Molecular mechanisms of avian neural crest cell migration on fibronectin and laminin

DEVELOPMENTAL

BIOLOGY

136,222-238 (1989)

Molecular Mechanisms of Avian Neural Crest Cell Migration on Fibronectin and Laminin ROBERTOPERRIS,**~MATS PAuLssoN,t AND MARIANNE BRONNER-FRASER* *Developmental

Biology Center, University of Cal~ornia Irvine, Irvine, Calijbrnia 92717; and tDepartment Biocenter of the University of Base& CH-4056, Basel, Switzerland

of Biophysical Chemistry,

Accepted June 27, 1989

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative “cell-binding domain,” neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by >‘70% after addition of heparin. The El’ fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive PI fragment were entirely nonpermissive for cell movement. Preincubation of laminin-nidogen substrates with antisera against the E8 fragment, but not against the El’ or the E4 fragment, potently reduced migration on the complex, further suggesting that the E8 domain is the predominant motility-promoting region of laminin. We conclude that initial neural crest cell migration on fibronectin occurs primarily through an interaction with the RGDS site within the cell-binding domain, whereas other potential attachment/motility-promoting sites may act to stabilize cell-fibronectin linkages. Neural crest cell migration on laminin is primarily mediated by the E8 domain. The efficiency of this domain as well as the ability of other potential motility-promoting domains to stimulate cell movement may be influenced by the association of laminin with other extracellular matrix molecules. o 1989 Academic Press, Inc.

INTRODUCTION

pathways of the neural crest is consistent with the possibility that these matrix molecules are involved in the The extracellular matrix can serve as a substrate for process of migration (Newgreen and Thiery, 1980; Duneural crest cell migration and may provide contact band and Thiery, 1987; Krotoski, et al, 1986). Furtherguidance for the directionality of cell movement, as more, injection in ouo of antibodies or synthetic pepsuggested by the presence of oriented matrix structures tides that perturb interactions between cells and fibrocontaining specialized cell-matrix linkages in viva nectin or laminin matrices interferes with cranial along neural crest migratory pathways (Liifberg et al., neural crest morphogenesis (Boucaut et al, 1984; Bron1980; Brauer and Markwald, 1988; Newgreen, 1989). ner-Fraser, 1985; Bronner-Fraser and Lallier, 1988), Maturation of the extracellular matrix surrounding the suggesting that these molecules are required for normal neural crest has been shown to initiate cell movement migration. (Lofberg et ab, 1985, 1989). The spatiotemporal distriIt is well-documented that cultured neural crest cells bution of extracellular matrix molecules like fibronecmigrate extensively on both fibronectin and laminin tin and laminin in both cranial and trunk migratory (Newgreen and Erickson, 1986; Perris and BronnerFraser, 1989). Recent in vitro studies in the axolotl have ’ To whom correspondence should be addressed. 0012-1606189$3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

222

PERRIS, PAULSSON, AND BRONNER-FRASER

shown that the extent of neural crest cell migration on fibronectin is proportional to the substrate concentration of the molecule. In contrast, cell dispersion on laminin is maximal at relatively low concentrations and is progressively reduced at higher concentrations (Perris and Johansson, 1987). It has been suggested that neural crest cells interact with the cell-binding domain of fibronectin (Boucaut et al., 1984) via the Arg-GlyAsp-Ser (RGDS) sequence common to a number of extracellular matrix molecules. Other regions of the molecule, however, such as the principal heparin-binding (II) near the C-terminus and the CSl cell adhesive site within the alternatively spliced type III-connecting segment, may act synergistically with the cell-binding domain (Perris and Johansson, 1987; Dufour et ah, 1988). Previous investigators (Bilozur and Hay, 1987) have reported that neural crest cell dispersion within reconstituted basement membrane matrices abundant in laminin can be inhibited by a synthetic peptide GlyTyr-Ile-Gly-Ser-Arg (YIGSR), corresponding to a postulated tumor cell-binding sequence in the laminin molecule (Graf et al, 1987). On the basis of this finding, they proposed that neural crest cells could interact with the central region of the laminin molecule during migration in vitro. The cells’ interactions with numerous extracellular matrix molecules are mediated by low affinity surface receptors, which are members of the integrin family of transmembrane glycoproteins (Hynes, 1987; Ruoslahti, 1988). In avians, integrin receptors having a common p1 subunit linked to, as yet unknown, (Ysubunits seem to be involved in cell adhesion to fibronectin and laminin (Horwitz et al., 1985; Bronner-Fraser, 1985; Lallier and Bronner-Fraser, 1990). In addition to integrins, a variety of cell types utilize cell surface-associated heparan sulfate proteoglycans to attach to the extracellular matrix (Lark et ak, 1985; Couchman et al, 1988; Mugnani, et ah, 1988; Saunders and Bernfield, 1988; Vallen et ah, 1988). Although substantial information has been compiled regarding the interaction of neural crest cells with fibronectin, the detailed nature of this interaction remains to be elucidated. Similarly, the cell surface receptors and recognition sequences involved in neural crest cell binding to laminin are unknown. In the present study we have examined the molecular interactions of neural crest cells with human plasma fibronectin and EHS tumor laminin during their initial migration from the neural tube in vitro. We have employed synthetic peptides as probes for receptor specificity and proteolytic fragments of fibronectin or laminin for the localization of motility-promoting sites within the glycoproteins. In addition, heparin and platelet factor 4 were employed, respectively, as a broad spectrum competitor

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Crest Cell Migration

and simulator of heparan sulfate-protein interactions. Our results are consistent with the idea that multiple adhesive/motility-promoting mechanisms operate for the regulation of neural crest cell migration on fibronectin and laminin. We infer that these mechanisms involve recognition of attachment/motility-promoting sites by specific receptors and by cell surface-associated heparan sulfate proteoglycans. MATERIAL

AND

METHODS

ExtraceZZuZarMatrix Molecules and Synthetic Peptides The matrix molecules used in this study were obtained as follows: human plasma fibronectin and collagen type IV from Collaborative Research, Inc.; heparin (bovine lung) from Sigma; four proteolytic fragments of human plasma fibronectin, 105,40,70, and 31 kDa (Fig. l), from Dr. Staffan Johansson (Biomedical Center, Uppsala, Sweden). These fragments were generated according to previously described procedures (Perris and Johansson, 1987; Woods et aZ.,1986). Two adjacent subfragments of the cell-binding fragment, one 11.5 kDa and one 50 kDa, were purchased from Telios Pharmaceuticals and Research Products (San Diego, CA). The fragments were generated by pepsin digestion of the 120-kDa cell-binding fragment. The 11.5-kDa fragment is known to correspond to a minimal RGDS-containing, fibronectin-derived fragment supporting significant cell attachment, whereas the 50-kDa fragment corresponds to the remaining portion of the arbitrarily defined cell-binding domain (65-kDa) (Pierschbacher et ah, 1981, 1982). The latter fragment also contains an antigenic site recognized by the 4B2 monoclonal antibody which is common for the 50-kDa fragment and the 45-kDa collagen-binding fragment (Pierschbacher et al, 1981). EHS mouse tumor laminin free of nidogen (Dziadek et ah, 1985) was obtained from Dr. Rupert Timpl (Max-Planck Institut fur Biochemie, Martinsried, West Germany). Synthetic peptides with the amino acid sequence GYIGSR-NH2 were obtained from Drs. Hynda Kleinman (National Institute of Dental Research, NIH, Bethesda, MD) and Staffan Johansson. Synthetic peptides containing the sequences GRGDSPASSK, with both L and D enantiomers of the Arg and Ser residues, GRGESPASSK, GRGDSP, GRGDTP, GRGDNP, GRADSP, and RGD-NH2 were kindly provided by Dr. Michael Pierschbacher (Cancer Research Foundation, La Jolla, CA) and purchased from Telios Pharmaceuticals and Research Products (San Diego, CA). A laminin-nidogen complex (1:l ratio) was isolated from the EHS mouse tumor according to previously published procedures (Paulsson et aa, 1987; Paulsson, 1988). Proteolytic fragments of the EHS tumor laminin

224

VOLUME1361989

DEVELOPMENTALBIOLOGY fibrin / heparin I

collagen

heparid

cell

IUCS

fibrina

COOH

NH2 L

/ L

+

4

CSl

RGDS

7OkDa

\ _-

40kDa

/

105kDa ----50 kDa

L__ 31 kDa

11.5 kDa

FIG. 1. Schematic illustration of human plasma fibronectin. The type I, II, and III homologies are illustrated collectively for the two chains of the molecule. The approximate localization of the characterized domains, fibriniheparin-binding I (29 kDa), collagen-binding (45 kDa), cell-binding (65 kDa), heparin-binding II (27 kDa), fibrin-binding II (23 kDa), and the alternatively spliced type III connecting segment (IIICS) are indicated by dashed bars (not in scale) on the top according to the conventional determination on the basis of enzymatic cleavage of the intact molecule (Skorstengaard et oL, 1986). The RGDS sequence within the cell-binding domain occurs between residues 1493-1496 (Pierschbather et al, 1982). Within this domain, the presence of a synergistic, 35- to go-amino acid-long binding site has recently been discovered, over 200 NH,-terminal amino acids away form the RGDS sequence (Obara et al., 1988). There is also a stipulated adhesive site (CSl) recognized by neural crest cells (Dufour et al, 1988) within the alternatively spliced region. The approximate extension (not in scale) of the proteolytic fragments used in this study is shown below the molecule by brackets with corresponding molecular mass designations. The fragments correspond to the following residues: 105 kDa = 692-1593 (McDonald et al, 1987); 70 kDa = l-599 (Skorstengaard et al., 1984); 40 kDa = 261-599 (Skorstengaard et uL, 1984); 31 kDa = 1595/1600-1867 (Skorstengaard et al., 1986; McDonald et al, 1987); and 11.5 kDa = 1416-1524 (Pierschbather et al., 1982).The amino acid sequence of the 50-kDa fragment has not been determined but it is known that the fragment comprises the N-terminal portion of the 65-kDa cell-binding domain adjacent to the 11.5-kDa fragment (Pierschbacher et al, 1981).

(Fig. 2) were generated by differential enzymatic di- Solid-Phase Binding Assays gestions of laminin as previously described (Ott et ab, Binding of various intact extracellular matrix compo1982; Paulsson et al, 1985; Aumailley et aZ.,1987). These nents and their proteolytic fragments to the plastic of fragments comprise El’ (530 kDa), Pl (290 kDa), E3 (50 culture dishes was assessed by using direct solid-phase kDa), E8 (280 kDa), and E4 (70 kDa). Nidogen and the binding assays involving biotinylated molecules (Perris low density heparan sulfate proteoglycan (LDPG) were and Johansson, 1987). The binding assays included (a) purified from the basement membrane matrix of the measurement of the relative binding of laminin, lamiEHS tumor according to published protocols (Paulsson nin-nidogen, El’, E8, nidogen, collagen type IV, and et al, 1986, 198’7;Paulsson, 1988). LDPG to untreated or polylysine-coated tissue culture Purified platelet factor 4 (PF4) was kindly obtained plastic; (b) measurement of the relative binding of lamfrom Dr. Daniel Carson (Department of Biochemistry inin-nidogen to a plastic surface precoated with deand Molecular Biology, University of Texas System creasing concentrations of nidogen; and (c) measureCancer Center, Houston, TX). ment of the binding of laminin-nidogen to collagen type IV or LDPG previously immobilized onto the plastic. Antibodies A modification of the previously published protocol Antisera against the laminin domains El’, E4, and E8 (Perris and Johansson, 1987) consisted of the use of peroxidase instead of strepwere raised in rabbits according to previously published streptavidin-horseradish procedures (Ott et ah, 1982; Edgar et al., 1984, 1988; tavidin-/!?-galactosidase. Wells were incubated with 200 peroxidase complex Paulsson et ab, 1985; Aumailley et aZ.,1987). The speci- ~1 of the streptavidin-horseradish diluted 1:2000 in 50 mM HCl-Tris buffer, pH 8.0, conficity of such antibodies for the corresponding laminin taining 150 mM NaCl, 20 mM CaClz, and 0.1% BSA. domains has previously been demonstrated by inhibition assays involving radioactively labeled ligands. Subsequently, the wells were washed and incubated with 200 ~1 of a precooled substrate for the peroxidase Comparative ELISA indicated that the particular batches of antisera used in this study bound 8-128 times enzyme (Ngo and Lenhoff, 1980) for 15 min at room more efficiently to the appropriate antigen than to temperature under vigorous shaking. The substrate was fragments corresponding to other domains of the lami- produced by mixing two solutions, at a ratio of 1:30, nin molecule (data not shown). The efficiency of the containing the following reagents: (I) 500 mg S-dimethantisera in blocking the adhesive sites within the cor- ylaminobenzoic acid in 300 ml of a 0.1/0.2 M citrate/ responding domains has been asserted in a variety of phosphate buffer, pH 7.2; and (II) 1.4 mg/ml 3-methylprevious experiments on cell-substrate attachment and 2-benzothiazolinone hydrazone-HCl in H,O. To this neurite outgrowth (Aumailley et ak, 1987; Edgar et all, mixture 0.0036% H202 and 1 mM EDTA were added. The enzymatic reaction was interrupted by addition of 1984,1988).

PERRIS, PAULSSON,AND BRONNER-FRASER

ET,--e.!,

FIG. 2. The EHS tumor laminin, in a complexed form with nidogen, is depicted in its characteristic cross-shape composed of three coiledcoil a-helical and cysteine-rich subunits, A, Bl, and B2, with eight globular structures (Sakai et aL, 1988). An RGD sequence (arrow) is found at residues 1123-1125. The stipulated cell-attachment/chemotactic peptide of the Bl chain corresponds in its long form to the sequence CDPGYIGSR, within residues 925-933 (Graf et al, 1987). The approximate extension of fragments El’-E8 is indicated by brackets with corresponding designations. The E8 fragment (280 kDa) comprises 226 residues from the Bl chain, 246 residues from the B2 chain, and 223 residues from the A chain. The El’ (530 kDa) fragment is illustrated by the large boxed area and includes the entire short arms of the A and B chains and a portion of the short arm of the Bl chain. The E3 fragment (50 kDa) corresponds to two of the five globules forming the large heparin-binding complex at the N-terminus. The Pl fragment (290 kDa; small boxed area) corresponds to the central portion of the molecule (Aumailley et al., 1987). The E4 (70 kDa) fragment corresponds to the distal arm of the Bl chain adjacent to the El’ fragment. Nidogen is structured by EGF-like repeats and cysteine-rich regions with two end globules. One RGD sequence (arrow) occurs in central domain II of the molecule (Durkin et al, 1988; Mann et aL, 1989).

50 pi/well 1 N HCl. The optical density of the indamine dye that formed under catalytical influence of the peroxidase was estimated using a microplate reader (BioTee Instruments) equipped with an interference filter for light of 600 nm wavelength. Binding assays involving substrates with different relative proportions of nidogen and the laminin-nidogen complex were carried out by coating wells with 0.01-10 pg/ml nidogen followed by 20 pg/ml biotinylated laminin-nidogen complex. This coating concentration of laminin-nidogen was chosen because it corresponded to that giving optimal neural crest cell dispersion in our cell migration assays. Areas of the well surface that remained uncovered by any of the two mol-

Neural Crest Cell Migration

225

ecules were blocked with 2% BSA/l% ovalbumin. The relative binding of the biotinylated molecules to the plastic was determined as described above. Control wells were coated with each of the biotinylated matrix molecules alone. Incubation with 1% ovalbumin after the first coating abolished binding of the second molecule, indicating that the laminin-nidogen complex did not bind to isolated nidogen. Binding of laminin-nidogen to collagen type IV or LDPG previously immobilized onto plastic was determined as follows. Microtiter plates were coated with collagen type IV (10 Fg/ml) or LDPG (10 pg/ml) as described above and subsequently incubated with ovalbumin. Plates were then incubated overnight at 4’C with 0.1-1000 pg/ml of biotinylated laminin-nidogen dissolved in PBS and further processed as in other assays. Control wells were incubated in the first coating step with one of the following components: buffer, blocking solution, or each of the nonbiotinylated ligand molecules, instead of LDPG or collagen type IV. In the second coating, control wells with buffer were incubated with the biotinylated ligands or, following coating with the blocking solution, with biotinylated laminin-nidogen. Direct competition assays were carried out by applying a mixture of biotinylated and nonbiotinylated laminin-nidogen in various proportions to collagen type IV or LDPG substrates. The mixtures of biotinylated/nonbiotinylated laminin-nidogen were diluted to the appropriate coating concentrations in 0.05 M Tris-HCl with 0.15 mM NaCl/O.Ol M phosphate buffer, pH 7.4. The procedure for the coating was identical to that used for other assays, and binding of the biotinylated laminin-nidogen was monitored as described above. Culture Substrates Culture substrates containing the various matrix molecules to be tested were prepared in 35-mm tissue culture dishes (Nunc, Denmark) according to previously published procedures (Perris and Johansson, 1987). Fibronectin was used at a coating concentration of 100 pg/ml, which has previously been shown to allow maximal neural crest cell movement under serum-free conditions (Perris and Johansson, 1987). However, in our serum-free assays, lower coating concentration of fibronectin (10 pg/ml) supported quail neural crest cell movement to comparable extents (data not shown). Collagen type IV supports maximal dispersion at 10 pg/ml coating concentration (Perris and BronnerFraser, in preparation). Mixed substrates of nidogenlaminin-nidogen, and substrates of laminin-nidogen coupled to polylysine, collagen type IV or LDPG were produced by a two-step coating as described for the

226

DEVELOPMENTALBIOLOGYV0~~~~136,1989

solid-phase binding assays. On the basis of results from these assays, ligand molecules and laminin-nidogen were used at 10 and 200 pg/ml, respectively, which correspond to their optimal binding concentrations. Areas of the plastic that remained uncovered by the matrix molecules were blocked with 2% BSA/l% ovalbumin solution. This treatment allows neural tube attachment to the substrate, but prevents nonspecific migration of neural crest cells as demonstrated by the inability of the cells to locomote on plastic coated with blocking solution alone (Perris and Johansson, 1987; Newgreen, 1989; data not shown). In a set of experiments, laminin-nidogen substrates were incubated overnight at 4°C with antisera to El’, E4, E8, or a combination of El’ and E4 fragments, following blockage with BSA/ovalbumin. The antibodies were used at dilutions of l:lO-1800 (‘7.6-0.29 mg/ml) in PBS containing 0.1% BSA. After the overnight incubation, the antibody solution was removed and the substrates were extensively rinsed. Substrates with parallel tracks of two different molecules were generated according to previously published procedures (Perris and Johansson, 1987). Cell Culture and Assessment of Cell Migration

Quail neural crest cell cultures were prepared according to previously published procedures. Neural tube-neural crest explants were grown for 16 hr in serum-free medium containing 0.1% ovalbumin. In some “recovery experiments,” the medium containing the synthetic peptides was replaced with serum-free or serum-supplemented medium with or without heparin after the 16 hr of culture, and the cells were further incubated for 6-12 hr. All explants were photographed at time of evaluation. The extent of neural crest cell dispersion from the neural tube after 16 hr of culture was assessed using the Sigmascan (Scientific Measurement System; Jandel Scientific) computer program for morphometric analysis. Phase-contrast negatives (24 x 35 mm) of explants to be analyzed were magnified 123.5 times, and the contours of the neural tube explant and neural crest cell outgrowth (Fig. 3) were traced on 28 X 43-cm paper sheets. The sheets were then oriented on a digitizing pad (Model 2210-0.43.C; Jandel Scientific) and the area occupied by the migrated neural crest cells was traced with a cursor. Area calculations and most of the statistical analyses were accomplished using functions provided by the Sigmascan program. Migration of the neural crest cells from the neural tube occurred initially from the dorsal side of the explant, but later cells could be observed on the ventral side as well. This resulted in an elliptic pattern of outgrowth, with the predominant portion of the cells on the

dorsal side of the neural tube explant (Fig. 3). The extent of ellipticity of the neural crest cell outgrowth varied according to the length and shape of the neural tube explant. We chose to assess the neural crest cell outgrowth fronting the dorsal edge of the neural tube in explants measuring 960-1370 pm in length. When comparing the extent of migration on two different substrates we established the probability limit for statistical significance for difference versus equality at P < 0.005 within a 95% confidence interval, according to Student’s t tests. RESULTS Domain Preference on Fibronectin

of Neural

Crest Cell Migration

Under serum-free conditions, quail neural crest cell migration on substrates composed of the 105-kDa cellbinding fragment (which embodies the defined 65-kDa cell-binding domain) was as pronounced as that on intact fibronectin (Fig. 4). In contrast, the 11.5-kDa subfragment encompassing the RGDS sequence supported migration to about 65% of that observed on intact fibronectin. Moreover, the adjacent 50-kDa subfragment from the cell-binding domain was unable to support neural crest cell movement (Fig. 4). When neural tubes were explanted onto two adjacent tracks composed of the 11.5- and 50-kDa subfragments, migrating neural crest cells were observed exclusively along the tracks containing the 11.5-kDa subfragment (Fig. 5A). Furthermore, addition of the soluble 50-kDa subfragment (at molar equivalents corresponding to 9 mg/ml fibronectin) did not alter migration on the 105-kDa cellbinding fragment. Proteolytic fragments encompassing the major heparin-binding domain II (31-kDa fragment), the collagenbinding domain (40-kDa fragment), and both the collagen-binding and fibrin/heparin-binding I domains (70kDa fragment; Fig. 1) also supported migration at 65, 35, and 39%, respectively, of that observed on intact fibronectin (Fig. 4). The absolute extent of dispersion on the isolated fibronectin fragments decreased in the order 105 kDa > 11.5 kDa > 31 kDa > 70 kDa > 40 kDa > 50 kDa, where equivalence indicates P > 0.005. The relative binding of intact fibronectin and its proteolytic fragments to plastic has been determined in a previous study (Perris and Johansson, 1987). Eflects of RGD Peptides and Heparin on Neural Cell Migration on Fibronectin and Its Proteolytic Fragments

Crest

Neural crest cell migration on the intact fibronectin molecule was blocked by a synthetic decapeptide con-

PERRIS, PAULSON, AND BRONNER-FRASER

Neural

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Crest Cell Migration

FIG. 3. Phase-contrast micrographs of representative elliptic neural crest cell outgrowth on fibronectin under serum-free conditions. The predominant area of outgrowth chosen for morphometric quantitation of neural crest cell dispersion is represented by the boxed area. X51. NT, neural tube.

taining the RGDS sequence, with a concentration of 150 PMyielding 54% inhibition (Fig. 6a). The same concentration of peptide was even more effective (81%) in inhibiting migration of the 105kDa fragment encompassing only the cell-binding domain (Fig. 6a). Substitutions in the RGDS-containing peptide of the Ser residue with Thr or Asn resulted in a significantly reduced ability to inhibit neural crest migration compared to that of the parent peptide (Fig. 7). The efficiency of inhibition was reduced by 4.6 for GRGDNP and 5.5 for GRGDTP (P < 4.5 X 10m5).Moreover, the small RGD-NH2 peptide lacking the Ser residue was >30 times less efficient than the hexapeptide encompassing the complete RGDS sequence (Fig. 7). The importance of correct positioning of the Arg, Gly, and Asp residues within the tetrad has been demonstrated previously (Boucaut et al., 1984, Pierschbacher and Ruoslahti, 1987; Dufour et al, 1988, Hautanen et al, 1989) and was confirmed here by substitution of Glu for Asp and Ala for Gly which resulted in completely ineffective peptides (Fig. 8). Enantiomeric substitution of the Ser or Arg residues did not significantly affect the inhibitory activity of RGDS-containing peptides (P = 0.414 and 0.02, respectively; data not shown). We used high mo1ecu1ar weight

competitive inhibitor

heparin

of the interactions

as a potential

between cell

surface heparan sulfate proteoglycans and the heparinbinding domains of fibronectin and laminin. Addition of heparin to neural crest cells migrating on intact fibronectin did not affect their locomotory ability. How1.500

T % g p g p t

0 KI I E9

1.200

NO addition RGDS (430 PM) Heparin (200 pg/ml) 50 kLM1 mglml)

o.900 0.600

0.300 0.000 Intact FN

105

11.5

50

70

Size of fragment

40

31

(kDa)

FIG. 4. Effects of an RGDS-containing decapeptide peptide and heparin on neural crest cell migration on proteolytic fragments of fibronectin. Bars indicate mean values with corresponding standard errors from a total of 11-32 neural crest explants examined per experimental condition. The synthetic decapeptide and heparin were added at the start of the culture at maximal inhibitory concentrations (430 pM and 200 /*g/ml, respectively). In one set of experiments, a soluble 50-kDa fragment was added at the given concentration to neural crest cells plated on a substrate of a 105-kDa cell-binding fragment.

FIG. 5. (A) Tracks composed of the 11.5-kDa, RGDS-containing fragment from the cell-binding domain of fibronectin on a background substrate of the 50-kDa fragment from the adjacent N-terminal portion of the cell-binding domain. The phase-contrast micrograph shows that only the 11.5-kDa fragment can support locomotion. X210. (B) Neural crest cell migration on a substrate of laminin-nidogen containing maximal amounts of the complex adsorbable onto plastic. X70. (C) Neural crest cell migration on similarly high concentrations of laminin-nidogen coupled to LDPG. X70. (D) Lack of neural crest cell migration on isolated nidogen. X70. (E) Limited neural crest cell migration along delimited paths of El’ fragment on an E4 substrate. X90. (F) Parallel paths composed of the E8 and Pl laminin fragments; neural crest cells locomote exclusively on the E8 fragment. X200. (G) Representative neural crest cell dispersion on a substrate of the E8 fragment. X70. (H) Inhibition of neural crest cell migration on the E8 fragment by addition of heparin. X70.

PERRIS, PAULSSON,AND BRONNER-FRASER b 6lFN I 105 kDa

%

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1 so0

[23 FN I 105 kDa

1.200

& sE

0.900

e m 2

0.600

% h:

o-0.02

0.15

Peptide

1.5

15.0

concentration

150.0

0.300

0

430.0

10 Concentration

&M)

200

50 heporin

&g/ml)

FIG. 6. Combinatory effect of RGDS peptides and heparin on neural crest cell migration on fibronectin and the cell-binding 105-kDa fragment (b). The effect caused by addition of RGDS peptides alone is also indicated for comparison (a). Heparin was added at the indicated concentrations in conjunction with 150 WM of peptide causing >50% inhibition on intact fibronectin and 81% inhibition on the 105kDa fragment (a). The mean values with corresponding standard errors were derived from 14-20 explants per data point.

ever, heparin in conjunction with RGDS peptides at a concentration of 150 PM, corresponding to that causing more than 50% inhibition of movement (Fig. 6a), resulted in a reduction of migration 41% greater than that caused by addition of the peptides alone (Fig. 6b). In contrast, simultaneous addition of heparin and RGDS peptides to neural crest cells migrating on the 105-kDa fragment resulted in an inhibition indistinguishable from that observed with the peptides alone (Fig. 6b). The inhibitory effects of RGD-containing peptides on neural crest cell migration were reversible. Removal of the peptide solution after 16 hr of culture restored the migratory ability of the cells up to ‘71%, within 6 or more hr of incubation (Fig. 8). Inclusion of heparin in the culture medium did not alter the rate or extent of recovery, suggesting that restoration of migration did not occur prevalently through an interaction with the heparin-binding domain(s). RGD-containing peptides did not alter irreversibly the ability of neural crest cells to proliferate since replacement of the peptide-containing medium with serum-supplemented medium induced pronounced cell division (data not shown). Six hours after plating, neural crest cells on fibronectin substrates retained their susceptibility to RGDS peptides (Fig. 8); the peptides arrested migration and caused a progressive detachment of the cells from the substrate. Under these conditions, the number of dispersed neural crest cells was reduced by >60% (as assayed by cell counts of 20 explants examined by morphometric analysis). RGDS peptides did not significantly affect migration on the 31-, 70-, or 40-kDa proteolytic fragments of fibronectin (~10%; P = 0.015-0.598; Fig. 4). In contrast, addition of heparin to the medium specifically blocked

quail neural crest cell movement on the 31-kDa heparin-binding fragment. Addition of heparin to neural crest cells plated on the 105-, 70-, and 40-kDa fragments resulted in minimal disturbance of their locomotory ability (6-14%; P = 0.008-0.235; Fig. 4). Diflerential Cell Migration on Isolated Laminin and Complexed Forms of Laminin Avian neural crest cells dispersed more extensively on low substrate concentrations of isolated laminin than on higher concentrations. Migration was reduced by 38% (P = 1.5 X 10m7)at maximal substrate-bound

1.500

l -. GRGDSP - 0 GRGDTP Q- .. 0 GRGDNP

q

1.200

0.900

T

--

0.600 --

0.300

--

10.0

Peptide

100.0

concentration

(#J)

FIG. 7. Inhibition of neural crest cell migration by addition of RGD peptides with various compositions. The mean values plus standard errors were derived from analysis of 13-24 explants. The dose-dependent perturbation of neural crest cell migration caused by the prototype peptides is included for direct comparison. Enantiomeric substitutions of the RGDS key amino acids in alternative peptides had little or no effect on the efficiency of the peptides to perturb cell movement when compared to the parent peptide (data not shown).

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DEVELOPMENTALBIOLOGY

1.200 5 E

0.900

B :

0.600

% uo 2

0.300

0.000 Control

RGDS

RGDS

RGDS

a~t~rreco”ery

RGDS

RADS

recovery +

RGES

Heparin

Heparin

FIG. 8. Effects of synthetic peptides and heparin on neural crest cell migration on fibronectin. Each bar represents mean values with corresponding standard errors for 13-20 explants examined. Unless indicated synthetic peptides and heparin were added at the start of the culture. Control, no addition; RGDS, addition of maximal inhibitory concentration of 430 pM (see Figs. 4 and 6) of a decapeptide comprising the cell attachment sequence; RGDS after 6 h, addition of the same concentration of peptide 6 hr after plating of the cells; RGDS recovery, replacement of the peptide-containing medium after 16 hr of culture and further incubation of the cells for >6 hr; RGDS recovery + heparin, same as in RGDS recovery, but in the presence of 200 pg/ml heparin; RADS and RGES, deca- and hexapeptides known to be inactive and used as controls.

laminin (200 pg/ml coating concentration; Fig. 9) when compared with the amount adsorbed onto plastic at a lOO-fold-lower coating concentration (Figs. 9 and 10a). A laminin-nidogen complex was similarly dose-dependent in its ability to promote movement, but was found to be 34% (P = 1.8 X lo-*) more favorable for neural crest cell dispersion at the highest substrate-bound concentration (200 pg/ml coating). In addition, at optimal migration-stimulatory concentration (20 pg/ml coating), laminin-nidogen was 39% (P = 4.2 X 10e6) more efficient as a migratory substrate than were equimolar amounts of pure laminin (Figs. 9 and 10a). According to our binding assays the adsorbance capacities of laminin and the laminin-nidogen complex to plastic were comparable (Fig. lOa), suggesting that this apparent increase in motility-promoting ability was not due to differential incorporation of laminin-nidogen into the substrate. Furthermore, the difference in molecular weight between isolated laminin and the complex is <20%, which excludes the possibility that a discrepancy in molecular size could be responsible for the differential effect on migration. Precoating of culture dishes with polylysine did not alter the relative amount of laminin-nidogen complex that became bound to the substrate (Fig. 10). Laminin-nidogen attached to polylysine, however, was a 50% less efficient migratory substrate than laminin-nidogen adsorbed directly onto the plastic (Fig. 11).

VOLUME136,1989

Isolated nidogen was unable to support neural crest cell migration (Table 1; Fig. 5D), despite its ability to promote attachment of certain cell types through an RGD sequence (Mann et ah, 1989). Substrates with relative proportions of nidogen and the laminin-nidogen complex were produced to simulate a situation where cells are confronted with a substrate containing laminin and nidogen in a ratio different than that provided by the specific laminin-nidogen complex employed. Augmenting the nidogen concentration proportionally reduced, but did not eliminate, the ability of the laminin-nidogen-containing substrates to support migration. In fact, at maximal concentrations of nidogen in the substrate (Fig. lob), the extent of neural crest cell dispersion still reached 42% of the maximal dispersion on the laminin-nidogen complex alone (Fig. 9). Solid-phase binding assays indicated that, at the highest coating concentration (200 pg/ml), similar amounts of laminin-nidogen bound to untreated plastic versus to collagen type IV or LDPG preimmobilized onto the plastic (Figs. 10a and 12a). Binding of biotinylated laminin-nidogen to collagen type IV or LDPG could be competed dose-dependently by nonbiotinylated laminin-nidogen (Fig. 12b). Moreover, preincubation of laminin-nidogen with an excess of collagen type IV or LDPG (about 1:2 in molar ratio) reduced the binding of laminin-nidogen to the corresponding ligand by >80% (data not shown). Coupling of 200 /*g/ml of lamininnidogen to collagen type IV or LDPG enhanced neural crest cell migration by 43% (P = 1.3 X 10-13)and 50% (P = 8.4 X 10-13), respectively, compared to 200 pg/ml of the complex bound directly to plastic (Figs. 11 and 5B-5C). In these cases, the extent of cell dispersion was comparable to that observed at the strongest migra-

0-o .-

.

o.-.

0 ni*ogenLN-N

P’

0.000 : d 0.01

LN N-N (2Opghl)

T

‘_‘. P ‘.

, 0.1

Cooting

1.0

10

concentration

bg/ml)

100

FIG. 9. Dose-dependent neural crest cell migration on isolated laminin (LN), the Iaminin-nidogen complex (LN-N), and a substrate containing different proportions of isolated nidogen and the laminin-nidogen complex (see Fig. 10). Each data point denotes mean values from analysis of 14-38 neural crest explants.

PERRIS, PAULSSON,AND BRONNER-FRASER 1.200

T

b

New-al

1.2 -

231

Crest Cell Migration

IJ-

Nidogen.

- q

0 -0

Nii

+ LN-t+ (20 fig/ml)

I

l.O-

5

--__

--

,’

--A

8’ 0.6 -

0.600 -0.400 --

,’ ,’

.-...LN

.-

v LN-N - m palylysine + LN-N Q--O El-4

0.200 --

0.000-I 0.03

n

iyyyy

,

n-0 ES

0.10

1 .oo

Cooting

concentration

10.00

100.00

0.01

Coating

(pg/ml)

10.0

1.0

0.1

concentration

nidogen

100.0

kg/ml)

FIG. 10 (a) Binding of biotinylated laminin (LN), laminin-nidogen (LN-N), and El’ and E8 laminin fragments to untreated and polylysinecoated plastic. The values for each curve are means of triplicates and error bars indicate the standard deviation. (b) Binding of laminin-nidogen (LN-N), applied at 20 rig/ml, to plastic precoated with increasing concentrations of isolated nidogen. The corresponding binding of nidogen to plastic is also given. Asterisk denotes biotinylated molecules. Values represent means of triplicates plus standard deviations. Laminin-nidogen did not bind to isolated nidogen as demonstrated by the lack of detectable enzymatic activity when, following coating with nidogen, the available sites on plastic were saturated with ovalbumin. Similar curves for the binding of laminin to plastic have been observed using radioactively labeled laminin (Lallier and Bronner-Fraser, 1990). Moreover, detachment of laminin-nidogen over the period of the migration assay was less than 20% (data not shown).

tion-permissive coating concentration (20 pg/ml) of laminin-nidogen bound to plastic (P = 0.012-0.037; Fig. 11). Isolated LDPG was unable to stimulate migration and the extent of cell movement on collagen type IV alone was comparable to that observed on laminin-nidogen bound to plastic (Table 1). Migration on laminin-nidogen was not affected by addition of RGDS-containing peptides or a peptide including the YIGSR sequence derived from the Bl chain of laminin (Fig. 11). Heparin inhibited neural crest cell migration on laminin-nidogen dose-dependently, with a maximal reduction of 35% (P = 7.1 X 10-14;Fig. 13).

(Fig. 2) since the proteolytic subfragment Pl encompassing this region was entirely nonpermissive as a migratory substrate (Table 2; Fig. 5F). The E4 fragment corresponding to the distal portion of the short arm of the Bl chain (Fig. 2) was similarly unable to support cell movement (Table 2). When encountering alternate lanes coated with the El’ or E4 fragment, neural crest

1.200 5

Laminin Domains Involved in Neural Crest Cell Migration Neural crest cells migrated poorly on substrates containing the proteolytic El’ fragment (Table 2; Fig. 5E) of laminin which has been postulated to comprise a heparin/cell-binding site (Fig. 2) (Charonis et ak, 1988). The poor migration on the El’ fragment was not due to an effect dependent on substrate concentration as in the case of intact laminin since a lo-fold-lower coating concentration generated an entirely ineffective substrate. When estimated at molar equivalents after adjustment for the relative binding of the El’ fragment and the laminin-nidogen complex to plastic, the El’ domain of laminin accounted for less than 10% of the migration on the intact molecule. Moreover, the weak motility-promoting ability of the El’ fragment was independent of the central region of the laminin molecule

s

0.900

El b 2

0.600

% 0 e a

0.300

W-N coating

Polyiysine LN-N

Cd IVLN-N

LDPCLN-N

cont. !A-N: 200 w/ml

LN-N

IN-N

+

IN-N+

RODS nc9R coating cont. LN-N: 20 &ml

FIG. 11. Neural crest cell migration on laminin-nidogen (LN-N) alternatively coupled to polylysine, collagen type IV (Co1 IV), or the low density heparan sulfate proteoglycan from EHS (LDPG). The migration observed on laminin-nidogen adsorbed directly onto plastic at the corresponding coating concentration (200 pg/ml; 25 rg/cm’) is included for comparison. Addition of RGDS or YIGSR peptides did not reduce the neural crest cell migration observed on laminin-nidogen at its optimal motility-promoting coating concentration (20 pg/ml; 2.5 pg/cm’). A total of 11-38 explants was examined for each experimental case.

232

V0~~~~136,1989

DEVELOPMENTAL BIOLOGY

crest cell migration by about 20% (P = 2.8 X 10e5). Presumably, this alteration was due to nonspecific interactions, rather than to a synergistic effect of the two antisera, since a threefold dilution of the antibody mixMolecule No. of Area of outgrowth ture abolished their inhibitory action. (coating concn) explants (mm2)a Comparison at molar equivalents after correction for LDPG 19 0.019 + 0.006 the differential binding of the El’ and E8 fragments to (10 r&ml) plastic indicated that the E8 fragment was >4 times Nidogen 25 0.056 f 0.014 more efficient than El’ in supporting neural crest cell (10 e/ml) migration. The motility-promoting activity of the E8 Collagen type IV 22 0.709 f 0.202 (10 dml) domain did not appear to involve the terminal, heparin-binding region of the A chain since the E3 fraga Values are means f SD. ment, comprising that portion of the laminin molecule (Fig. 2), was unable to support cell locomotion (Table 3). At a coating concentration yielding optimal neural cells markedly favored the El’ paths (Fig. 5E). Addition of heparin to neural crest cells plated on El’ did not crest cell migration on laminin-nidogen, only 45% of activity of laminin could be atsignificantly inhibit their dispersion (11% decrease; P the motility-promoting tributed to the E8 domain. In contrast, at a coating = 0.043). In contrast, addition of RGDS peptides comconcentration giving maximal adsorbance of lamininpletely blocked neural crest cell migration on the El’ nidogen onto plastic, and causing a corresponding defragment (Table 2). crease in cell movement, the E8 fragment was about 1.3 Neural crest cell migration on the E8 fragment was times more efficient in supporting neural crest cell disproportional to the quantity of the fragment in the persion than the intact complex. It was not possible to substrate (Table 2). Migration on E8 was also inhibited directly test the motility-promoting ability of the El’ in a dose-dependent manner by addition of heparin, which restricted dispersion up to 77% (Figs. 5 and 13). fragment in association with the potential ligands for laminin-nidogen since it did not bind significantly to The extent of neural crest cell dispersion on laminincollagen type IV or LDPG (data not shown; Mann et al., nidogen was virtually eliminated by preincubation of 1989). Neural crest cells demonstrated a strong preferthe laminin-nidogen substrates with an antiserum ence for movement on the E8 fragment versus Pl fragagainst the E8 domain (87% inhibition), which was efment when presented with adjacent tracks of the two fective at a dilution of 1:800 (Table 3). Preincubation of substrates (Fig. 5). laminin-nidogen substrates with antisera to the El’ or E4 laminin domain, at concentrations higher than those Neural Crest Cell Migration on Platelet Factor 4 used for the E8 antiserum, did not affect cell migration. Substrates of PF4 have been useful for examining the Preincubation of laminin-nidogen substrates with a mixture of the two antisera to El’ and E4 altered neural possibility that cell surface heparan sulfate proteoglyTABLE

1

NEIJRALCRESTCELLMIGRATIONON ISOLATED EXTRACELLULARMATRIXMOLECULES

a

0.7 0.6 -

b o-o q -

q

Cdl”+ LOW

0.600

LN-N. + LN-N.

1 E c

1.0

10.0

Added laminin-nidogen

100.0

@g/ml)

-

Cd I” i LN-N.

~100pg/md)lLt+N

-

LDPG + LN-N.

( lOO,did)/Lt+N

0.640 h

0.0004 0

100

300

500

Cont. non-biotinyloted

700

900

LN-N &/ml)

FIG. 12. (a) Binding of laminin-nidogen to collagen type IV (Co1 IV) or LDPG previously immobilized onto the plastic at their maximal concentrations of adsorbance (Perris and Bronner-Fraser, in preparation). Asterisk denotes biotinylated molecules. (b) Direct competition assays involving relative proportions of biotinylated and nonbiotinylated laminin-nidogen. These assays were carried out in the absence of Ca*+ and at low temperature to avoid self-aggregation of the laminin-nidogen complexes (Yurchenco et aa, 1985; Paulsson, 1988).

PERRIS, PAULSSON,AND BRONNER-FRASER TABLE 2 NEURAL CRESTCELLMIGRATIONONLAMININFRAGMENTS No. of explants

Fragment El-4 (15 rg/ml)b El-4 (1.5 pg/ml) El-4 (15 pg/ml) + heparin (200 rg/ml) El-4 (15 pg/ml) + RGDS (427.8 PM) Pl (0.1-10 rg/ml) E8 (85 pg/ml) E8 (8.5 pg/ml) E8 (0.85 pg/ml) E8 (0.085 @g/ml) E3 (10 #g/ml) E4 (10 pg/ml)

Area of outgrowth (mm’)’

15 11 13

0.109 + 0.045 0.033 f 0.006 0.097 f 0.037

15

0.013 f 0.006

22 16 16 16 15 13 14

0.022 1 0.007 1.122 f 0.164 0.824 f 0.156 0.369 2~0.136 0.020 f 0.006 0.024 k 0.013 0.018 + 0.007

a Values are means + SD. bCoating concentration except for RGDS peptides and heparin that were added to the medium.

233

Neural Crest Cell Migration 1.500

0 I

T

5

LN-N ES

-T

0.900

E cn 3

0.600

% z 2

0.300

50

10

0

Concentration

heparin

lL 200

@g/ml)

FIG. 13. Dose-dependent inhibition of neural crest cell migration on the laminin-nidogen complex (LN-N) and the E8 heparin-binding fragment by addition of heparin. Mean values represented by the bars derive from a total 15-31 neural crest explants analyzed. Heparan sulfate, chondroitin sulfate isomers, dermatan sulfate, and keratan sulfate were considerably less efficient or entirely ineffective in inhibiting neural crest cell migration (data not included). DISCUSSION

cans or equivalent molecules may act as potential receptors (Mugnani et CAL,1987, 1988). Neural crest cells dispersed in a dose-dependent fashion on PF4. The extent of cell dispersion was comparable to that observed on the 31-kDa heparin-binding fragment of fibronectin and corresponded to 59% of that observed on the E8 laminin fragment (Tables 2 and 4; Fig. 4). Neural crest cell movement on PF4 was markedly inhibited by addition of heparin at a concentration significantly lower than that perturbing migration on laminin-nidogen, the E8 laminin fragment, or the 31-kDa fibronectin fragment (Table 4, Figs. 4 and 12).

The cell-binding 105kDa fragment of fibronectin, lacking the CSl adhesion site of the alternatively spliced IIICS, supported avian neural crest cell migration to an extent equal to that of the intact molecule. This result, which is in agreement with previous findings using axolotl neural crest cells (Perris and Johansson, 1987), emphasizes the importance of the RGDS attachment site and further suggests that interaction with the RGD-independent CSl site may not be primarily involved in the stimulation of migration, but rather may participate in the stabilization of the cellfibronectin linkage in stationary cells. Previous investigators have observed that CSl peptides do not alter speed of locomotion but have some effect on the persisTABLE 3 INHIBITIONOFNEURALCRESTCELLMIGRATION ONLAMININ-NIDO- tence of directional movement in neural crest cells disGENBYPREINCUBATIONOFTHESUBSTRATEWITHANTI~ERAAGAIN~T persing on fibronectin substrates (Dufour et al, 1988).

VARIOUSLAMININ DOMAINS Antiserum (dilution)

No. of explants

Anti-El’ (1:lO) Anti-E4 (1:lO) Anti-El’ + anti-E4 (1:lO) (1:30) Anti-E8 (1:lOO) (1:200) (1:800) a Values are means + SD.

Area of outgrowth (mm’)

TABLE 4 Inhibition (%I

15

0.779 k 0.123

0

17

0.970 f 0.093

0

16 12

0.767 + 0.119 0.958 t- 0.117

19.5 0

19 11 12

0.013 f 0.063 0.388 + 0.065 0.870 2 0.074

86.7 59.3 18.6

NEURALCRESTCELLMIGRATIONONPLATELETFACTOR~(PF~) Coating cone PF4 (pg/ml) 50 5 0.5 0.05 50 50 50 50

Addition heparin (dml) 200 50 10 1

a Values are means f SD.

No. of explants

Area of outgrowth (mm2)a

22 16 19 14 12 18 15 19

0.665 zk0.099 0.528 AZ0.067 0.377 _+0.093 0.036 + 0.012 0.026 + 0.010 0.300 Yk0.084 0.398 + 0.096 0.580 f 0.076

234

DEVELOPMENTALBIOLOGYV0~~~~136,1989

Consistent with previous cell attachment studies (Obara, et al, 1988) and binding assays involving isolated fibronectin receptors (Hautanen, et ah, 1989), we observed that the 105kDa fragment was considerably more effective as a migratory substrate than the 11.5kDa cell attachment subfragment containing the RGDS recognition site and then an adjacent 50-kDa fragment from the cell-binding domain could not itself support neural crest cell migration. At present, we cannot exclude the possibility that the interaction of neural crest cells with the RGDS cell attachment site could be reinforced by a synergistic adhesive site within the cellbinding domain (Obara et aZ.,1988). However, this possibility seems unlikely since an excess of soluble 50-kDa fragment comprising the proposed synergistic adhesive site was ineffective in perturbing migration on the 105kDa cell-binding fragment. Thus, as suggested by others (Hautanen et al, 1989), it is conceivable that the RGDS site alone can support maximal neural crest cell movement when contained within a fragment of sufficient size to maintain its recognizable conformation (Chierniewski et al, 1988) after adsorption onto plastic. It has been shown previously that migration of neural crest cells on fibronectin can be entirely interrupted by high concentrations of RGDS-containing peptides (Boucaut et al, 1984). However, similarly high concentrations of RGDS peptides have also been reported to accelerate the speed of movement of individual neural crest cells dispersing on fibronectin substrates in the presence of serum (Dufour et ah, 1988). We find that under serum-free conditions, concentrations of RGDS peptides considerably lower than those previously reported inhibit by more than 80% initial neural crest cell migration on fibronectin. Addition of RGDS peptides during early phases of neural crest cell movement on fibronectin markedly reduces the number of cells that migrate away from the neural tube and decreases the total distance that the cells are able to disperse. This observation further corroborates the role of the RGDS recognition sequence as the primary motility-promoting site. Receptor specificity for fibronectin versus vitronectin can be determined by stereochemistry of the Arg-GlyAsp-Ser sequence in synthetic peptides, as demonstrated by selective enantiomeric substitution of the Arg-Gly-Asp-Ser residues or by replacement of the end Ser residue (Pierschbacher and Ruoslahti, 1987). In agreement with previous observations on kidney cell attachment to fibronectin, we find that the Arg-GlyAsp sequence is required for inhibition of neural crest cell movement and that enantiomers of these key amino acids do not alter the ability of the peptides to block the avian “fibronectin receptor.” In contrast, replacement of the Ser residue with Thr, Asn, or an amide group,

which causes unaltered (Thr), stronger (Asn), or weaker (amide group) perturbation of kidney cell attachment to fibronectin (Pierschbacher and Ruoslahti, 1987), decreases the efficiency of the peptide to inhibit neural crest cell migration on fibronectin. It is likely that amino acid substitutions in the hexapeptide GRGDSP result in conformational changes of the soluble peptide, presumably by influencing its secondary structure (Reed et ah, 1988). Nevertheless, taken together these findings suggest that the fibronectin receptor of neural crest cells, in contrast to the human fibronectin receptor, has a stringent structural requirement for ligands containing the complete RGDS sequence. In addition to the cell-binding region, we find that avian neural crest cells migrate in a RGDS-independent manner on fragments corresponding to the principal heparin-binding region (Hep II) (Benecky et ah, 1988) and to a lesser extent on the collagen-binding/aminoterminal regions of fibronectin. This finding confirms the existence of at least one additional RGD-insensitive mechanism of neural crest cell interaction with fibronectin, though these sites may not be exposed in intact fibronectin. In the case of the N-terminal fragments, it is possible that binding of cell surface collagens to these fragments could activate receptor-like molecules. Alternatively, absorbance of these and other proteolytic fragments to plastic may expose potential cell-binding sites which are normally cryptic. Cell surface-associated heparan sulfate proteoglycans have been shown to participate in both initial attachment and focal contact formation in a variety of cell types exhibiting strong binding affinity for fibronectin (Stamatoglou and Keller, 1983; Lark et al., 1985; Saunders and Bernfield, 1988; Couchman et aZ., 1988, Vallen et al., 1988; Mugnani, et ab, 1987, 1988). The inhibitory effect of heparin on neural crest cell movement on the heparin-binding 31-kDa fragment is consistent with these previous findings as well as with our earlier observations in the axolotl (Perris and Johansson, 1987). Complementary binding of cell surface heparan sulfate proteoglycans to fibronectin could represent a mechanism that acts cooperatively or as an alternative to integrin receptors. Evidence for cooperative binding is provided by the observation that heparin potentiates the inhibitory effects of the synthetic peptides on intact fibronectin, but not on the 105-kDa cell-binding region which lacks significant heparin-binding activity (Benecky et al., 1988). Furthermore, the migration of axolot1 neural crest cells on intact fibronectin is partially blocked by antibodies against the cell-binding region of fibronectin, but is completely blocked by addition of antibodies against both the cell-binding and heparinbinding II domains (Perris and Johansson, 1987). Differential glycosylation of fibronectin in various embry-

PERRIS, PAULSSON, AND BRONNER-FRASER

onic regions and/or phases of development could possibly determine the availability of binding sites within the various motility-promoting domains (Jones et al, 1986), thereby influencing the mode of neural crest cell-fibronectin interaction. Overabundance of matrix molecules characteristic of basement membranes has been suggested to be inhibitory for neural crest cell migration in viva (Payette et aZ., 1988). Supporting this notion, we have previously shown that relatively high concentrations of isolated EHS tumor laminin are inhibitory for amphibian neural crest cell movement in vitro (Perris and Johansson, 1987). In the present study, we provide evidence that the differential dose-effect of this particular laminin may be due to differences in its configuration. First, laminin complexed with one of its natural ligands, nidogen (Dziadek et aL, 1985; Paulsson et cd., 1986, 1987; Paulsson, 1987,1988), displays significantly greater motility-promoting ability than laminin alone, despite the finding that nidogen itself is unable to support migration. Second, linkage of laminin-nidogen to the other potential matrix ligands, collagen type IV and LDPG, which codistribute with laminin along neural crest cell migratory pathways (Perris et ab, 1989; Duband and Thiery, 1987), markedly augmented the permissiveness of the complex as a migratory substrate. Conformational changes in the laminin molecule, plausibly induced by interactions with other molecules, may lead to exposure of covert adhesive sites or to altered affinity for already available sites. Alternatively, independent interaction of neural crest cells with motility-promoting sites on collagen type IV and LDPG could act synergistically to give an elevated rate of locomotion. In the latter case, close contact between the two motilitypromoting molecules would still be required to evoke optimal cell migration. We observed that neural crest cells were unable to utilize LDPG as a migratory substrate and that the extent of cell movement on collagen type IV and laminin-nidogen was virtually indistinguishable. These observations suggest that the enhanced neural crest cell migration on collagen type IV and LDPG-associated laminin-nidogen does not result from a complementary stimulation of movement by the ligand molecules. The association of laminin with other basement membrane constituents may reflect a natural way to organize the molecule in its optimal attachment/motility-promoting configuration (Schittny et al, 1988). This does not preclude, however, that certain forms or concentrations of laminin may directly inhibit cell motility (Woodley et ah, 1988). We find no evidence that neural crest cell migration on the laminin-nidogen complex is connected to the putative YIGSR cell attachment sequence identified in the Bl chain of laminin (Graf et ah, 1987). In fact, a syn-

Neural Crest Cell Migration

235

thetic peptide containing the YIGSR sequence failed to inhibit neural crest cell locomotion on laminin-nidogen, and the Pl fragment of laminin encompassing this recognition site and possessing cell-adhesive properties (Aumailley et ah, 1987; Nurcombe et al., 1989;Johansson et al, in preparation) was unable to support movement. On the other hand, the larger El’ fragment, which is thought to comprise a cell/heparin-binding site (F9/ Hep-2) (Charonis et al, 1988) in the short arm of the Bl chain and an RGD sequence in the short arm of the A chain (Sakai et al., 1988), supported neural crest cell migration to a minimal extent. The ability of the El’ fragment to support cell migration could be attributed to the F9 potential motility-promoting site and/or to the RGD sequence. Our observation that RGDS peptides, but not heparin, completely blocks migration on the El’ fragment supports the latter assumption. Whether this RGD attachment site on the A chain of laminin is recognized by neural crest cells migrating on the intact molecule remains an open question. The observation that RGDS peptides do not affect cell locomotion on intact laminin suggests that the RGD site may function complementarily, possibly by being recognized by the “fibronectin receptor” (Lallier and Bronner-Fraser, 1990). Our data suggest that the motility-promoting site(s) for neural crest cell migration on laminin is localized in the long arm of the molecule within the E8 domain. The site of neural crest cell attachment on the E8 domain is likely to be similar to that involved in promotion of neurite outgrowth (Edgar et ah, 1984,1988, Rogers et ab, 1988) and the attachment of other cell types (Aumailley et al., 1987; Goodman et aZ, 1987; Dillner et al, 1988, Rogers et cd, 1988; Nurcombe et al, 1989). It is still unclear whether the effective sequence(s) within this domain is expressed on the B or A chains, but the nonpermissiveness of the E3 fragment as a migratory substrate precludes a direct interaction with the aminoterminal, heparin-binding domain of the A chain. The occurrence of a postulated heparin-binding site with cell-binding characteristics in the laminin molecule (Charonis et aZ.,1988) highlights the possibility that the E8 domain may similarly embody a dual heparin/cellbinding site. Support for this idea derives from the findings that exogenous heparin inhibits neural crest cell migration on the E8 fragment, whereas other glycosaminoglycans were virtually ineffective (data not shown). It is also well-documented that heparin inhibits neurite elongation on laminin substrates in several experimental systems, implying a role for cell surface-associated heparan sulfate proteoglycans in the process (Lander et aZ., 1982; Cole et al., 1985; Chernoff, 1988). However, in light of the conspicuous involvement of integrins in neural crest cell attachment to laminin

236

DEVELOPMENTALBIOLOGY

(Lallier and Bronner-Fraser, 1990), it is most likely that binding of heparin in the vicinity of cell attachment sites directly interferes with the function of cell surface receptors and/or disturbs the cooperative action of cell surface receptors and heparan sulfate proteoglycans. The potential ability of neural crest cells to utilize cell surface heparan sulfate proteoglycans or related molecules for their migration was further corroborated by the ability of neural crest cells to locomote on PF4 in a dose-dependent and heparin-sensitive fashion. In conclusion, our results demonstrate that neural crest cell migration on fibronectin and laminin occurs through complementary mechanisms that involve recognition of multiple motility-promoting sites. Neural crest cell locomotion on fibronectin depends on efficient binding of RGDS-reactive receptors to their cognate ligand. Our data suggest that an RGDS-dependent integrin may function in a cooperative manner with cell surface heparan sulfate proteoglycans or related/dependent molecules for migration on fibronectin. In the case of laminin, there is evidence that both the CSAT antibody, against a /3i subunit of a chick integrin, and the HNK-1 antibody inhibit neural crest cell attachment (Bronner-Fraser, 1985; Lallier and BronnerFraser, 1990). These observations in conjunction with our present data suggest that the migration of avian neural crest cells on laminin involves members of the integrin family of receptors and occurs via an RGDS-independent mechanism. In addition, we show that neural crest cell motility on laminin is somewhat sensitive to heparin, again implying the participation of cell surface molecules related to or dependent on heparan sulfate proteoglycans. In general, we find that laminin is less efficient than fibronectin in promoting neural crest cell motility in vitro and that its efficiency may be determined by the conformation/orientation of the molecule. As previously suggested (Perris and Johansson, 198’7), the difference in the motility-promoting capacity of the two glycoproteins could be related to a difference in their overall cell-adhesive properties. Attachment of neural crest cells to fibronectin is virtually constant over a broad range of substrate concentrations (Lallier and Bronner-Fraser, 1990). Attachment of neural crest cells to laminin, on the other hand, increases with increasing amounts of the protein in the substrate. Interestingly, the extent of neural crest dispersion on laminin actually decreases with increasing substrate concentrations, highlighting the possibility that a delicate balance exists between adhesion and motility. Our data support the idea that domains that are either permissive or nonpermissive for cell motility exist in laminin, and the predominance of one or the other may be dependent upon the configuration of the molecule. Alternatively, latent cell attachment/motility-promoting

VOLUME136.1989

sites within laminin (Nurcombe et aZ., 1989) could become exposed as a result of differential assembly of the molecule and may complement the primary motilitypromoting sites. We are grateful to Katrina Saladin for assistance in the preparation of laminin fragments and to Dr. Staffan Johansson for valuable suggestions and the contribution of fibronectin fragments and YIGSR peptides. We thank Dr. Hynda Kleinman for generously donating YIGSR peptides, Dr. Michael Pierschbacher for kindly providing various RGD synthetic peptides and information on fibronectin fragments, and Dr. Daniel Carson for the donation of PF4. This study was supported by USPHS HD-15527 and a Basic Research Grant from the March of Dimes Birth Defects Foundation (to M.B.-F.), the Swiss National Science Foundation, and the Maurice Mtiller Foundation (to M.P.). M.B.-F. is a Sloan Foundation Research Fellow. REFERENCES AUMAILLEY, M., NURCOMBE,V., EDGAR,D., PAULSSON,M., and TIMPL, R. (1987). The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites. J. BioL C&m. 262, 11,532-11,538. BENECKY,M. J., KOLVENBACH,C. G., AMRANI, D. L., and MOSESSON, M. W. (1988). Evidence that binding to the carboxyl-terminal heparin-binding domain (Hep II) dominates the interaction between plasma fibronectin and heparin. Biochemistry 27,7565-7571. BILOZUR,M. E., and HAY, E. D. (1987). Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin and collagen. Dev. BioL 125,19-33.

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