areas of cecell contacts (Fig. 8C), while α6 staining was mostly cyto-plasmic around the nuclei and almost absent from the csurface (Fig. 8D).

These results then indicate that neural crest cells culturedlaminin-1 express multiple integrins, including α1β1, α3β1,α6β1, αvβ3 and possibly one or more β8 integrins, as pre-

the

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2736

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Fig. 7. Immunofluorescencedetection of the integrin subunitsβ1 (A), α1 (B), α3 (C), α6 (D),αv (E) and β3 (F) in neural crestcells cultured for 18 hours onlaminin-1. Cells were fixedusing different proceduresbefore immunofluorescencestaining. For the β1, α6 and αVsubunits, cells were fixed in PBScontaining 3.7% formaldehyde,0.5% Triton X-100 and 5%sucrose for 5 minutes andpostfixed in 3.7% formaldehydefor 1 hour. For the α3 and β3subunits, cells were first fixed in3.7% formaldehyde for 1 hourand permeabilized with 0.5%Triton X-100 for 5 minutes. Forthe α1 subunit, cells were fixedin 3.7% formaldehyde for 1hour. Arrows point at focaladhesion sites. Bar, 25 µm.

sumptive laminin-1 receptors. In addition, these integrins distributed in remarkably distinct cellular compartmentwhich may vary over time in culture. In contrast, α2β1 andα6β4 are apparently not synthesized by neural crest cellsleast when cultured on laminin-1.

Role of integrins in neural crest cell adhesion andmigration on laminin-1Since a number of potential laminin-1 receptors appearedbe expressed by neural crest cells, their possible involvemin cell adhesion to and migration on laminin-1 was evaluausing function-blocking antibodies. The mAb CSAT to the β1subunit exhibited a potent inhibitory effect on attachmespreading and migration of neural crest cells on laminin(Fig. 9A). Spreading was entirely abolished even at loantibody concentrations, and cell attachment was reduceless than 50% of the control at higher doses. The effectcell migration was dose-dependent between 1 and 10 µg/mland became maximal at higher concentrations. At 20 µg/ml,for example, cells migrated only 20% of the control after hours in the presence of the antibody; they were round formed clusters that remained in proximity to the neural tu(Fig. 10A). The polyclonal antibody 178 to α1β1 alsoaffected spreading and migration of neural crest cells indose-dependent manner and, at high concentration of

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antibody, both spreading and migration were entirely anihilated (Fig. 9B). After 15 hours in the presence of thantibody, the cell population covered less than 100 µm, andcells displayed the same morphology as when treated wmAb CSAT (Fig. 10B). Interestingly, attachment of neuracrest cells to laminin-1 was not perturbed by the antibody 1even at high doses. Videomicroscopy analyses revealed the effects of the mAb CSAT and of the polyclonal antibod178 were almost immediate. Cells became round within lethan 30 minutes, and both their speed of migration and psistence of movement were substantially reduced during subsequent 10-15 hours (Fig. 11).

The mAb P1B5 to α3β1 and the polyclonal antibody α6-EXdid not show any detectable effect on cell attachmespreading or motility at any of the concentrations tested (F9C,D). In the presence of these antibodies, neural crest cexhibited a cellular morphology similar to that in controlwithout antibody, except that with mAb P1B5, cells werslightly less spread and cohesive (Fig. 10C,D). In addition, tantibodies P1B5 and α6-EX used in combination did not showany additive effect, nor did they potentiate the action antibody 178 on cell attachment (not shown). The mAb LM60to αvβ3, in contrast, caused a certain enhancement in neucrest cell adhesion and spreading to laminin-1 (Fig. 9EHowever, at high doses, this antibody reduced cell migrati

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2737Neural crest cell migration on laminin

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Fig. 8.Surface distribution ofintegrins in early migratingneural crest cells on laminin-1. Immunofluorescencedetection of the integrinsubunits β1 (A), α1 (B), α3(C) and α6 (D). Cells werecultured for 5-10 hours andfixed in 3.7% formaldehyde,0.5% Triton X-100 in 5%sucrose for 5 minutes andpostfixed in 3.7%formaldehyde for 1 hour forthe β1 integrin subunit, and incold methanol for 5 minutesfollowed by cold acetone for1 minute for the α1, α3 andα6 subunits. Note that α3 isfound in the cell-cell contactareas and α6 is essentiallyseen as an intracellularstaining around the nucleus.Thick arrows point at focaladhesion sites and thin arrowsat cell-cell contact areas. Bar,25 µm.

significantly to about 50% of the control (Fig. 9E). Celbecame extremely cohesive and those situated at the fronmigration became round (Fig. 10E). Finally, the antibody β8-EX did not show any detectable effect on cell adhesion amotility even at high concentrations up to 300 µg/ml (data notshown).

The apparent lack of function of α3β1 and α6β1 might bedue to the fact that they were in an inactivated state. To addthis point, we analyzed the effect of the mAb TASC on neucrest cell adhesion to laminin-1. TASC is an anti-β1 antibodyknown to promote adhesion of retinal neurons to laminin-1 acollagen IV with respect to attachment kinetics, and it is aable to activate resting β1 integrins (Neugebauer andReichardt, 1991). As shown in Fig. 9F, the time course neural crest cell attachment and spreading on laminin-1 wnot significantly modified by the mAb TASC, indicating thaβ1 integrins, such as α3β1 and α6β1, could not be activated inthose cells.

We also analyzed the effect of the antibodies on the abiof neural crest cells obtained after varying periods of timespread on laminin-1 (Fig. 9G). Although neural crest ceobtained after 5-10 hours of culture were generally more sceptible to mAb CSAT and polyclonal antibody 178 thawere cells obtained after 18-24 hours, there was no dramtemporal change in the functional specificities of integrinscell adhesion. In particular, the antibody α6-EX remainedineffective on cell spreading at all times of culture invesgated. Interestingly, antibodies to αvβ3 slightly inhibitedspreading of cells cultured for 5 hours, whereas thincreased spreading of cells cultured for longer periodstime (not shown).

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Binding specificities of integrins in neural crestcells cultured on laminin-1Finally, we evaluated the effect of anti-integrin antibodies ocrest cell adhesive and migratory properties on the E8 and ′fragments. As shown in Fig. 12A,C, the mAb CSAT to β1reduced dramatically crest cell attachment on both the E8 athe E1′ fragments. It is noteworthy that the antibody was evemore potent on the fragments than on laminin-1 (almost 90inhibition of attachment on E8, 60% on E1′ and only 50% onlaminin-1; compare with Fig. 9A). The mAb CSAT alsoblocked totally spreading on E8 (Fig. 12A). As for intaclaminin-1, the polyclonal antibody 178 to α1β1 abrogated cellspreading on the E8 fragment and it showed only a limiteeffect on cell attachment on both E8 and E1′ fragments (Fig.12B,D; compare with Fig. 9B). In migration assays, both thmAb CSAT and the polyclonal antibody 178 totally abolisheexpansion of the neural crest outgrowth on the E1′ fragment(Fig. 12C,D). On the E8 fragment, the same antibodies severreduced cell migration, but never caused a complete inhibiti(Fig. 12A,B), probably because cells were extremely cohesand flattened, thus reducing antibody access to integriFinally, the antibodies to the α6 and α3 chains did not perturbneural crest cell adhesion and migration on the E8 and E′fragments (not shown).

DISCUSSION

Numerous in vivo studies have revealed that, durinmigration, neural crest cells come in contact at all axial levewith a complex fibrillar extracellular matrix which is lined by

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2738

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Fig. 9. (A-E) Neural crest cellattachment (open circles), spreading(open squares), and migration (opentriangles) on laminin-1 adsorbed ontobacteriological plastic dishes at 25µg/ml in the presence of function-perturbing antibodies to integrins:mAb CSAT (anti-β1; A), antiserum178 (anti-α1β1; B), mAb P1B5 (anti-α3β1; C), antiserum α6-EX (anti-α6;D), and mAb LM609 (anti-αvβ3; E).(F) Kinetics of neural crest cellattachment (circles) and spreading(squares) on laminin-1 at 25 µg/ml inthe presence of mAb TASC at 100µg/ml. (G) Comparative effects ofmAb CSAT at 2.5 µg/ml, antiserum178 at 1/500 dilution, and antiserumα6-EX at 1/100 dilution on thespreading of neural crest cellscultured for varying periods of timeon laminin-1 at 25 µg/ml. In controlassays for cellular adhesion, at least50% of the cells deposited on thesubstratum were adherent. In assaysfor cellular migration, cells wereallowed to migrate from the neuraltube explants for 5 hours, andantibodies were then added to theculture medium for the subsequent 15hours in serum-containing medium.

the basal laminae of epithelial tissues (Krotoski et al., 19Duband and Thiery, 1987; Mackie et al., 1988; Perris et 1991a,b, 1993a; Delannet et al., 1994). The ability of neucrest cells cultured in vitro to locomote efficiently on variouextracellular matrix elements, e.g. fibronectin, vitronectlaminin-1 and collagens either fibrillar or non-fibrilla(Newgreen et al., 1982; Rovasio et al., 1983; Tucker aErickson, 1984; Perris et al., 1993a,b; Delannet et al., 199and the observation that antibodies to fibronectin or lamin1 injected into trunk regions of avian embryos fail to pertuneural crest cell migration (Bronner-Fraser, 1993), suggthat these cells show no particular preference for a givextracellular matrix molecule over the others for migratioConsequently, this would mean that the extracellular matmaterial onto which cells move would only play a permissi

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role for migration and that the path-finding mechanism neural crest cells would be essentially driven by negative cuthat impede cell movement in defined areas of the embr(Rickmann et al., 1985; Tosney and Landmesser, 198Newgreen et al., 1986; Loring and Erickson, 1987; Teillet al., 1987; Pettway et al., 1990; Wang and Anderson, 199However, a careful examination of the mode of interaction neural crest cells with extracellular matrix components aof the organization of the migratory pathways suggests thfibronectin, laminin-1 and vitronectin may not be entirelinterchangeable for neural crest cell migration, but instewould play different, specific roles. Indeed, our present stuclearly shows that neural crest cells travel longer migratodistances on laminin-1 than on fibronectin and vitronectin. addition, they develop adhesion to laminin-1 only progres

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2739Neural crest cell migration on laminin

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Fig. 10.Morphology and migration of neural crest cells on laminin-1 in the presence of function-perturbing antibodies to integrins. (A) mAbCSAT anti-β1 at 50 µg/ml, (B) antiserum 178 anti-α1β1 at 1/100 dilution, (C) mAb P1B5 anti-α3β1 at 100 µg/ml, (D) antiserum α6-EX anti-α6 at 1/25 dilution and (E) ascitic fluid LM609 anti-αVβ3 at 1/25 dilution. In A, B and E, the neural tube (nt) is to the left and in C and D, tothe bottom. Bar, 50 µm.

mAb mAb

mAb

ively during migration, whereas adhesion to fibronectin acquired prior to emigration. This greater efficiency laminin-1 in promoting migration can be related to thmigratory behavior of trunk neural crest cells in vivo (Tosnet al., 1994). When reaching the somite, neural crest cellsnot move ventrally into the dissociating sclerotome, binstead follow the basal lamina of the myotome that is pgressively developing ahead of them. This preference for myotomal route is believed to facilitate a faster access toventral parts of the embryo, to prevent neural crest cdispersal into the sclerotome and to maintain the cell polation in a restricted environment, thereby ensuring thagreater proportion of cells to reach their target sites, aallowing them to avoid the impediments for migration thdevelop progressively in the sclerotome. Therefore, diffences in the relative efficacy of matrix components

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promoting rapid cell migration may provide positiveguidance cues for migrating neural crest cells.

Under in vitro culture conditions, neural crest cells expremultiple laminin-1-binding integrins, as judged by immunoprecipitation and immunofluorescence techniques. Curiousonly few of them exhibit any function in cell motion. Indeedonly antibodies to the α1β1 heterodimer and to the β1 subunittotally abolished cell spreading and motility on laminin-1. Athe cellular level, α1β1 is distributed in high amounts, both infocal contacts and uniformly on the cell surface. Thereforα1β1 can be considered as the major laminin-1-bindinintegrin involved in neural crest cell migration. Consistent withis finding, α1β1 has been demonstrated to be present in vion the surface of migrating neural crest cells (Duband et 1992). The recent discovery that α1β1 can activate the MAPkinase pathway via the adaptor protein Shc, thereby promot

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2740

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v = 117.5 ± 10 µm/hrp = 0.30 ± 0.04

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v = 40 ± 6 µm/hrp = 0.17 ± 0.03

t0hr

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t0hr

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v = 48 ± 11 µm/hrp = 0.16 ± 0.04

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Time (hrs) after addition of the antibody

0

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Vel

ocity

(µm

/hr)

mAb CSATanti-β1

Ab 178anti-α1β1

B

50 µm

Fig. 11.(A) Migration tracks and morphologies of neural crest cellson laminin-1 at 25 µg/ml in the presence of mAb CSAT anti-β1 at 50µg/ml and antiserum 178 anti-α1β1 at 1/100 dilution. Neural crestcells were allowed to migrate from the neural tube explants for ab5 hours before addition of the antibodies (defined as t0hr). Cellmigration was subsequently recorded using time lapsevideomicroscopy. Migration tracks of several cells as well as theirpositions and shapes every hour were plotted, and the total distanof migration was measured. A typical track and values of cellvelocity (v) and persistence of movement (p) are indicated. (B)Velocity of neural crest cells on laminin-1 in the presence of mAbCSAT at 50 µg/ml (circles), antiserum 178 at 1/100 dilution(triangles) and in controls (squares) over time in culture afteraddition of the antibodies.

cell division and preventing apoptosis, emphasizes the critrole of this integrin in neural crest cell migration and prolifeation (Wary et al., 1996). As also observed previously (Lalland Bronner-Fraser, 1992; Lallier et al., 1994), we found tantibodies to α1β1 did not affect cell attachment to laminin-while antibodies to the β1 subunit showed a greater inhibitoreffect, suggesting that other β1 integrins are involved in cellattachment. Candidate integrins are α3β1, α6β1, α7β1 andα9β1. Antibodies to α3β1 and α6β1, alone or in combination,did not significantly affect adhesion and migration on lamin1, indicating that these integrins are not part of the laminin

icalr-ierhat1y

in--1-

adhesion machinary in neural crest cells. Although we counot determine whether α9β1 is expressed by neural crest cellsit is unlikely that this integrin could be involved in theiradhesion to laminin-1 as it has so far only been describedmuscles and epithelia (Palmer et al., 1993). In contrast, α7β1has been recently reported to be expressed at low levels osubset of migrating neural crest cells (Kil and Bronner-Frase1996) and, even though its function has not been directaddressed, it is the best candidate so far for mediating neucrest cell attachment to laminin-1.

Beside α1β1, the only other characterized integrin thatplayed a role in neural crest cell migration on laminin-1 iαvβ3. Although it has been found that αvβ3 could function asan RGD-independent laminin-1 receptor for human microvacular endothelial cells (Kramer et al., 1990), this integrin hato be considered as a secondary receptor for laminin-1 as it hbeen shown to bind the cryptic RGD sequence in the fragmeE1′ and not intact laminin-1 (Sonnenberg et al., 1990b). Thinhibitory effect of antibodies to αvβ3 on neural crest cellmigration on laminin-1 would then suggest that, duringmigration, laminin-1 molecules are possibly cleaved bproteases released by neural crest cells, thereby exposing cryptic RGD site accessible to the αvβ3 integrin. In support ofthis hypothesis, it has been suggested that neural crest cmay modify their laminin-1 substrata in vitro (Lallier et al.,1994), but our present data would tend to contradict thstatement. Alternatively, αvβ3 may interact with laminin-1indirectly through associated extracellular matrix componentsuch as nidogen and fibulin (Ekblom and Timpl, 1996)However, to our knowledge, there is no direct proof so far thαvβ3 binds fibulin or nidogen with significant affinity. Finally,it cannot be excluded that αvβ3 may also function indepen-dently of laminin-1 as a signaling integrin, activated througbinding to extracellular matrix molecules that are secreted bneural crest cells themselves. In this respect, it is notewortthat neural crest cells have been shown to synthesize vronectin and tenascin, which are both ligands for αvβ3 (Tuckerand McKay, 1991; Delannet et al., 1994).

The absence of α6β1 involvement in laminin-1 recognitionby migrating neural crest cells is not surprising. Indeed, α6β1is not detected in vivo on the surface of neural crest cells unthey differentiate into cells of the peripheral nervous syste(Bronner-Fraser et al., 1992). Interestingly, neural crest cewere found to express in vitro the same variant of the α6 chainas Schwann cells, one of the numerous neural crest derivativ(Hogervorst et al., 1993), and this would possibly reflect theprecocious commitment into peripheral lineages. In contrast α1β1, α6β1 does not activate the MAP kinase pathwayresulting in exit from the cell cycle progression, an evennecessary for cell differentiation (Wary et al., 1996). Thuswhile α1β1 would be involved in neural crest cell migrationand proliferation, α6β1 would play a critical role in their differ-entiation into cells of the peripheral nervous system. On thother hand, although α6β1 is synthesized in high amounts, asjudged by immunoprecipitation analyses of metabolicallylabelled cells, it is essentially maintained intracellularly and ionly secondarily expressed on the cell surface at late stagesmigration. In addition, the pool of α6β1 receptors that is foundon the cell surface could not be activated by antibodies. Thsituation is reminiscent of recent data showing that the integrα6 and β1 chains associate with the chaperone calnexin in th

out

ce

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2741Neural crest cell migration on laminin

in

m;

dmy

hedof

to-

lls,

chmi-

entndd,erd

l

A B

Concentration of antibody(serum dilution)

α1β1 integrin(rabbit 178 serum)

10000 5000 1000 500 100

0

20

40

60

80

100

0

20

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100

Concentration of antibody(µg/ml)

0.25 0.5 1 2.5 5 10 25

β1 integrins(mAb CSAT)

% o

f co

ntro

l

0.1 50

% o

f co

ntro

l

C

E8 E8

D

Concentration of antibody(serum dilution)

α1β1 integrin(rabbit 178 serum)

10000 5000 1000 500 100

0

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0

20

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Concentration of antibody(µg/ml)

0.25 0.5 1 2.5 5 10 25

β1 integrins(mAb CSAT)

% o

f co

ntro

l

50

% o

f co

ntro

l

AAAA

AAAAAAAA

A

AAAA

E1' E1'

AA

Fig. 12.Effect of anti-integrin antibodieson the adhesion and migration of neuralcrest cells on the laminin-1 fragments E8(A,B) and E1′ (C,D) both at 25 µg/ml.Effects of the mAb CSAT anti-β1 (A,C)and of the antiserum 178 anti-α1β1 (B,D)on neural crest cell attachment (circles),spreading (squares) and migration(triangles).

endoplasmic reticulum prior to heterodimer assembly (Lenand Vestweber, 1994) and that, during terminal differentiatiof human epidermal keratinocytes, newly synthesized integrundergo a prolonged association with calnexin and are tretained in the endoplasmic reticulum instead of being traferred to the cell surface (Hotchin et al., 1995). Therefore ilikely that, at least in vitro, α6β1 expression is regulated posttranscriptionnally in migrating neural crest cells and that newsynthesized heterodimers are also prevented from accumuing at the cell surface by a calnexin-dependent process. Ssynthesis and maturation of integrins is a long and compprocess that lasts over several hours (Akiyama and Yama1987), such a regulation is believed to ensure flexibility durineural crest development, allowing, for example, rapid recrument of α6β1 to the cell surface as cells undergo neural diffeentiation and grow neurites (de Curtis and Reichardt, 1993

In contrast to α6β1, α3β1 is abundantly expressed on thsurface of neural crest cells, but its function is still to bdefined. It has been found that, in carcinoma cells, α3β1 bindslaminin-5 but not laminin-1 (Rousselle and Aumailley, 1994In agreement with this finding, our preliminary data show thα3β1 may constitute a functional laminin-5 receptor in neurcrest cells. Interestingly, α3β1 is initially concentrated in theareas of cell-cell contacts at the onset of migration, as cdisplay a round morphology and are cohesive. Thereafwhen cells are fully spread and migratory, α3β1 becomesessentially diffuse over the cell surface. In addition, the timcourse of changes in α3β1 distribution on the surface of neuracrest cells correlates precisely with the progressive acquisiof laminin-1 adhesion by early migrating neural crest celVarious studies have shown that α3β1 is present in cell-cell

teroninshusns-t is-lylat-

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ngit-r-).ee

).atal

ellster,

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contacts, and it has been proposed that it may play a rolecell-to-cell adhesion either by a homophilic bindingmechanism or by a possible heterophilic binding mechanisinvolving α2β1 (Larjava et al., 1990; Sriramarao et al., 1993Symington et al., 1993). If α3β1 is able to mediate intercellu-lar adhesion in neural crest cells, it is likely that this woulrestrain cell motion to some extent and that its exclusion froregions of cell-cell contacts would allow cells to move freelon the substratum. In support of this possible function of α3β1in mediating intercellular adhesion in neural crest cells is tfact that antibodies to α3β1 caused cells to become less spreaand cohesive, although they did not modify the extent migration of the cell population.

It appears from our study that neural crest cells are ablerecognize the two major cell-binding domains of the laminin1 molecule situated in the E1′ and E8 fragments and that thisinteraction is essentially mediated by α1β1. This findingcontrasts to previous reports showing that neural crest cealso using α1β1, interact exclusively with the E8 fragment(Perris et al., 1989; Lallier et al., 1994). The reasons for sua discrepancy have not yet been elucidated. Possible contanation of the E1′ fragment by E8 in our experiments can bexcluded for several reasons. First, we used two differesources of fragments that had been purified independently athat did not show any contaminant by SDS-PAGE. Seconantibodies to the E8 fragment did not affect cell migration ovthe E1′ fragment. On the other hand, it should be mentionethat recognition of the E1′ fragment by neural crest cells waspredictable to a certain extent, given that the α1β1 integrin isthe major laminin-1-binding integrin that functions in neuracrest cells and this integrin is known to bind primarily the E1′

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2742

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N. Desban and J.-L. Duband

domain in most cell types (Hall et al., 1990; Tomaselli et a1990; Goodman et al., 1991; Colognato-Pyke et al., 199Thus, neural crest cells would not be an exception, but raresemble rat hepatocytes, in which α1β1 has also been shownto bind the two distinct domains of laminin-1 (Forsberg et a1990).

Most interestingly, the E1′ and E8 fragments were found toelicit cellular responses in neural crest cells that were chateristically different for each fragment and distinct from thoobtained with the whole, intact laminin-1 molecule. While, othe E1′ fragment, cells were poorly spread, they were contrast extremely flattened and cohesive on the E8 one. Csistent with this, in adhesion assays, the E1′ fragment onlypermitted cell attachment and the E8 fragment promoted battachment and spreading. In migration assays, the E1′ and E8fragments could substitute for laminin-1 to some extent, migration was generally lower. This would suggest that bindito the E1′ domain may play a regulatory, anti-adhesive rocausing reduction in cellular spreading and cohesion, therallowing faster cell locomotion. Recently, it has been proposthat a suppressor site may exist in the E1′ domain of laminin-1 that selectively interferes with the neurite outgrowtpromoting activity of the E8 fragment and may be uncoverby antibodies to E1′ (Calof et al., 1994). Such a mechanism unlikely to operate for neural crest cells, as the E1′ fragmentitself showed migration-promoting activity and antibodies the E1′ fragment did not enhance cell migration on laminin-A more plausible explanation is that, depending on the lamin1 domains with which it interacts, the α1β1 integrin transducesdistinct intracellular signals and is differently associated wthe cytoskeleton. Thus, when bound to the E8 domain, α1β1would induce a highly structured organization of the cytosketon, promoting excellent spreading, but perhaps too rigidpermit rapid changes in cell shape for active cell movementaddition, it is also conceivable that the integrin-activatsignals allowing release of adhesion (e.g. see Lawson Maxfield, 1995) would not be elicited by α1β1 upon bindingto E8, causing the cell to be stuck on the substratum. Cversely, when bound to the E1′ domain, α1β1 would not beable to organize transiently the actin cytoskeleton into strfibers necessary to generate the tension to allow cell motbut would transduce the signals necessary for cell-substradetachment. On intact laminin-1 molecules, a combinationboth signals would ensure rapid and efficient cell migrationit is true, this process would be the first example listed soof a possible regulation of the signaling activity of an integrby the binding domains of its corresponding extracellumatrix ligands. At the molecular level, this process would driven by differential conformational changes of the integrreceptor induced upon binding to E1′ and E8 and affectingassociation of the intracellular domains of α and β chains withcytoplasmic elements (Dedhar and Hannigan, 1996).

In conclusion, our study provides evidence for a critical roof α1β1 in neural crest cell migration in vitro through interaction with distinct binding domains of the laminin-1 moleculeHowever, the exact function of α1β1 in neural crest cell devel-opment in vivo remains to be established, particularly in tlight of the recent demonstration that mice carrying nmutations in the integrin α1 gene develop normally and presenno overt phenotype (Gardner et al., 1996). Likewise, mstudies have so far concerned laminin-1 and it is not known

l.,5).

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whether neural crest cells remain in contact only with this paticular type of laminin throughout migration or if theyencounter other laminin isoforms. In this respect, it will be ointerest to analyze how these cells interact with the laminisoforms (i.e. laminin-5, 6 and 7) that lack the α1β1-bindingdomain at the N terminus of the α chain.

We are extremely grateful to Ken Yamada, Peter Yurchenco, JeClaude Lissitzky, Yvan de Curtis, and Josef Syfrig for useful discusions and advice, and for providing antibodies or laminin-1 fragmenso generously. We also thank Louis Reichardt, Mats Paulsson, MHayashi, David Cheresh, Clayton Buck, Guido Tarone and KeMcNagny for providing antibodies or hybridoma cell lines, anSandrine Testaz for providing data about β8 integrins. This work wassupported by the Centre National de la Recherche Scientifiq(Programme ATIPE), the Ministère de la Recherche et de la Tenologie (91.T.0011), the Association pour la Recherche contreCancer (ARC-6517), the Institut National de la Santé et de Recherche Médicale (CRE 910705), the Ligue contre le Cancer, Association Française contre les Myopathies, and the Fondation pla Recherche Médicale.

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(Received 14 February 1997 - Accepted 29 July 1997)