Environmental influences on neural crest cell migration

Environmental Influences on Neural Crest Cell Migration

Marianne Bronner-Fraser

Developmental Biology Center, University of California, Irvine, California 9271 7

SUMMARY

Neural crest cells migrate extensively and interact with numerous tissues and extracellular matrix components during their movement. Cell marking techniques have shown that neural crest cells in the trunk of the avian embryo migrate through the anterior, but not posterior, half of each sclerotome and avoid the region around the notochord. A possible mechanism to account for this migratory pattern is that neural crest cells may be inhibited from entering the posterior sclerotome and the perinoto-

chordal space. Thus, interactions with other tissue may prescribe the pattern of neural crest cell migration in the trunk. In contrast, interactions between neural crest cells and the extracellular matrix may mediate the primary interactions controlling neural crest cells migration in the head region. 0 1993 John Wiley & Sons, Inc. Keywords: neural crest cell migration, extracellular matrix molecules, cell marking techniques, notochord inhibitory effects.

INTRODUCTION

The neural crest is a migratory cell population, arising within the dorsal aspect of the neural tube. After closure of the neural tube, neural crest cells emigrate from its dorsal margin, migrate along characteristic pathways, and differentiate into widely varied derivatives. The neural crest has several unique properties that make it an ideal system for studying cell migration and differentiation. First, these cells migrate extensively in a stereo- typic manner. Second, neural crest cells give rise to diverse and numerous derivatives, ranging from melanocytes and cranial cartilage to adrenal chromaffin cells and the ganglia of the peripheral nervous system. Third, neural crest cells are accessible to surgical, immunological, and biochemical ma- nipulations during both initial and certain later stages in their development.

As a population, the neural crest is regionalized cells derived from different axial levels follow dis- tinct migratory pathways to their final destinations

Received September 8, 1992; accepted September 8, 1992 Journal ofNeurobiology, Vol. 24, No. 2, pp. 233-247 (1993) 0 1993 John Wiley & Sons, Inc. ccc 0022-3034/93/020233-15

and there give rise to diverse progeny (LeDouarin, 1982; Noden, 1975). In the cranial region, neural crest cells migrate subjacent to the cranial ectoderm. Some cranial neural crest cells enter the branchial arches, where they will form many of the cartilagenous elements of the facial skeleton, whereas others contribute to the ciliary ganglion of the eye and the cranial sensory ganglia. Vagal neural crest cells migrate under the ectoderm and into gut, where they move along its anterior to posterior extent to populate the enteric nervous system. Trunk neural crest cells migrate in two direc- tions: ventrally through the somites and dorsally under the ectoderm. Those cells following the ventral route give rise to dorsal root ganglia, sympathetic ganglia, and adrenal chromaffin cells; those cells following the dorsal route give rise to melanocytes of the skin (Weston, 1963; LeDouarin, 1982).

Neural crest cells migrate through or adjacent to numerous tissues including the neural tube, somites, and gut. Their migratory pathways often contain abundant quantities of extracellular matrix (ECM) molecules. In addition, neural crest cells possess receptors for ECM molecules as well as numerous cell surface molecules which may be important for cell-cell adhesion to themselves or

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other cell types. Thus, a variety of cell-cell and cell-substrate interactions may influence the migration and localization of neural crest cells. In order to understand these interactions, one must have a detailed knowledge of the pathways followed by neural crest cells, the potential ECM li- gands available along these pathways, and the tissues with which neural crest cells may interact. This review summarizes recent experiments from our laboratory regarding the tissue and substrate interactions that may influence the pattern of neural crest cell migration, primarily in the trunk region, although some other populations are con- sidered as well.

ANALYSIS OF NEURAL CREST MIGRATORY PATHWAYS USING CELL MARKING TECHNIQUES

The pathways of neural crest cell migration have been studied using a variety of cell markers. The initial analysis of neural crest cell migratory pathways in avian embryos involved transplantation of neural tubes marked with either [ 3H] thymidine (Weston, 1963) or derived from quail embryos (for review, see LeDouarin, 1982) into unlabelled chick hosts. By fixing animals at various stages after operation and looking at the distribution of labelled cells, it was possible to follow both the routes and derivatives of neural crest cells. These experiments demonstrated that neural crest cells migrate along two predominant pathways in the trunk region: either ventrally toward the dorsal aorta or dorsolaterally under the ectoderm. Such transplantation experiments have been particu- larly useful for establishing the progeny arising from neural crest cells at various axial levels.

Because transplants require some time to heal and may cause scarring, neural tube grafts have been less useful for describing the early pattern of neural crest cell migration. Recently, new techniques have emerged that circumvent these diffi- culties. First, monoclonal antibodies that recognized migrating neural crest cells and some of their derivatives have become available. NC- 1 and HNK- 1 antibodies (Tucker, Aoyama, Lipinski, Tursz, and Thiery, 1984) both recognize a carbohydrate epitope that is present on the surface of neural crest cells and some of their derivatives. By examining the HNK- 1 immunoreactivity in fixed sections through embryos, it was discovered that neural crest cells that move along the ventral pathway migrate in a segmental fashion, moving through the anterior, but not the posterior half of

each somitic sclerotome, and then ventrally toward the dorsal aorta (Rickmann, Fawcett, and Keynes, 1985; Bronner-Fraser, 1986a) (Fig. 1). The cells following the ventral route will form the dorsal root and sympathetic ganglion cells, as well as adrenomedullary cells. Interestingly, their metameric pattern of migration is later reflected in the segmental arrangement of neural crest-derived peripheral ganglia (Teillet, Kalcheim, and Le Douarin, 1987; Lallier and Bronner-Fraser, 1988). Although the results obtained with antibodies are intriguing, the pattern of cell migration can be only inferred from observations of sections through fixed embryos. Furthermore, these antibodies are not entirely specific; they recognize several non- neural crest cell types (Kruse et al., 1984) and do not recognize all neural crest cells (Teillet et al., 1987).

An alternative and more direct method for examining the pathways of neural crest cell migration is to inject the lipophilic dye, DiI, into the neural tube ( Serbedzija, Bronner-Fraser, and Fraser, 1989). Because the dye is hydrophobic and inter- calates into cell membranes, injection of DiI into the neural tube marks all neural tube cells including premigratorq neural crest cells within its dorsal margin. By examining embryos at various times after injection, it is possible to trace directly the routes followed by neural crest cells. Similar to the results using the HNK-1 antibody, early DiI-labelled migrating trunk neural crest cells were found to migrate ventrally in a segmental fashion. By altering the time of injection, it also was possible to establish the order in which neural crest cells contribute to their derivatives. By injecting DiI at progressively later stages of development, we labelled only those neural crest cells that were premi- gratory at the time of injection. Later emigrating cells were found to contribute to progressively more dorsal derivatives (Fig. 2). Thus, the contribution of neural crest cells to their derivatives be- comes restricted in a ventral-to-dorsal order. The last cells to exit the neural tube are precursors to pigment cells, which migrate dorsolaterally under the ectoderm where they eventually form pigment cells (Serbedzija et al., 1989). An advantage of DiI- labelling of neural crest cells is that DiI can be ap- plied to a variety of axial levels and any species. For example, this approach has been used successfully to map the pathways of sacral neural crest cell migration in the chick and mouse (Pomeranz and Gershon, 199 1; Serbedzija, Burgan, Fraser, and Bronner-Fraser, 199 1 ) and trunk and cranial neural crest cell migration in the mouse (Serbed- zija et al., 1990; 1992, submitted).

Neural Crest Cell Migration 235

Figure 1 Schematic diagram illustrating the early pathways of trunk neural crest cell migration. Neural crest cells emerge from the neural tube ( N T ) and proceed either ventrally (indicated by large curved arrow) through the somitic sclerotome (Scl) or dorso-laterally (indicated by small curved arrow) between the ectoderm (Ec) and dermomyotome (DM). The ventrally migrating cells only move through the rostra1 ( R ) half of each sclerotome and are not observed in the caudal (C) half. Neural crest cells avoid the region around the notochord (No). Ao = dorsal aorta.

The conclusion that neural crest cells migrate in an orderly pattern is in agreement with the results of Weston and Butler ( 1966), who transplanted tritiated thymidine-labelled neural tubes from the trunk region of “older” avian embryos to trunks of “younger” hosts (Weston and Butler, 1966). They found that the neural crest cells emerging from both the older and young neural tubes could give rise to the same range of neural crest derivatives. In contrast, neural crest cells emerging from “young” neural tubes transplanted to “older” embryos con- tributed only to dorsal derivatives, such as dorsal root ganglia and pigment cells. Thus, neural crest cells and/or the migratory paths available to them may change during the course of their migration and the occupation of their derivatives.

These results suggest that the time at which neural crest cells exit the neural tube may contrib-

ute to the range of derivatives available to them. This could be a result ofthe extrinsic changes occur- ring in the environment, intrinsic changes within the neural crest population, or a combination of the two. We tested whether late emigrating neural crest cells are more restricted in developmental potential than early migrating cells by culturing neural crest cells prepared from the neural tubes of embryos at various stages of neural crest cell migration (Artinger and Bronner-Fraser, 1992). Later emigrating neural crest cells displayed a more limited repertoire of cell fates than early emigrating neural crest cells. Although both populations gave rise to pigment cells and neurons, only early emigrating neural crest cells formed adrenergic cells. These results suggest that late emigrating neural crest cells have a more restricted developmental potential than do early migrating neural crest cells.

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Figure 2 (Top) Trunk neural crest cell migration pathways and derivatives. There are two major pathways: ( A ) The dorso-lateral pathway between the somite and the ectoderm, and (B) the ventral pathway through the rostral half of each somite. Trunk neural crest give rise to (A’) pigment cells, (B’) dorsal root ganglia, ( C ’ ) sympathetic ganglia, and (D‘) cells around the dorsal aorta. (Bottom) A schematic representation of the rostral to caudal distribution of DiI in the neural crest derivatives of a single embryo injected at stage 19 and fixed at stage 2 1. (a-f) Represents levels along the rostrocaudal axis from which transverse sections were taken. Be- cause development in a single embryo occurs in a rostrocaudal sequence, different stages of neural crest migration exist within the same embryo. ( a ) At the level of the ninth somite, Dil-labelled cells were observed along the dorso-lateral pathway. (b) At the level of the 15th somite, DiI labelled cells were observed along the dorso-lateral pathway and in the dorsal root ganglia. (c ) At the level of the 22nd somite, DiI-labelled cells were seen along the dorso-lateral pathway, in the dorsal root ganglia, and in the sympathetic ganglia. (d-f) From the level of the 38th somite to the caudal end of the embryo, DiI-labelled cells were observed in all truncal neural crest derivatives. Thus, neural crest cells appear to fill their derivatives in a ventral-to- dorsal order. (From G. Serbedzija et al., Development 1062306-816, 1989.)


Thus, although the orderly pattern of migration may restrict the range of derivatives open to later emigrating neural crest cells, other factors such as time spent within the neural tube also may contribute to restriction of these cells' fate.

ROLE OF SURROUNDING TISSUES IN DETERMINING THE PATTERN OF NEURAL CREST MIGRATION

Neural crest pathways are lined with a variety of tissue including the somites, ectoderm, neural tube, notochord, and dorsal aorta. During their migration, neural crest cells contact a number of these tissues. Described below are experiments examining the role that some of these tissues may play in establishing the pattern of neural crest cell migration.

Segmental Information within the Somites

Like motor nerves (Keynes and Stern, 1984), migrating neural crest cells (Rickmann et al., 1985; Bronner-Fraser, 1986a; Teillet et al., 1987; Loring and Erickson, 1987) are restricted to the anterior half of each sclerotome. The conclusion that the somite contains segmental information is based on the result of a simple experiment: if the segmental plate (which gives rise to the somites) is rotated 180" about its anterior-posterior axis, the migratory pattern of neural crest cells through the sclerotome is reversed such that they now traverse the posterior (original anterior) halves of the rotated sclerotomes (Bronner-Fraser and Stern, 199 1 ) ( Fig. 3 ) . This pattern is similar to that observed for the navigation of motor axons (Keynes and Stern, 1984). The results suggest that the segmented pattern of movement is due to cues inherent to the somite. Thus, there may be inhibitory cues in the posterior sclerotome, attractive cues in the anterior sclerotome. or both.

Inhibitory Effects of the Notochord

Avian neural crest cells migrating along the trunk ventral pathway are distributed throughout the anterior half of the sclerotome with the exception of a neural crest cell-free space of approximately 85 pm in width surrounding the notochord. In tissue culture experiments, Newgreen found that neural crest cells avoided the region surrounding notochords with which they were co-cultured, suggesting that the notochord produces a substance that inhibits neural crest migration (Newgreen, Scheel,

Figure 3 Fluorescence photomicrographs of longitu- dinal sections through operated embryos stained with the HNK-1 antibody. ( a ) An embryo in which the segmental plate was rotated 180" about its rostrocaudal axis. By the time of fixation, the segmental plate had differentiated into mature somites with dermomyo- tomes and sclerotomes. Neural crest cells are always observed in the original rostra1 half of each sclerotome and are absent from the original caudal halves. The arrow indicates a small somite which formed at one of the junc- tions between graft and host tissue. Abbreviations as per Figure I . (From M. Bronner-Fraser and C. Stern, Dev. Biol. 143:2 13-2 17, 199 1 .)

and Kastner, 1986). To determine if the neural crest cell-free space in vivo results from the notochord inhibiting neural crest cell migration, we implanted a length of quail notochord lateral to the neural tube along the neural crest ventral migratory pathway of 2-day-old chicken embryos (Fig. 4). The subsequent distribution of neural crest cells was analyzed in embryos fixed 2 days after grafting. When the donor notochord was isolated using collagenase, neural crest cells avoided the ectopic notochord and were absent from the area im- mediately surrounding the implant (mean distance of 43 pm) (Figs. 4 and 5 ) . The neural crest cell-free space was significantly less when notochords were isolated using trypsin or chondroitinase digestion and was eliminated by fixation of notochords with paraformaldehyde or methanol prior to implanta- tion. These results suggest that the notochord pro-

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UNOPERATED OR SHAM EMBRYOS

DISTRIBUTION NEURAL CREST CELLS

EMBRYOS IMPLANTED WITH AN EXTRA NOTOCHORD

- D I STR I BUT I ON NEURAL CREST CELLS

Figure 4 Schematic diagram illustrating the pattern of neural crest cell migration in normal embryos (top) and the effects on neural crest cell migration of implanting an extra notochord adjacent to the neural tube. Abbreviation as per Figure 1.

duces a trypsin and chondroitinase-labile substance that can inhibit neural crest cell migration (Pettway, Guillory, and Bronner-Fraser, 1990). A likely candidate for the inhibitory molecule is a chondroitin sulfate proteoglycan which bears the HNK- 1 epitope (Henning and Schwartz, 199 1 ). The perinotochordal space also appears to be inhibitory for motor axons (Oakley and Tosney, 199 1 ) .

Dorsoventral Patterning of Neural Crest Derivatives by the Neural Tube

Neural crest cells have a characteristic pattern of migration in the dorsoventral plane. Weston

( 1963) demonstrated that when the neural tube is rotated dorsoventrally by 180" to cause neural crest cells to emerge ventrally, neural crest cells migrate in two streams: one dorsally, in reverse direction to that taken normally and the other ventrally towards the aorta. Weston concluded that the neural crest cells are probably not directed to their targets by chemoattractants but rather that they ex- ploit all the spaces available to them. Because these experiments were conducted before it was known that trunk neural crest cells migrating through the sclerotome are restricted to the anterior half, this experimental question was reexamined using mod- ern cell markers.

Netirul Crest Cell Migration 239

Figure 5 Fluorescent photomicrographs of transverse sections through chick embryos implanted with quail notochords that were isolated by collagenase digestion. The sections were stained with the HNK- 1 antibody, which recognizes a surface epitope on neural crest cells and the perinotochordal matrix. ( a ) An embryo fixed at stage 16. HNK-I immunoreactive neural crest cells approach but do not contact the implanted notochord (arrow), which is located in a ventrolateral position. (b, c ) Low and high magnifications of the same section through an embryo fixed at stage 18 in which the implanted notochord lies lateral to the neural tube. Neural crest cells (curved arrow) appear to be in a ball-like aggregate dorsal to the ectopic notochord (eNo). Abbreviations as per Figure 1. (From Z. Pettway et al., Dev. Bid. 142:335- 345, 1990.)

Using transplantation approaches, we explored the factors that control the polarity, position, and differentiation of the neural crest-derived sympathetic and dorsal root ganglion cells. The neural tube, with or without the notochord, was rotated by 180” dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated and in others a second notochord was implanted. Neural crest cells emerging from an inverted neural tube migrate in a ventral- to-dorsal direction through the sclerotome, where they become segmented by being restricted to the anterior half of each sclerotome. The dorsal root ganglia always form adjacent to the neural tube, and their dorsoventral orientation often follows the orientation of the grafted neural tube. Simi- larly, the ventral roots emanated from the dorsal portion of the neural tube (originally “ventral” prior to rotation) (Fig. 6) ; however, differentiation of sympathetic neurons only occurs near the aorta/ mesonephros and requires the proximity of

either the ventral neural tube or the notochord. The results suggest that the dorso-ventral polarity of the neural tube, dorsal root ganglia, and ventral roots appear to follow the orientation ofthe neural tube. Thus, the neural tube and notochord exert important effects on neural crest cells, influencing the direction of their migration as well as being required for differentiation (Stern, Artinger, and Bronner-Fraser, 199 1 ) .

DISTRIBUTION OF EXTRACELLULAR MATRIX AND ADHESION MOLECULES ALONG NEURAL CREST PATHWAYS

Numerous glycoproteins, proteoglycans, and gly- cosaminoglycans have been described along neural crest migratory pathways. The most prevalent glycoproteins appear to be fibronectin (Newgreen and Thiery, 1980), laminin (Krotoski et al., 1986), tenascin /cytotactin (Tan, Crossin, Hoffman, and

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Figure 6 Fluorescence photomicrographs of transverse sections through an embryo in which the neural tube was rotated 180" in the dorsoventral plane in the absence of a notochord. The embryo was fixed at stage 25, which is well after gangliogenesis normally occurs. The neural tube forms normally but with inverted dorsoventrally polarity. In most sections, the dorsal root ganglia (D) form normally relative to the inverted neural tube. In a few cases, supernumerary dorsal root ganglia form, as illustrated in (c ) . The ventral roots ( V R ) project from the dorsal portion of the neural tube and appear to find their targets in the limb, as seen in (a) and (b). HNK- 1 immunoreactive nerve roots sometimes appeared to cross the midline, as shown in (d) . This is probably due to the absence of the notochord. G = gut; other abbreviations as per Figure 1. Scale bar = 100 pm. (From C. D. Stern et al., Developmerzt 113:207-216, 1991 .)

Edelman, 1987 ) , and various collagens (Duband and Thiery, 1987; Perris et al., 199 1 b). Fibronec- tin, in particular, has been suggested to play a major role in the adhesion and motility of neural crest cells (Thiery, Duband, and Delouvee, 1982). In tissue culture, neural crest cells migrate avidly on both fibronectin (Rovasio, Delouvee, Timpl, Ya- mada, and Thiery, 1983) and laminin (Newgreen, 1984) substrates; however, the role of fibronectin and laminin in vivo is not yet clear. Of the proteoglycans present within the embryo, heparan sulfate proteoglycans (Pems et al., 1991b) appear on neural crest cell pathways, whereas chondroitin sulfate proteoglycans are generally present in regions from which neural crest cells are absent (Tan

etal., 1987; Pemsetal., 1991b), suchastheperino- tochordal space. Chondroitin sulfate proteoglycans tend to inhibit neural crest cell migration in vitro, consistent with the idea that they may restrict or inhibit migration in the embryo.

The metameric distribution of neural crest cells within the somite may be caused by molecular differences between the anterior and posterior halves of the somite. A monoclonal antibody discovered by Tanaka and colleagues (Tanaka, Agata, and Obata, 1989) recognizes a molecule in the anterior half of the somite prior to neural crest cell entry into this tissue and whose pattern is unaltered by neural crest cell ablation. Tenascin /cytotactin is also in the anterior half of the somite, although its

N t w u l Crest Cell Migration 241

distribution there appears to require the presence of neural crest cells (Stern et al., 1989; but see Tan et al., 1987, 1991). Other substances have been identified in the posterior half of each somite during neural crest cell migration, such as molecules bearing a moiety that binds peanut lectin (Stern and Keynes, 1987), a chondroitin sulfate proteoglycan that binds cytotactin (Tan et a]., 1987) and T-cadherin (Ranscht and Bronner-Fraser, 199 1 ). A functional role for a peanut lectin-binding glycoprotein fraction derived from posterior sclerotome is suggested by experiments in which liposomes containing these molecules inhibit growth cones in vitro (Davies, Cook, Stern, and Keynes, 1990). In addition, collagen types I and 111 and a keratan sulfate proteoglycan become restricted to the posterior half of the sclerotome during advanced stages of neural crest cell migration (Penis, Krotoski, and Bronner-Fraser, 199 1 a). Because the reorganiza- tion of these molecules occurs relatively late, it is likely that they are a consequence, rather than a cause of the neural crest migratory pattern. Thus, the ECM components along neural crest pathways go through dynamic rearrangements at the time of neural crest cell migration.

Because of its presence within the posterior sclerotome, we have examined in detail the distribution of T-cadherin (truncated-cadherin), a novel member of the cadherin family of cell adhesion molecules, during trunk neural crest cell migration (Ranscht and Bronner-Fraser, 199 l ). T- cadherin was selectively expressed in the posterior half of each sclerotome at all times examined (Fig. 7). T-cadherin immunostaining appeared graded along the anterior-posterior axis, with increasing levels of reactivity in the posterior halves of progressively more mature somites. The earliest T-cadherin expression was detected in a small population of cells in the posterior portion of the somite three segments anterior to the last-formed somite, concomitant with the initial invasion of neural crest cells into the anterior portion of the same somite. When neural crest cells were ablated surgi- cally prior to their emigration from the neural tube, the pattern of T-cadherin immunoreactivity was unchanged compared to unoperated embryos, suggesting that neural crest cells are not required for the metameric distribution of T-cadherin. This distribution is consistent with the possibility that T-cadherin plays a role in maintaining somite polarity and in influencing the pattern of neural crest cell migration (Ranscht and Bronner-Fraser, 199 1 ) ; however, its functional significance re- mains to be tested.

Figure 7 Fluorescence photomicrograph of a sagittal section through the rostral somites of a stage-1 7 embryo. A double exposure showing that that neural crest cells (red) appear in the rostral half of each somite, whereas T-cadherin (green) appears in the caudal half. Abbrevia- tions as per Figure 1. (From B. Ranscht and M. Bronner- Fraser, Development 11 1: 15-22, 199 1 .)

In contrast to molecules that are selectively distributed within either the anterior or posterior half of the sclerotome, other matrix components including fibronectin, laminin, and collagen IV re- main uniformly distributed throughout the sclerotome. Hence, differential distribution of permissive and nonpermissive ECM molecules, together with changes in the cells’ ability to interact with the matrix, may determine the migratory pattern of trunk neural crest cells. Although the distribution of extracellular matrix molecules has not been studied as extensively in cranial regions as in the trunk, such molecules as fibronectin, laminin, tenascin, and heparan sulfate proteoglycans are abundant along cranial neural crest pathways (Duband and Thiery, 1987; Krotoski, Domingo, and Bronner- Fraser, 1986; Bronner-Fraser, 1988 ). Perturbation experiments (see below) suggest that these molecules may play a functional role in cranial neural crest migration.

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ANALYSIS OF CELL-MATRIX INTERACTIONS IN VITRO

Neural crest cells can be grown in vitro by explant- ing neural tubes prior to neural crest migration. They emigrate from the dorsal side of the ex- planted neural tube and form a monolayer on the substrate (Cohen, 1977). Neural crest cells cultured in this way migrate avidly on a variety of substrates, including fibronectin (Rovasio et al., 1983; Perris et al., 1989), laminin (Newgreen, 1984; Lallier and Bronner-Fraser, 199 1 ), collagen (Penis et al., 1991a), and other molecules. On fibronectin substrates, neural crest cell migration and attachment is progressively enhanced with increasing fibronectin concentration. In the case of laminin, however, higher concentrations of substrate can actually inhibit cell migration (Perris et al., 1989).

Antibodies that perturb cell-substrate adhesion have been useful for defining the interactions important for cell movement. The CSAT monoclonal antibody recognizes the PI integrin subunit on avian cells (Honvitz, Duggan, Greggs, Decker, and Buck, 1985); in vitro, this antibody inhibits chick neurite outgrowth on laminin, fibronectin, and type-IV collagen (Bozyczko and Honvitz, 1986; Hall, Neugebauer, and Reichardt, 1987; Le- tourneau et al., 1988; Neugebauer, Tomaselli, Li- lien, and Reichardt, 1988). The CSAT antibody against PI integrin can be added to neural crest cells in vitro to test the possible function of the recognized epitopes under defined conditions; it disrupts neural crest cell adhesion to fibronectin and laminin substrates (Rovasio et al., 1983; Bron- ner-Fraser, 1985; Lallier and Bronner-Fraser, 199 1 ). The effects are rapid, causing many cells to detach from the dish or aggregate with other cells within 15 min, and are readily reversible upon re- moval of the antibody. These results suggest that binding and dissociation of integrin receptors to matrix molecules occur rapidly. Because not all neural crest cells are affected equally by the antibody, CSAT may recognize receptors differentially expressed on subsets of neural crest cells or only expressed at certain times. HNK-1 is another cell surface epitope that is present on migrating neural crest cells, neural crest-derived neurons, and some other cells (Tucker et al., 1984). Addition of HNK- 1 antibody to neural crest cells in tissue culture causes detachment and aggregation of the cells grown on laminin substrates ( Bronner-Fraser, 1988).

In order to assess the interactions of neural crest cells with laminin substrates in a quantitative manner, we have utilized the cell attachment assay of

McClay ( McClay, Wessel, and Marchase, 198 1 ), which yields highly reproducible results for small numbers of cells. The results show that an increasing percentage of neural crest cells adhere to laminin with increasing substrate concentrations. This adhesion was inhibited by CSAT antibodies against the chick 0, subunit of integrin, suggesting that &-integrins mediate neural crest cell interactions with laminin (Fig. 8) . The HNK- 1 antibody, which recognizes a carbohydrate epitope, inhibits neural crest cell attachment to laminin at low-substrate-coating concentrations (> 1 pg/ml; Low- LM) (Fig. 8) , but not at high-coating concentration of laminin ( 10 pg/ml; High-LM) . Surpris- ingly, attachment to Low-LM occurs in the absence of divalent cations. In contrast, attachment to High-L,M requires Ca2+ or Mn2’. These results suggest that neural crest cells have at least two integrins that recognize laminin: one that requires divalent cations for binding; the other that can function without divalent cations. These appear to recognize different conformations of laminin.

We have adapted biochemical techniques utilized by von Boxberg, Wutz, and Schwartz, ( 1990) to biotinylate the cell surface of neural crest cells, making it possible to analyze some of their biochemical properties for the first time. Using this method, we have performed preliminary biochemical characterization of the HNK-1 epitope on neural crest cells. The HNK- 1 antibody recognizes a 165 kD protein which is also found in immuno- precipitates using antibodies against the /3, subunit of integrin, which precipitates both the 0, and associated a bands. Our results suggest that the HNK- 1 epitope on neural crest cells is present on or associated with a novel or differentially glycosylated alpha integrin subunit, which interacts with laminin in the apparent absence of divalent cations (Lallier and Bronner-Fraser, 199 1 ) . In order to determine the identity of this novel 01 subunit, we have obtained various antibodies specific to avian 01 integrin subunits. Interestingly, an antibody against the chick a, subunit of integrin (Syfrig, Mann, and Paulson, 1991) recognizes a 165 kD protein. In preliminary experiments, this antibody inhibits attachment of neural crest cells to laminin in the absence of divalent cations, similar to the effects of HNK-1 antibody.

ANALYSIS OF CELL-MATRIX INTERACTIONS IN VIVO

We have utilized antibody perturbation experiments to “knock-out’’ selected cell-matrix interac-


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Figure 8 Neural crest cell attachment to various concentrations of laminin, ranging from 0.1 to 10 pg/ml, in the presence ofthe CSAT and HNK- 1 antibodies. CSAT and HNK-1 antibodies (50 pg/ ml) were added to test their ability to perturb cell adhesion over a range of substrate/ coating concentrations. Points represent the mean of at least six experiments and the error bars represent the S.E.M.

tions along neural crest migratory pathways. These experiments have been performed primarily along cranial neural crest pathways because numerous extracellular matrix molecules are present along these pathways at times that correlate with initial and active migration of this population. Antibod- ies have been introduced along cranial neural crest pathways by microinjection lateral to the mesence- phalic neural tube. Embryos ranging from the neural fold stage to the 9-somite stage are used for injection (Fig. 9). Embryos with greater than 10 somites at the time of injection had no detectable abnormalities, suggesting that they were sensitive to the injected antibodies for only a limited time during their development. After injection, antibody molecules diffused freely on the injected side of the embryo, but were barely detectable on the uninjected side, as if they did not readily cross the midline.

By microinjecting antibodies that bind and functionally inactivate the P, subunit of the integrin, we sought to test the role of the integrin receptor in cranial neural crest cell migration in situ. Integrin antibodies caused major defects including reduced numbers of the neural crest cell on the injected side, neural crest cells within the lumen of the neural tube, ectopic neural crest cells external to the neural tube, and neural tube anomalies (Bronner-Fraser, 1985, 1986b). Similar results were obtained with synthetic peptides containing the fibronectin cell binding sequence (Boucaut et al., 1984) or antibodies against fibronectin (Poole and Thiery, 1986). In contrast to the antibodies

that block cell-ECM interactions, several control monoclonal antibodies that bind to integrins but do not block cell-matrix interactions had no detectable affect on cranial neural crest or neural tube development. These findings support the notion that antibody-induced perturbations in cranial morphogenesis result from a functional block of the integrin receptor and suggest that the receptor

4 - 9 Somite Embryo

\ I I / 1. Inject Ab that blocks

0 1 cell interactions

2. Incubate for 24 -48 hr

3. Section and stain with HNK-1 Ab

Controls: Non-blocking Ab

Figure 9 Schematic diagram illustrating the procedure for injecting antibodies into the cranial mesenchyme adjacent to the mesencephalon. Neural crest cells in this region migrate through the mesenchyme underneath the surface ectoderm.

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complex is important in the normal development of the cranial neural crest and neural tube.

The results of experiments using integrin antibodies or synthetic decapeptides that competi- tively inhibit cell binding to fibronectin taken together suggest an important function for fibronectin in cranial neural crest migration; however, these reagents are not entirely specific. For example, the cell binding sequence of fibronectin is also present in numerous other extracellular matrix molecules. Likewise, integrin antibodies block neural crest cell adhesion to laminin, fibronectin, and collagens. In addition to using antibodies that interfere with cell binding to fibronectin, we have used an antibody that functionally perturbs cell adhesion to laminin as a first attempt to distinguish between the respective roles of these matrix molecules.

Laminin is thought to occur in a complex with heparan sulfate proteoglycan (HSPG) in its native state. The inhibitor of neurite outgrowth (INO) antibody recognizes and functionally blocks cell interactions with this laminin-heparan sulfate proteoglycan complex (Chiu, Matthew, and Patter- son, 1986). We have injected I N 0 antibody along neural crest pathways in the mesencephalon in order to examine the possible role of laminin in cranial neural crest migration ( Bronner-Fraser and Lallier, 1988). At 1 day after injection, the embryos had severe abnormalities in cranial neural crest migration including ectopic neural crest cells external to the neural tube, neural crest cells within the lumen of the neural tube, and neural tube de- formities (Fig. 10). In contrast, embryos injected with antibodies against laminin or heparan sulfate proteoglycan were unaffected. These results indi- cate that functional blockage of a laminin-heparan sulfate proteoglycan perturbs cranial neural crest migration, providing evidence that laminin / HSPG is involved in aspects of neural crest migration in vivo.

Antibodies against several extracellular matrix molecules have been shown to inhibit cranial neural crest migration. In addition to I N 0 and CSAT antibodies described above, antibodies against tenascin (Bronner-Fraser, 1988), the HNK- 1 epitope ( Bronner-Fraser, 1987; which may be present on a subset of integrins), and fibronectin (Poole and Thiery, 1986) inhibit cranial neural crest cell migration. In addition to antibodies that block cell-matrix interactions, the galacto- syltransferase inhibitor, alpha lactalbumin, inhibits neural crest cell spreading on laminin, indi- cating a possible role for this enzyme in modulating cell attachment by the alteration of cell

Figure 10 Fluorescence micrograph through the cranial region 1 day after injection of I N 0 antibody. Neural crest cells can be recognized by their HNK- 1 immunoreactivity. In this embryo, ectopic aggregaters of neural crest cells (arrows) were observed adjacent to the neural tube. The neural tube also appeared deformed. Abbrevia- tion as per Figure 1. (From M. Bronner-Fraser and T. Lallier, J. Ce// Bid. 106:1321-1330, 1988).

surface carbohydrates ( Runyan, Maxwell, and Shur, 1986). Thus, cell-matrix interactions may be multivalent or may occur as an interrelated sequence of interactions, such that interfering with any step disrupts the process. These results suggest that multiple interactions are necessary for normal migration of cranial neural crest cells and high- lights the fact that complex interactions may be important dunng complicated morphogenetic events.

Interestingly, none ofthe function-blockinganti- bodies that affect cranial neural crest cell migration have been shown to be functional in the trunk region. Because the tissue and extracellular matrix environments appear very different in the head and trunk, it is possible that different strategies may be important for the migration of cranial ver- sus trunk neural crest cells. In the cranial region, it has been established that integrins, laminin, fibronectin, and tenascin are involved in some aspects of neural crest migration. In the trunk region, neural crest cell guidance may result from either tissue-derived cues such as differences between the anterior and posterior halves of the somites or inhibitory substances from the notochord. Thus, cell-matrix interactions may play a permissive rather than an instructive role in the migration of trunk neural crest cells.

CONCLUDING REMARKS

The experiments discussed in this chapter illustrate the important role of interactions between cells


and the extracellular environment in determining the routes of neural crest cell migration. Using a variety of cell marking techniques, it has been possible to document the pathways of neural crest cell migration in the chick and other species. Once the pattern of migration was established, we examined the role of tissue interactions in the patterning of neural crest cell movement. In the trunk region, tissues that line neural crest migratory pathways may dictate the patterns of cell migration. For example, the metameric pattern of neural crest cell migration through the anterior but not the posterior sclerotome appears to be dictated by the somites themselves. The absence of neural crest cells from the perinotochordal space appears to arise from an active inhibitory process of the notochord on neural crest cells in its vicinity. In addition, the neural tube is responsible for much of the patterning of neural crest cells along the dorsoventral axis.

Neural crest cells from different regions of the embryo may utilize different mechanisms of cell migration. Both cranial and trunk neural crest cells possess numerous receptors for extracellular matrix molecules, including several integrins. In vitro, these integrins mediate neural crest cell attachment to fibronectin, laminin, and collagens. Fur- thermore, cell-matrix interactions appear to be required for proper migration of cranial neural crest cells in vivo; however, there is no evidence to indi- cate that a particular cell-matrix interactions is es- sential for dictating the pattern of neural crest cells migration in the trunk region in v i v a Thus, although extracellular matrix molecules may serve as a permissive migratory substrate throughout the embryo, they may not necessarily provide specific guidance cues. This suggests that the interactions involved in guidance of neural crest cells are complex and often multivalent. The neural crest offers a good experimental system for testing the role of various interactions in cell movement because of the diverse nature of the populations and the range of tissues and molecule with which they can interact.

Parts of the work described in this review were sup- ported by USPHS grant HD-15527.

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