Basic fibroblast growth factor promotes adhesive ... - Semantic Scholar

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Development 119, 943-956 (1993) Printed in Great Britain © The Company of Biologists Limited 1993

Basic fibroblast growth factor promotes adhesive interactions of neuroepithelial cells from chick neural tube with extracellular matrix proteins in culture Yoshito Kinoshita, Chizuru Kinoshita, Josef G. Heuer1 and Mark Bothwell* Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, Washington 98195, USA 1Present

address: Howard Hughes Medical Institute, Department of Biology, Indiana University, Bloomington, Indiana 47405, USA *Author for correspondence

SUMMARY Fibroblast growth factors have been increasingly assigned mitogenic and trophic roles in embryonic and postnatal development of the nervous system. Little is known, however, of their functional roles in early embryonic neural development at the neural tube stage. We have examined the effect of basic fibroblast growth factor (bFGF) on the adhesive behavior in culture of dissociated brachio-thoracic neural tube cells from 26- to 30-somite stage chick embryos. Cells plated on collagencoated substratum at a low density attach to the substratum but show poor cell spreading. Addition of bFGF markedly promotes cell spreading, yielding an epithelial morphology. This effect becomes discernible 6-8 hours after cell plating with bFGF and is completed by 24 hours, with half-maximal and maximal effects attained at around 0.4 and 10 ng/ml, respectively. The number of cells remains largely constant up to 24 hours, and then cell survival and/or mitogenic effects of bFGF become apparent. The cell spreading effect is abolished by cycloheximide treatment, inhibited by the anti- 1-integrin antibody CSAT, and accompanied by about twofold increases in the expression of 1-integrin and vinculin,

INTRODUCTION Fibroblast growth factors (FGFs), initially characterized as mitogens for mesoderm- and neuroectoderm-derived cells, are now known to play vital roles in various proliferative and differentiative aspects of embryonic development ranging from mesoderm induction and formation of muscle, cartilage and bone to vascular development (Slack, 1990; Baird and Walicke, 1989; Gospodarowicz et al., 1986; Gospodarowicz 1990; Wagner, 1991). The FGF family consists of 8 members, acidic FGF (aFGF), bFGF, int-2 (Dickson and Peters, 1987), hst/ks (kFGF) (Delli-Bovi et al., 1987; Taira et al., 1987; Yoshida et al., 1987), FGF-5 (Zhan et al., 1988), FGF-6 (Marics et al., 1989), keratinocyte growth factor (Finch et al., 1989) and androgen-induced growth factor (Tanaka et al., 1992). While the biological functions of aFGF

components of focal adhesion complexes. Cells cultured with bFGF for 24 hours exhibit enhanced cell attachment and cell spreading with little time lag following cell plating. In earlier embryonic stages, developmentally less mature cells depend much more on bFGF for their cell spreading and survival, while in later stages the cell spreading response to bFGF becomes undetectable as neural tube develops to spinal cord. The cell spreading effect of bFGF is realized on specific extracellular matrix proteins including laminin, fibronectin and collagen, but not on vitronectin, arg-gly-asp peptide (PepTite-2000), poly-L-ornithine or others. These results suggest that, in an early stage of neural tube development, bFGF is involved in the developmental regulation of adhesive interactions between neuroepithelial cells and the extracellular matrix, thereby controlling their proliferation, migration and differentiation. Key words: basic FGF, FGF receptor, neural tube, neuroepithelial cell, cell adhesion, cell attachment, cell proliferation, cell spreading, cell survival, extracellular matrix, β1-integrin, vinculin

and bFGF have been studied most extensively, it is likely that other members of this expanding growth factor family also play various roles in the context of embryonic development. The multiplicity of FGFs is paralleled by FGF receptors (FGFRs), which also constitute a gene family, with four FGF receptor genes presently identified (reviewed by Johnson and Williams, 1993; designated here as FGFR-1, FGFR-2, FGFR-3, FGFR-4). Additional functional complexity is presented by the existence of alternatively spliced transcripts for both FGFs and FGFRs, generating functionally distinct proteins (Westermann et al., 1990; Acland et al., 1990; Giordano et al., 1992; Johnson and Williams, 1993). Although still poorly characterized, the variety of possible combinational interactions between FGFs and FGFRs has the potential to provide highly refined and flexible regulatory interactions during embryonic development.

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Y. Kinoshita and others

In the past several years, FGFs have been increasingly implicated in the development and regeneration of the nervous system (for reviews, see Westermann et al., 1990; Wagner, 1991). aFGF and bFGF stimulate proliferation of neuronal precursor cells (Gensburger et al., 1987; Murphy et al., 1990) and glial cells (Morrison and De Vellis, 1981; Eccleston and Silberberg, 1985), and also support the survival and differentiation of developing and regenerating neuronal cells of various origins in the central and peripheral nervous systems (Togari et al., 1985; Morrison et al., 1986; Wagner and D’Amore, 1986; Walicke et al., 1986; Unsicker et al., 1987; Walicke 1988; Dreyer et al., 1989; Birren and Anderson, 1990; Eckenstein et al., 1990; Grothe et al., 1991). Thus, mitogenic and trophic effects of FGFs on the nervous system are now evident at least at certain developmental stages. However, these studies have not specifically addressed possible functions of FGFs early in the development of the central nervous system following neural tube formation. Development of the central nervous system at the neural tube stage is particularly interesting in relation to putative FGFs’ functions because this stage involves both determinative/differentiative (for neurons) and self-replicating (for stem or precursor cells) events progressing in parallel within a relatively homogeneous neuroepithelial cell population. In situ hybridization studies have revealed a relatively high level of expression of FGFR-1 mRNA in the neural tube of chick (Heuer et al., 1990) and murine (Reid et al., 1990; Wanaka et al., 1991; Orr-Urtreger et al., 1991; Peters et al., 1992a, 1993) embryos. These results, together with the presence of bFGF at this stage (Kalcheim and Neufeld, 1990), suggest that bFGF has a functional role in neural tube development before and around the period of overt neuronal differentiation. bFGF has been shown to exert effects on cell adhesive properties in various cell types (Gospodarowicz et al., 1986), and recent studies in our (Heuer et al., 1990) and other (Murphy et al., 1990; Drago et al., 1991a) laboratories revealed an adhesion-promoting effect of bFGF on early neural tube cells in culture. Since cell-to-extracellular matrix (ECM) interactions are of great importance in embryonic development of the nervous system (Sanes, 1989; Reichardt and Tomaselli, 1991), we sought to characterize further the bFGF effect to gain insight into possible bFGF functions in the development of early neural tube in vivo. We report here that early neural tube cells in culture respond to bFGF by showing markedly improved cell spreading. This effect is dependent on the presence of specific ECM proteins and the function of integrin, is accompanied by increased expression of β1-integrin and vinculin and is developmentally regulated. MATERIALS AND METHODS Materials Materials were obtained from the following sources: Nunclon 4well multidishes from Nunc (Naperville, IL); 24- and 96-well culture plates from Corning (Corning, NY); Ham’s F12 nutrient mixture, mouse laminin, trypsin (1:250), trypsin-EDTA and penicillin G-streptomycin sulfate from Gibco (Grand Island, NY); fetal

bovine serum from JRH Bioscience (Lenexa, KS); Vitrogen (type I collagen) from Celtrix Laboratories (Palo Alto, CA); poly-Llysine hydrobromide (Mr 150,000-300,000), poly-L-ornithine hydrobromide (Mr 30,000-70,000), and bovine plasma fibronectin from Sigma Chemical Company (St. Louis, MO); bovine vitronectin from ITOHAM FOODS Inc. (Ibaraki, Japan); RGD peptide (PepTite-2000) from Telios Pharmaceuticals, Inc. (San Diego, CA); Noble agar from Difco (Detroit, MI); CSAT monoclonal antibody, horse radish peroxidase (HRP)-conjugated goat anti-mouse Ig and Enhanced Chemiluminescence (ECL) reagent from Amersham (Arlington Heights, IL), V2E9 and VN3-24 monoclonal antibodies from Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA), biotinylated horse anti-mouse IgG from Vector (Burlingame, CA), and HRP-streptavidin from Zymed (South San Francisco, CA). Nitrocellulose membrane was from Schleicher & Schuell (Keene, NH). bFGF used in the present study was human recombinant bFGF which was a gift from Chiron Corp. (Emeryville, CA). Transforming growth factor-beta 1 (TGFβ1) was provided by Dr M.B. Sporn, National Cancer Institute. βnerve growth factor (NGF) was purified from mouse saliva according to Burton et al. (1978) and Davies et al. (1980). Human recombinant epidermal growth factor (EGF; amino acid 1-51) was obtained from Gibco, and human recombinant insulin-like growth factor-I (IGF-I) and aFGF were from United States Biochemicals (Cleveland, OH). Other chemicals were from Sigma. White Leghorn chicken eggs were obtained from H & N International (Redmond, WA). Coating of culture substratum Culture plastic was routinely coated with fibrillar type I collagen by covering each well of culture plates with a thin film of a diluted Vitrogen solution (1.2 mg/ml; 0.2 mg/ml was also used for some experiments yielding slightly better cell spreading in control cultures), according to the manufacturer’s instruction, and then by allowing it to gel at 37°C. In some experiments where cell detachment was to be minimized to reduce death of nonspread but otherwise viable cells in control cultures, culture plastic was precoated with poly-L-ornithine followed by collagen coating (abbreviated as PORN/collagen). After drying and washing with distilled water, the coated dishes were stored dry. For coating with polycationic materials, culture plates were incubated at least for 4 hours at room temperature with a solution (0.5 ml/15 mm well) of poly-L-lysine at 50 µg/ml, poly-L-ornithine at 100 µg/ml or protamine sulfate at 250 µg/ml. Plates were then washed five times with distilled water and allowed to dry. For experiments with ECM proteins other than collagen, plates were incubated with a solution (0.2-0.5 ml/15 mm well) of fibronectin (10-50 µg/ml), laminin (10-20 µg/ml), vitronectin (10-100 µg/ml) or PepTite-2000 (RGD (arg-gly-asp) peptide chemically modified for surface coating; 20-100 µg/ml) for 2-4 hours at 37°C, washed twice with F12 nutrient mixture and then used immediately. Preparation of dissociated neural tube cells Eggs were incubated at 39°C in a humidified forced-draft incubator until embryos reached the appropriate developmental stages. Embryos were staged according to Hamburger and Hamilton (HH stage; 1951). Embryos at the 26- to 30-somite stage (H-H stage 16-17, approx. 54 hours) were routinely used, except for experiments comparing responses to bFGF of neural tube cells at different developmental stages. Trunk regions containing somites 9 to 16 (counting from the most caudal somite) or as otherwise specified were dissected out, pooled in Hanks’ balanced salt solution (HBSS), and treated with 1% trypsin (1:250) in HBSS for 20-30 minutes at 4°C. Neural tubes were freed from surrounding tissues in HBSS using a pair of tungsten needles; care was taken to remove sclerotomal cells as completely as possible and those not cleaned of these cells satis-

bFGF-promoted neural tube cell adhesion factorily were discarded. Isolated neural tubes were then chopped into small pieces, transferred into a solution of 0.25% trypsin (1:250) in Ca 2+- and Mg 2+-free HBSS (CMF-HBSS) and digested for 5 minutes at 37°C; note that this condition is not completely Ca2+- and Mg2+-free because the chopped neural tubes were not washed with CMF-HBSS and because EDTA was not employed. The digestion was terminated by adding 10% serum-containing culture medium. Cells were sedimented by centrifugation, washed once with culture medium and dissociated to single cells in a small volume (usually 200 µl) of culture medium by trituration through a standard yellow micropipetter tip. Typically, about 1×105 cells were obtained from one neural tube segment and the cell viability was higher than 95% as assessed by trypan blue dye exclusion test. Neural tube cells from younger embryos were prepared by essentially the same procedure. When older embryos (embryonic day (E) 3.5, E5 and E8) were employed, spinal cords were non-enzymatically dissected from the brachial region using tungsten needles, meninges removed when recognizable, and dissociated cells were obtained by trypsin treatment as described above. Neural tube cells thus prepared were counted with a hemocytometer, diluted into culture medium with or without bFGF and plated, usually on the collagen substratum, at different cell densities depending on the types of assays (see below). The culture medium was Ham’s F12 nutrient mixture supplemented with 10% fetal bovine serum, 25 units/ml penicillin G and 25 µg/ml streptomycin sulfate, and cultures were incubated at 37°C in a humidified atmosphere of 95% air-5% CO2. Growth factors were freshly diluted into culture medium for each experiment. bFGF was used at 10 ng/ml, unless otherwise noted. To obtain bFGF-treated cells for assessing their cell spreading and attachment ability after replating and aggregation behavior, cells were seeded onto collagen substratum at 2-2.5×105 cells/cm2 and cultured with or without bFGF (10 ng/ml) for 24 hours. Cultures were rinsed several times with an appropriate buffered saline and harvested with EDTA or trypsin as specified below. Cell yield was only slightly higher (122 ± 5%, mean ± s.e.m. of four experiments) in bFGF-treated cultures than in untreated ones. After washing several times with culture medium, cells were suspended in an appropriate culture medium with or without bFGF, adjusted for cell number and processed for the assays as described below. Cell viability of the cultured cells thus obtained was 90-95%. Cell spreading assay Cell spreading assays were performed using 4-well multidishes (well diameter 15 mm) that had been coated with collagen unless otherwise indicated. Freshly dissociated or cultured neural tube cells (1-2×104 cells in 48 µl) were introduced into a ring placed at the center of each well. The rings (approx. 1.5 mm in height and approx. 5 mm in internal diameter) were made by cutting off the bottom portion of yellow tips for micropipetters. After the completion of cell attachment culture medium (550 µl) was added gently and then the floating rings were removed. In experiments with substrata coated with ECM proteins other than collagen, rings were attached to the bottom of wells with silicone vacuum grease (Dow Corning, Midland, MI) before receiving cell suspension and usually they were not removed after medium addition. Cell spreading index was determined by counting the numbers of total (at least 400, usually 600-800 cells/culture) and spread cells in two or three representative microscopic fields each, of duplicate or triplicate cultures, under a phase-contrast microscope with 200× magnification, and expressed as ‘percent spread cells’. Spread cells included partially spread cells which were moderately phase-dark and flattened around cell perimeter with little membrane blebbing, in addition to unambiguously well-spread cells which usually spread flat enough so that their nuclei are readily recognizable. Caution was taken against prolonged observation of individual dishes, which retarded or even reversed the cell spreading process

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due to the cooling of cultures, and also against the time elapsed while scoring all cultures; to this end, culture dishes were scored in turn for only one well of each dish at a time and in alternating orders for the second and third experimental sets of cultures to average the ‘time effect’ on cell spreading. Cell attachment assay Collagen-coated 96-well plates received 2-3×104 freshly dissociated or cultured cells in 100 µl per well. After incubation for indicated periods of time, cultures were gently washed with CMFPBG (Gey’s PBS) to remove unattached cells, and attached cells were harvested with 0.25% trypsin-1 mM EDTA. Cells were counted using a hemocytometer. Cell proliferation assay Freshly prepared cells were plated into rings at 1×104 cells/well as for cell spreading assays with or without bFGF. At indicated times, cultures were rinsed with CMF-HBSS and cells detached with 0.25% trypsin-1 mM EDTA. All cells, including those in the culture medium withdrawn and in the rinses, were combined and counted with a hemocytometer. Phase-bright cells were counted as viable cells. In this type of experiment, PORN/collagen substratum was also used. In some experiments to determine bFGF’s long-term (days) effects, freshly prepared cells were plated into collagen-coated 96well plates at 3×104 cells/well in normal medium. Medium was changed to either normal or bFGF (10 ng/ml)-supplemented medium about 20 hours after cell plating and then replenished with fresh medium every other day. After the periods of days indicated, cell number was determined as above. Cell proliferation was also monitored by labeling cells in the Sphase, started at the time of cell plating, with 5-bromodeoxyuridine (BrdU, 20 µg/ml), followed by immunocytochemical visualization of incorporated BrdU with anti-BrdU antibody. Cell aggregation assay 24-well plates were coated with 1% agar in HBSS (250 µl/well) to prevent cells from attaching to the plastic surface and to give an appropriate concave surface for cell gathering toward the centers of wells and rinsed with HBSS three times. Cells cultured with or without bFGF for 24 hours and harvested with 0.1 mM EDTA in CMF-HBSS or 1% trypsin (1:250) in HBSS were inoculated into the plate at 2.5×104 cells in 250 µl per well. The plate was then rotated on a gyratory shaker (New Brunswick Scientific Co., Inc., Edison, NJ) at 80 revs/minute for 60 minutes at room temperature (approx. 22°C) in the ambient atmosphere. Cell aggregation was terminated by addition of 5% glutaraldehyde in HBSS and documented by taking photographs. Western immunoblotting Cells were prepared from the level of somite 5-16 (counting from the most caudal somite) and were cultured at 4×105 cells/well (diameter, 15 mm) with or without bFGF for 12 hours. After 2 to 3 rinses of cultures with HBSS, electrophoresis sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS and 10% glycerol) containing protease inhibitors (1 mM benzamidine, 1 mM N-ethylmaleimide and 0.1 mM phenylmethylsulfonylfluoride) was added directly to the cultures. The cell lysates were collected, then boiled for 5 minutes and stored at −20°C until use. Proteins from cell lysates (5×104 or 1 ×105 cells/lane) were separated by electrophoresis on SDS-polyacrylamide gels (7%) (Laemmli, 1970) under nonreducing conditions and electrophoretically transferred to nitrocellulose membrane. Membranes were stained with India ink (Hancock and Tsang, 1983) to confirm uniform and equally efficient protein transfer for samples of control and bFGF-treated cells and then processed for immunodetection. Mouse monoclonal antibodies V2E9 (Hayashi et al., 1990) and VN3-24 (Saga et al., 1985) were

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used to detect β1-integrin and vinculin, respectively. After blocking with 5% nonfat dry milk and 0.2% Tween 20 in phosphate-buffered saline (Glenney, 1986), membranes were incubated with primary antibodies diluted in the blocking buffer overnight, followed by incubation with a HRP-conjugated secondary antibody for VN324 or with biotinylated secondary antibody and then HRP-conjugated streptavidin for V2E9. HRP activity was detected by using the ECL detection system (the staining with India ink did not interfere with immunodetection with the ECL reagent) and recorded on Kodak X-OMAT AR5 X-ray films with the aid of glass mirrors to increase the sensitivity and linearity (BMBiochemica (Boehringer Mannheim), vol. 10, No. 1). Semiquantitative determinations of antigen bands on X-ray films were made with a scanning densitometer (Biomed Instruments, Fullerton, CA).

RESULTS Cell spreading on collagen substratum is markedly improved by bFGF For assessing cell spreading behavior in culture, neural tube cells from 26-30 somite stage embryos were routinely plated on a collagen-coated substratum at a sparse cell density (12×104 cells/20 mm2). Cell attachment was largely completed in 6 hours, yet only a very few cells started spreading by this time. These early spreading cells may represent neural crest cells and possibly contaminating sclerotomal cells. Addition of bFGF at 10 ng/ml markedly improved cell spreading; this effect became discernible, as compared to control cultures, during the period from 6 to 8 hours after cell plating (Fig. 1A,B). By around 15 hours bFGF-treated cultures reached a nearly maximal level of cell spreading (usually around 70% spread cells) while spreading in control cultures remained poor (usually below 20% spread cells) (Fig. 1C,D). Subsequently, bFGF-treated cells became more extensively spread and formed island-like flat cell sheets, whereas in control

cultures only a small fraction of cells showed a comparable extent of cell spreading (cell spreading index reached a level of 20% at most) and the rest of the cells remained only partially spread or barely attached, or started detaching from the substratum (Fig. 1E,F). Finally, at day 2 and later, the resulting cultures without bFGF were far sparser than those with bFGF which eventually formed dense cell sheets composed mainly of cells with astroblastic cell morphology and scattered neuronal and neural crest-derived (melanocytic and Schwannic) cells (Fig. 1G,H). When control cultures were exposed to bFGF after 8 hours in culture, they still responded to bFGF by showing cell spreading to an extent similar to that seen with cultures that were treated with bFGF from the time of plating (% spread cells on PORN/collagen at 21 hours, mean±s.e.m. of triplicate cultures: control, 45.7±0.6%; bFGF 8-21 hours, 72.8±0.8%; bFGF 0-21 hours, 82.9±0.7%). In these cultures, a more adhesive substratum (collagen applied over poly ornithine) was employed, yielding greater cell spreading in control than observed in other experiments employing collagen alone. It was of some concern that the proteolysis routinely employed for cell dissociation might affect the ability of cells to respond to bFGF. Consequently, we examined the response to bFGF of cells prepared using no proteolysis (0.5 mM EDTA in CMF-HBSS), or proteolytic conditions of varying degrees of harshness (0.25% trypsin in HBSS, 0.25% trypsin in CMF-HBSS (CMF-trypsin, the routine procedure), and 0.25% trypsin plus 0.5 mM EDTA in CMFHBSS (trypsin-EDTA)). While the completeness of cell dissociation varied among these protocols, the extents and time courses of bFGF-promoted cell spreading were comparable (data not shown). Also, cells prepared in the continuous presence of bFGF (bFGF was added to all solutions for cell preparation) showed no accelerated cell spreading (data not

Fig. 1. bFGF-induced cell spreading of dissociated neural tube cells on collagen-coated substratum. (A,C,E,G) control, (B,D,F,H) bFGFtreated. Photographs were taken at 7 hours (A,B), 15 hours (C,D), 21 hours (E,F) and 2.5 days (G,H) after cell plating. bFGF, 10 ng/ml. For details, see text. Bar, 100 µm.

bFGF-promoted neural tube cell adhesion shown). Thus, bFGF-dependent cell spreading reflects neither regeneration from proteolytic damage caused by cell dissociation nor retrieval of adhesion mechanisms lost due to bFGF-deprivation during cell preparation. The cell spreading response was detected at 0.1-0.2 ng/ml bFGF, was half maximal around 0.4 ng/ml and was maximal around 10 ng/ml (Fig. 2). This dose-dependence is consistent with the range of bFGF concentrations reported to be effective for mitogenic or neurotrophic actions in other systems (Morrison et al., 1986; Walicke et al., 1986; Gensburger et al., 1987; Unsicker et al., 1987; Walicke, 1988; Eckenstein et al., 1990; Grothe et al., 1991). In contrast to the conspicuous bFGF effect on cell spreading, bFGF did not affect the rate of initial cell attachment of freshly dissociated cells plated on collagen; e.g., the percent attached cells in the presence or absence of bFGF was 34.8±1.3% and 38.0±0.7% at 1.5 hours after plating, and 70.0±0.5% and 70.4±1.3% at 3 hours, respectively (mean±s.e.m. of triplicate cultures). This indicates that bFGF by itself does not act as a cell attachment factor under the assay condition employed. bFGF is a mitogenic/survival factor but the effect is demonstrable only after 1 day To examine whether the cell spreading effect of bFGF is accompanied by a mitogenic/survival effect, the numbers of cells in control and bFGF-treated cultures were determined during the time course of cell spreading assays (up to 1 day). As shown in Fig. 3, there were only very small changes in the number of cells both in the absence and presence of bFGF up to 14 hours, well beyond the time (usually about 8 hours after cell plating) when bFGF-promoted cell spreading was already obvious. During the following time period up to 24 hours, the number of cells was essentially constant and independent of added bFGF. BrdU labeling indices during the time course of cell spreading were comparable between control and bFGFtreated cultures (at 10 hours: control 33.8%, bFGF-treated 28.3%; at 20 hours: control 41.3%, bFGF-treated 42.7%;

Fig. 2. Dose-response of bFGF-induced cell spreading. Cell spreading was determined at two different time points after cell plating. The dotted line represents a rough estimation of the halfmaximal concentration for the data obtained at 17 hours. Values are means of duplicate cultures.

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more than 800 cells were counted). At 10 hours there was still little sign of extensive cell division. The number of BrdU-labeled cells appearing as pairs resulting from recent mitoses (as judged by their close apposition and similar extents of immunostaining intensity) was less than 10% of total cells. At 20 hours there may be a balance of cell proliferation and cell death as the number of cells remained largely unchanged in spite of a BrdU labeling index of about 40%. These results suggest that the bFGF effect on cell spreading is unlikely to be merely a result of bFGF-induced selective and extensive expansion of a cell population with better cell spreading. Effects of bFGF on cell survival and proliferation became apparent after longer culture periods. In the following 24 hours period, the number of cells increased sharply with bFGF, while without bFGF cell number fell substantially below the number initially plated (Fig. 3). It should be also noted that, in the absence of bFGF, the cell survival was much poorer on collagen alone than on PORN/collagen, suggesting that an increased cell-to-substratum contact, even if it is physically achieved through altered substratum, enhances cell survival. While the results described above suggest that bFGF has mitogenic activity, bFGF may merely enhance the survival of cells which proliferate in a bFGF-independent manner. To assess bFGF’s mitogenic effect with the minimal contri-

Fig. 3. Effect of bFGF on the number of cells during the cell spreading response and subsequent cell survival and proliferation. Open symbols, control; closed symbols, bFGF-treated cultures. Cells were plated at 1×104/well in quadruplicate, and counted at times indicated after combining two cultures each. To rescue those cells that detach from the substratum and die simply because they cannot attach firmly to or spread on the substratum, PORN/collagen substratum (without *) was also used in addition to collagen alone (with *). Data from three experiments, represented by circles, squares and triangles, are presented. (inset) Ratio of the number of cells in bFGF-treated versus control cultures. *, as above.

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bution of its effect on cell survival, cells were initially cultured for 20 hours without bFGF to eliminate cells whose survival was most strongly dependent on bFGF, after which cells were cultured in medium containing bFGF, or in control medium without bFGF. As shown in Fig. 4, without bFGF, cell number decreased to a minimum at around day 2 and increased slowly thereafter. In contrast, following bFGF addition, cell number decreased slightly over the first day, and then increased rapidly, with a doubling time of about 24 hours, over the next 2 days. While we cannot exclude the possibility that a portion of the increased cell number in cultures with bFGF still results from enhanced cell survival, it appears that bFGF must also substantially stimulate cell proliferation. bFGF-treated cells show enhanced cell attachment and cell spreading Cells were first cultured with or without bFGF for 24 hours and then harvested and replated on collagen. In spite of no effect of bFGF on cell attachment of freshly dissociated cells as mentioned above, bFGF-treated cells that were harvested with EDTA (0.5 mM in CMF-HBSS, 20 minutes) and replated showed an increased rate of cell attachment as compared to untreated cells (Fig. 5A). bFGF-treated cells also exhibited markedly enhanced cell spreading, which was seen with little time lag following cell plating (Fig. 5B) in sharp contrast to freshly dissociated cells which took several hours to start spreading. This result confirms that a prolonged exposure to bFGF endows cultured cells with tenacious cell-to-substratum adhesive interactions, which are not seen with freshly prepared cells. Also, the bFGFtreated cells similarly showed enhanced cell spreading in the absence of bFGF in replating medium (Fig. 5B), implying that bFGF probably does not directly act as an adhesive substance. Comparable extents of improved cell spreading were observed with cells harvested by 5 minutes treatment with either CMF-trypsin (i. e., the condition routinely employed for preparing freshly dissociated cells) or trypsin-EDTA,

Fig. 4. Stimulation of cell proliferation by bFGF. For details, see Materials and Methods and the text. The values are means ± s.e.m. of triplicate cultures. In some data points, s.e.m. is within the size of symbols.

instead of EDTA treatment as above. Cells harvested by a more vigorously proteolytic method (trypsin-EDTA, 20 minutes) resulted in slightly impaired cell spreading (data not shown). Thus, the adhesion mechanisms established by bFGF action are relatively resistant to the method (CMFtrypsin) we have routinely used to prepare dissociated cells. Treatment with bFGF did not appear to affect cell-to-cell adhesiveness significantly as little difference in cell aggregation ability was seen between bFGF-treated and untreated cells (Fig. 5C). The bFGF effect therefore appears to be directed mainly toward cell-to-substratum (or ECM) interactions. Developmental change in cell spreading ability and its dependence on bFGF To gain insight into possible functional roles of the cell spreading-promoting effect of bFGF, we examined developmental changes in the ability of neural tube cells to spread with or without added bFGF (we will refer to spreading in the absence of added bFGF as ‘bFGF-independent’ cell spreading, but in doing so we do not mean to exclude the possibility that this cell spreading might be the result of endogenously expressed bFGF). First, cell spreading behavior was assessed in relation to the rostrocaudal gradient of development along the neuraxis; four rostrocaudally different neural tube cell preparations were made from 32-somite stage embryos (H-H stage 18, approx. 66 hours), each representing the first to fourth neural tube segments of 8-somite-pair length. Both the bFGF-dependent and bFGFindependent cell spreading were found to be better rostrally (Fig. 6A), apparently corresponding to a developmental gradient that exists along the neuraxis at this stage of development. Similarly, more rostrally derived cells showed greater extents of cell survival without bFGF, while cells from the most caudal segment were extremely dependent on bFGF for cell survival (not shown). Next, to confirm that the rostrocaudal gradient of bFGF responsiveness reflects a gradient in developmental state, neural tube cells at the 8th-15th somite level from embryos at 35-somite stage (H-H stage 18-19, 68 hours), 24- to 25somite stage (H-H stage 15, 54 hours) or 16- to 18-somite stage (H-H stage 12-13, 48 hours) were prepared in parallel and examined for their differential cell spreading abilities. Cells from older embryos were again characterized by better bFGF-independent and bFGF-dependent cell spreading (Fig. 6B). Thus, cell spreading ability of neural tube cells shows a clear spatiotemporal developmental gradient. Interestingly, cells from the rostral cranial neural tube (prosencephalon and mesencephalon) of 8- to 10-somite stage embryos showed poorer and slower cell spreading response to bFGF than did the rest of the caudal neural tube cells (at 6 hours, 24.4% versus 65.0%: at 14 hours, 62.9% versus 81.7%; means of duplicate cultures). This may relate to the fact that, in the early embryonic brain, neural development proceeds in a caudal-to-rostral direction rather than in the rostrocaudal direction observed for the rest of the neuraxis (Windle and Austin, 1936). Neural tube cells from 4-somite stage embryos, the youngest stage examined, responded to bFGF similarly by showing highly bFGF-dependent cell spreading

bFGF-promoted neural tube cell adhesion

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Fig. 5. Adhesive properties of bFGF-treated neural tube cells. (A) Cell attachment. Open circles, untreated cells; closed circles, bFGFtreated cells. Cells were first cultured with or without bFGF (10 ng/ml) for 24 hours. After harvesting by 20 minute treatment with 0.5 mM EDTA in CMF-HBSS, 2.5×104 cells/well were inoculated into 96-well plates with or without bFGF. The percentage of the attached cells (attached cells/plated cells) was determined at the time points indicated and is presented as mean ± s.e.m. of triplicate cultures. (B) Cell spreading. Open circles, untreated cells replated without bFGF; closed circles, bFGF-treated cells replated with bFGF; triangles, bFGF-treated cells replated without bFGF. Cells were cultured and harvested as in A and plated at 1.5×104 cells/20 mm2 with or without bFGF. At the times indicated, % spread cells was determined. The values are means ± s.e.m. of triplicate cultures. (C) Cell aggregation. (a,c) Control cells, (b,d) bFGF-treated cells. Cells cultured with or without bFGF for 24 hours were harvested by 10 minutes treatment with 1% trypsin (1:250) in HBSS (a,b) or by 20 minutes treatment with 0.1 mM EDTA in CMF-HBSS (c,d). The cells were then allowed to aggregate in the corresponding medium with or without bFGF as described in Materials and Methods. Cell aggregation looked better for cells harvested with trypsin in HBSS compared to cells harvested with EDTA partly because the trypsin treatment in HBSS (containing Ca2+) did not completely dissociate cultured cells to single cells, leaving some cell clumps undissociated (up to 4-5 cells/clump). Bar, 100 µm.

and survival (data not shown), indicating early establishment of the responsiveness to bFGF. The cell spreading response to bFGF appears to be gradually lost as neural tube develops to spinal cord (Fig. 7). Spinal cord cells prepared from brachial regions of E3.54 (H-H stage 23), E5 (H-H stage 27) and E8 (H-H stage 34) embryos survived well without bFGF. While cells from E3.5 and E5 embryos still showed a moderate to weak, yet typical cell spreading response to bFGF, those from E8 embryos apparently lacked such responsiveness. The gradual loss of response to bFGF was accompanied by slower initiation of bFGF-induced cell spreading; the bFGF effect was already obvious at 10 hours after cell plating in E3.5-4 neural tube/spinal cord cell cultures (Fig. 7A,B), but only barely detectable at this time point in E5 cultures (Fig. 7E,F). The effect in the E5 culture became obvious after one day (Fig. 7G,H), but was not yet readily detectable in E8 cultures (Fig. 7I,J). Cells with neuronal morphology (possessing neurite-like processes) showed little morpho-

logical response to bFGF in either of these spinal cord cultures. FGFR-1 mRNA expression, as revealed by in situ hybridization, was detected uniformly over the entire neural tube at E2 (not shown). It became progressively confined to the ventricular germinal zone of spinal cord (not shown, see Heuer et al., 1990), concomitant with the gradual disappearance of responsiveness to bFGF. Thus, FGFR-1 expression and the resulting responsiveness to bFGF appear to be associated with neuroepithelial cells of neural tube/spinal cord. bFGF-induced cell spreading on different substrata As cell spreading is thought to be highly dependent on the nature of the substratum on which cells are plated, several different types of substrata were compared with collagen for their ability of supporting the bFGF-dependent cell spreading. Uncoated tissue culture plastic did not support

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Fig. 6. Developmental changes in spontaneous and bFGFpromoted cell spreading. (A) Rostrocaudal level-related changes within the neural tube. Rostrocaudal levels of the four cell preparations used are represented by somite numbers. (B) Agerelated changes. Ages of the embryos used are represented by their numbers of somites. Neural tube segments at the 8th to 15th somite level were used. Percentage of spread cells was determined at the times, as indicated, after cell plating with or without bFGF (5 ng/ml). Values are means of duplicate cultures (A) or means ± s.e.m. of triplicate cultures (B).

any cell spreading even in the presence of bFGF. Polycationic materials such as poly-L-lysine, poly-L-ornithine and protamine sulfate also failed in supporting bFGF-inducible cell spreading (Figs 8, 9). Thus, the cell-to-substratum interaction necessary for bFGF-promoted cell spreading is apparently not electrostatic in nature but is likely to be dependent on ECM proteins such as collagen. Therefore, we compared the abilities of collagen, fibronectin, laminin, vitronectin and RGD peptide to support cell spreading (Figs 8, 9). Laminin at 10-20 µg/ml promoted both bFGF-independent and bFGF-dependent cell spreading as effectively as did collagen. Fibronectin (10-50 µg/ml) was also supportive of bFGF-independent and bFGF-

dependent cell spreading, especially at higher concentrations, but was less effective than collagen or laminin (Fig. 9). RGD peptide (PepTite-2000, 20-100 µg/ml) and vitronectin (10-100 µg/ml) did not support any significant level of cell spreading. These results indicate that both bFGF-independent and bFGF-induced cell spreading is specifically dependent on certain types of ECM components. It should be noted that the bFGF requirement for optimal cell spreading was not eliminated by simply providing cells with substrata coated with appropriate ECM components, indicating that the cell spreading effect of bFGF does not primarily result from stimulated production/secretion of any of the ECM proteins examined here. These results raise the possibility that bFGF may modulate cells’ ability to recognize and interact with ECM proteins. Because of the well-established importance of the integrin class of receptors for cellular adhesion to collagen, laminin and fibronectin, we first examined whether bFGFpromoted cell adhesion involved integrin. The CSAT monoclonal antibody to β1-subunit of chick integrin (Neff et al., 1982; Buck et al., 1986) effectively blocked cell adhesion and spreading on collagen in the presence of bFGF (Fig. 10A,B) and in its absence as well (not shown). Fluorescent immunostaining with the same CSAT antibody showed typical fibrillar and/or punctate staining at the perimeters of spread cells, but failed to reveal any consistent difference in the pattern and intensity of staining between spread cells in control and bFGF-treated cultures (not shown). Accordingly, expression of β1-integrin was assessed by western immunoblotting probed with another anti-β1 integrin antibody, V2E9 (Fig. 11A). The level of β1-integrin was increased about twofold by bFGFtreatment as determined by densitometry. Expression of vinculin, an intracellular component of focal adhesion complexes, was also found augmented about twofold after bFGF treatment (Fig. 11B). Involvement of protein synthesis in cell spreading response to bFGF The results described above suggest that bFGF promotes cell spreading by inducing or maintaining the expression of certain adhesion mechanisms that utilize ECM proteins to allow cells to spread. To examine whether this effect might require protein synthesis de novo, the effect of inhibition of protein synthesis was examined. The protein synthesis inhibitor cycloheximide (1 µg/ml) completely abolished bFGF’s cell spreading effect on freshly dissociated neural tube cells (Fig. 10C,D), even when added 5 hours after cell plating (not shown). This indicates that continuous protein synthesis is necessary for bFGF to exert its cell spreading-promoting effect on freshly dissociated cells. We also examined the effect of cycloheximide on cells that were treated with bFGF for 24 hours. Upon treatment with cycloheximide, these cells, which were fully spread initially, started to round up within 2 to 3 hours. When the bFGF-treated cells were exposed to cycloheximide after replating, the effect of cycloheximide was profound for cells that were subjected to the most proteolytic harvest condition (trypsin-EDTA, 20 minutes) that was shown to slightly


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Fig. 7. Cell spreading response to bFGF of spinal cord cells from E3.5-4 (A-D), E5 (E-H) and E8 (I,J) embryos. Cells from a brachial region were cultured with (lower panels) or without (upper panels) bFGF at 5 ng/ml or 10 ng/ml (J). Photographs were taken at 10 hours (A,B,E,F), 23 hours (C,D,G,H) or 26 hours (I,J) after cell plating. Bar, 100 µm.

impair cell spreading (see above). Nevertheless, the inhibitory effect was also evident, though to a lesser extent, even under cell harvest conditions (EDTA, 20 minutes or CMF-trypsin, 5 minutes) shown to cause little proteolytic damage of adhesion mechanisms (see above). These results collectively suggest that the cell-to-substratum adhesion machinery is dynamically maintained, requiring continuing protein synthesis for its maintenance even after 24 hours treatment with bFGF. Effects of other growth factors Several growth factors in addition to bFGF were examined for their cell spreading and mitogenic effects, including aFGF, IGF-I, EGF, TGF-β1 and NGF. Cell spreading at 22 hours (30.9±0.3% in control culture, mean±s.e.m. of triplicate cultures) was slightly improved with 100 ng/ml EGF (39.4±3.4%) and substantially with 10 ng/ml aFGF (62.1±3.7%) and 100 ng/ml IGF-I (60.1±1.1%), yet bFGF at 10 ng/ml (74.1±1.5%) was most effective. In spite of their positive effects on cell spreading, neither EGF nor IGF-I served as cell survival and/or mitogenic factors: with 1×104 cells/culture initially plated in quadruplicate, the number of cells/culture at day 3, presented as an average of two values obtained by combining each two cultures, was 0.31×104 for EGF, 0.46×104 for IGF-I and 0.31×104 for control. aFGF showed a moderate cell survival/mitogenic effect (1.00×104), which was yet far smaller than that of bFGF (1.78×104). In other experiments, neither NGF (50 ng/ml) nor TGF-β1 (0.01-10.0 ng/ml) elicited any cell spreadingpromoting effect.

DISCUSSION We have shown in the present study that bFGF action is necessary for young neural tube cells to achieve optimal cell spreading on ECM proteins in culture, suggesting a fundamental role of bFGF in the regulation of cell-to-ECM adhesive interactions in the developing central nervous system. Since cell-to-ECM interactions have significant effects on cell proliferation, migration and differentiation in many developing systems including the nervous system (Sanes, 1989; Reichardt and Tomaselli, 1991), we would accordingly suggest that the bFGF-promoted interactions with ECM may underlie some aspects of the mitogenic and differentiative/survival effects of bFGF on neural development. Indeed, in addition to its effect on cell spreading, bFGF elicited cell survival and mitogenic effects on neural tube cells. Since the latter two effects were notable only after 24 hours in culture while the cell spreading effect was already clearly seen by 8 hours, it is unlikely that the cell spreading effect is a mere consequence of the improved cell survival with bFGF. It is also unlikely that the improved cell spreading with bFGF results from the proliferation of a selected cell population(s) which show bFGF-dependent cell spreading. While cell spreading is not a consequence of cell survival, the converse may be true to some extent, since a more adhesive substratum (PORN/collagen as compared to collagen) supported better cell spreading with improved cell survival. However, the ability of growth factors such as IGFI and EGF to promote cell spreading without enhancing cell survival or proliferation suggests that effects of bFGF on

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Y. Kinoshita and others proliferation and survival may be at least partly independent of effects on cell spreading. The effect of bFGF on cell spreading in our cultures appears to be primarily directed toward neuroepithelial cells, since there is progressive loss of the bFGF response and correspondingly restricted expression of FGFR-1 mRNA in the ventricular zone as neural tube develops to spinal cord. The cell spreading response of early neuroepithelial cells to bFGF appears relatively unique; Somite and neural crest cells which also express FGFR-1 (Heuer et al., 1990) do not respond in a similar manner. Spreading of neural crest cells is unaffected by bFGF while bFGF causes more rapid spreading of somite cells but does not influence the ultimate extent of spreading. The present study revealed that the bFGF-promoted cell spreading can be realized only on collagen, fibronectin or laminin substrata among those examined. All of these three ECM components are enriched in the basement membrane of neural tube/spinal cord throughout the embryonic stages examined here, yet intercellular distribution of these proteins seems to be limited (Duband et al., 1986; Krotoski et al., 1986; Duband and Thiery, 1987). Therefore, the bFGF effect described here is likely to be directed mainly to the interaction between neuroepithelial cells and the basement membrane. Proliferative neuroepithelial cells in the neural tube maintain constant contact with the overlying basement membrane at their basal surfaces. When cells traverse the mitotic phase of the cell cycle, however, this cell-to-ECM interaction must be modified to allow cells to temporarily terminate the cell anchoring to the basement membrane (Jacobson, 1991). This does not necessarily imply that bFGF-promoted anchoring of cells to the basement membrane may function anti-proliferatively. In fact, retinal neuroepithelial cells require interactions with ECM for their proliferation (Reh and Radke, 1988), and bFGF’s mitogenic activity on P19 embryonal carcinoma cells (Schubert and Kimura, 1991) and rat Schwann cells (Chen et al., 1991) depends on ECM protein-coated substrata. Also, cell spreading ability has been shown to be an important determinant of cell proliferation of anchorage-dependent cells in vitro (O’Neill et al., 1990), consistent with our data obtained here. Thus, optimally regulated proliferation of neuroepithelial cells in vivo appears to require, in addition to adequate growth factor action, an appropriate cell-to-ECM interaction, which is now recognized as a signal transductional event (Reichardt and Tomaselli, 1991; Hynes, 1992; Zachary and Rozengurt, 1992; Juliano and Haskill, 1993).

Fig. 8. Micrographs of neural tube cells spreading on different substrata with or without bFGF. TCP, uncoated tissue culture plastic; PORN, poly-L-ornithine (100 µg/ml); CL, collagen (type I, 1.2 mg/ml); FN, fibronectin (50 µg/ml); LN, laminin (20 µg/ml); RGD, RGD peptide (PepTite-2000, 100 µg/ml); VN, vitronectin (50 µg/ml). Photographs were taken at 12 hours after cell plating for collagen, laminin and fibronectin, and at 22 hours for the others. Note that collagen (CL), laminin (LN) and fibronectin (FN) already supported bFGF (10 ng/ml)-inducible cell spreading at 12 hours after cell plating, whereas neither of the other substrata supported cell spreading even after a prolonged incubation (for 22 hours) with bFGF. Bar, 100 µm.


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Fig. 9. ECM protein-dependent cell spreading response of neural tube cells to bFGF. (A) Cell spreading on non-ECM components, (B) Cell spreading on ECM components. PLL, poly-L-lysine (50 µg/ml), PS, protamine sulfate (250 µg/ml). The abbreviations and concentrations of the other agents used for substratum coating are as in Fig. 9. Percent of spread cells was determined at the times after cell plating as indicated. Values are means of duplicate cultures. *, not determined at 7.5 hours.

Our results suggest that this cell-to-ECM interaction is also regulated by the growth factor bFGF itself. The contact of neuroepithelial cells with the basement membrane is permanently lost once neuroepithelial cells enter their terminal mitoses to start neuronal differentiation. In this context, bFGF-promoted anchoring of neuroepithelial cells to the basement membrane may modify the proportion of cells that become neurons. Indeed, ECM components can directly influence neuronal determination/ differentiation (e.g. Reh et al., 1987). We have previously demonstrated that expression of FGFR-1 mRNA ceases as cells migrate out from the ventricular germinal zone (Heuer et al., 1990). Cessation of FGFR expression may terminate the FGF-promoted adhesion of cells to the basement membrane, thereby signaling those cells to withdraw from the mitotic cell cycle and to migrate from the ventricular germinal zone to form the mantle layer. The modification of cellular synthesis and secretion of ECM components is a primary consequence of FGF action in several types of cells (Gospodarowicz et al., 1986). Indeed, Drago et al. (1991a) reported bFGF-promoted laminin expression in E10 mouse brain neuroepithelial cell cultures. However, this mechanism is unlikely to play a significant role in bFGF-enhanced cell spreading observed in the present study as the effect was entirely dependent on exogenously added ECM components. Rather, it is likely that bFGF regulates the expression or functional state of receptor systems interacting with ECM proteins. The integrin family of α/β heterodimeric proteins are the best characterized receptors for ECM proteins (Reichardt and Tomaselli, 1991; Hynes, 1992). During development in vivo, integrin molecules, as identified by the CSAT antibody that recognizes chick β1-integrin (Neff et al., 1982; Buck et al., 1986), are already expressed relatively uniformly on the neural tube cell surface well in advance of the embryonic ages that we examined here (Krotoski et al., 1986). Using the CSAT antibody, we showed that the majority of spread cells in neural tube cell cultures express β1-integrin. We also demonstrated the functional involvement of integrin by showing that the CSAT antibody, which is functionblocking (Neff et al., 1982), completely inhibited cell spreading in both control and bFGF-treated cultures. Western immunoblotting analyses revealed an increase in β1-integrin expression as a result of bFGF treatment. In addition, expression of vinculin, a component of focal

adhesion complexes, which is preferentially expressed in neuroepithelial cells (Duband and Thiery, 1990) was also demonstrated to be increased to a similar extent by bFGF treatment. Thus, bFGF-stimulated expression of β1-integrin and vinculin provides, at least in part, a molecular basis for the bFGF-promoted cell spreading. It is unclear whether the observed two-fold increase in expression of these components of focal adhesion complexes is sufficient to account for the dramatic increase in cell spreading observed. Since the cell spreading response is likely to be a non-linear function of density of focal adhesion complexes, it is possible that a two-fold increase in such complexes could dramatically increase cell spreading. Alternatively, bFGF may also regulate the dynamic state of adhesion complexes through modification of the phosphorylation status of their components like integrin, vinculin and talin. This is in keeping with the recent identification of adhesion complexes as a site of signal transduction from the ECM and of a possible crosstalk with growth factor-mediated signal transduction pathways through phosphorylation (Hynes, 1992; Luna and Hitt, 1992; Zachary and Rozengurt, 1992; Juliano and Haskill, 1993). Is bFGF available to neuroepithelial cells in vivo at the developmental stages examined here? Kalcheim and Neufeld (1990) demonstrated the presence of bFGF immunoreactivity in 1-day cultures of E2 quail neural tube cells and in E3 neural tube extracts. However, recent results of in situ hybridization in this laboratory (P. Evers, manuscript in preparation) reveals the presence of bFGF mRNA in chick neural tube neuroepithelial cells at the early embryonic stages employed in the present study. Other studies have shown that an FGF activity is present in E2 chick embryos (Seed et al., 1988) and that functional bFGF is present even prior to mesoderm induction (Mitrani et al., 1990). Also, E10 mouse cephalic neuroepithelial cells express bFGF mRNA (Drago et al., 1991b). Collectively, these data indicate that bFGF is available to developing chick neural tube cells throughout the embryonic ages examined here, thus making our findings relevant in vivo as well. Although bFGF lacks a conventional secretory signal peptide sequence, a recent finding of naturally occurring cell death in the early neural tube at H-H stages 15-18 (Homma et al., 1990) suggests that bFGF might be released by cell lysis. Alternatively, an intracrine mode of action, as suggested earlier (Halaban et al., 1988; Neufeld et al., 1988;

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Fig. 10. Effects of CSAT antibody and cycloheximide on bFGFpromoted cell spreading. (A, B) Cells were cultured for 20 hours with 10 ng/ml bFGF in the absence (A) or presence (B) of 5 µg/ml CSAT monoclonal antibody. The control culture (A) received nonimmune mouse IgG and the antibody buffer solution including NaN3 (0.0025% at the final concentration), where cell spreading was almost indistinguishable morphologically from that in a parallel culture which was treated only with bFGF (not shown). (C, D) Cells were cultured for 20 hours with 10 ng/ml bFGF (C) or 10 ng/ml bFGF plus 1 µg/ml cycloheximide (D). Note that cells in both treated cultures (B, D) appear viable. Bar, 100 µm.

Yayon and Klagsbrun, 1990), might yield functional responses in the absence of bFGF release. Finally, however, there is evidence that bFGF may be secreted by some cells, employing a mechanism that remains to be characterized (e.g., Mignatti et al., 1991). The endogenous effector for neuroepithelial cell FGFRs may be another member of the FGF family rather than bFGF. Indeed, aFGF showed a moderate extent of cell spreading and cell survival effects. aFGF mRNA is expressed at a low level in E3.5 chick brain with a progressive increase later on (Schnürch and Risau, 1991). However, our preliminary results (P. Evers, manuscript in preparation) indicate that only very low levels of aFGF mRNA are present in neural tube and surrounding mesenchyme at the earlier developmental period examined in the present study. bFGF and int-2 have been shown to be functionally equivalent in promoting otic vesicle formation (Represa et al., 1991). int-2 expression is, however, restricted to a limited region of hindbrain (Wilkinson, 1990). FGF-4 mRNA has recently been localized to neuroepithelial cells in mouse (Drucker and Goldfarb, 1993). The expression of other FGF family members in neural tissue has not been characterized. Similarly, the FGFR type responsible for mediating the bFGF actions that we have characterized may not be FGFR1. In addition to FGFR-1 (Heuer et al., 1990; Wanaka et al., 1991), FGFR-2 is also expressed during early neural tube development (Reid et al., 1990; Orr-Urtreger et al., 1991; Peters et al., 1992a). FGFR-3 has recently been demonstrated to be expressed in the neural tube/spinal cord (Peters et al., 1993; P. Evers, manuscript in preparation). Work in progress in this laboratory (P. Evers, unpublished data)

Fig. 11. Western immunoblotting analyses of bFGF effect on β1integrin and vinculin expression. (A) β1-integrin. (Lane 1) E13 chicken breast muscle as a reference, (lane 2) control neural tube cells, (lane 3) bFGF-treated neural tube cells. The relative molecular mass of the immunoreactive band was determined to be 110×103, in good agreement with published data (Hall et al., 1987). The bands indicated by asterisks are nonspecific bands due to the biotinylated secondary antibody and/or HRP-streptavidin. They serve as an internal standard indicating that the amounts of proteins in the lanes for control (lane 2) and bFGF-treated (lane 3) are similar. (B) Vinculin. (Lane 1) bFGF-treated, (lane 2) control. The relative molecular mass of the band is 135×103, close to the value published (Saga et al., 1985). The same batch of cell lysate was used for the analyses in A and B.

demonstrates that in chick neuroepithelial subpopulations FGFR-3 expression appears to mark neuroepithelial cells in regions of the neural tube in which neuronal differentiation is actively underway. Thus complex interactions involving multiple FGFs and FGFRs may differentially regulate ECM adhesion, proliferation and differentiation. Cell-to-ECM adhesive interactions must be a key factor controlling cell proliferation, migration and differentiation in the developing central nervous system (Sanes, 1989; Reichardt and Tomaselli, 1991). Factors that regulate interactions of neural tube cells with ECM are, therefore, likely to be of substantial importance in histogenic differentiation of the neural tube into the spinal cord and brain. The present results of bFGF effects in culture suggest that bFGF or other FGFs are likely to be involved in this process by modulating the interaction of neuroepithelial cells with ECM. However, it is presently unclear whether the primary role of bFGF is to promote neuroepithelial cell proliferation, thereby suppressing neuronal differentiation, or to promote neuronal differentiation, or both. Murphy et al. (1990) demonstrated that bFGF stimulates neuronal differentiation of E10 mouse neural tube cells, although the differentiative effect is segregatable from a cell proliferative effect of bFGF in an immortalized neuroepithelial cell line. However, for chicken neuroepithelial cells, we have observed that bFGF suppresses initiation of neuronal differentiation (Y. Kinoshita et al., unpublished data). Thus, bFGF or other FGFs may be multifunctional in the developing nervous system. Such multifunctionality may derive from different modes of FGF action including; autocrine, intracrine and paracrine actions (Browder et al., 1989), signaling through

bFGF-promoted neural tube cell adhesion distinctive post-tyrosine kinase receptor pathways (Mohammadi et al., 1993; Peters et al., 1992b), and from the multiplicity of FGF and FGFR families. To examine these possible multiple functions of bFGF or other FGFs in embryonic development, it will be necessary to develop methods to modify FGF actions in vivo and also to establish in vitro culture systems that more faithfully reproduce the behavior of developing neural tube cells. This work was performed with the support of NIH grant NS29582 to M. B. We wish to thank Chiron Corporation for the gift of human recombinant bFGF, Dr T. Yoshihara, ITOHAM FOODS Inc., for a generous gift of vitronectin, Dr M. B. Sporn for providing us with TGF-β1, and Dr C. von Bartheld for critical reading of the manuscript. We also thank Drs M. Iwata and F. Peale for technical advice and thoughtful discussion. The V2E9 and VN3-24 monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biology, University of Iowa, Iowa City, IA, under contract NO1HD-2-3144 from the NICHD.

REFERENCES Acland, P., Dixon, M., Peters, G. and Dickson, C. (1990). Subcellular fate of the int-2 oncoprotein is determined by choice of initiation codon. Nature 343, 662-665. Baird, A. and Walicke, P. A. (1989). Fibroblast growth factors. Br. Med. Bull. 45, 438-452. Birren, S. J. and Anderson, D. J. (1990). A v-myc-immortalized sympathoadrenal progenitor cell line in which neuronal differentiation is initiated by FGF but not NGF. Neuron 4, 189-201. Browder, T. M., Dunbar, C. E. and Nienhuis, A. W. (1989). Private and public autocrine loops in neoplastic cells. Cancer Cells 1, 9-17. Buck, C. A., Shea, E., Duggan, K. and Horwitz, A. F. (1986). Integrin (the CSAT antigen): Functionality requires oligomeric integrity. J. Cell Biol. 103, 2421-2428. Burton, L. E., Wilson, W. H. and Shooter, E. M. (1978). Nerve growth factor in mouse saliva. J. Biol. Chem. 253, 7807-7812. Chen, J.-K., Yao, L.-L. and Jeng, C.-B. (1991). Mitogenic response of rat Schwann cells to fibroblast growth factors is potentiated by increased intracellular cyclic AMP levels. J. Neurosci. Res. 30, 321-327. Davies, R. L., Grosse, V. A., Kucherlapati, R. and Bothwell, M. (1980). Genetic analysis of epidermal growth factor action: Assignment of human epidermal growth factor receptor gene to chromosome 7. Proc. Natl. Acad. Sci. USA 77, 4188-4192. Delli-Bovi, P., Curatola, A. M., Kern, F. G., Greco, A., Ittman, M. and Basilico, C. (1987). An oncogene isolated by transfection of a Kaposi’s sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell 50, 729-737. Dickson, R. B. and Peters, G. (1987). Potential oncogene product related to growth factors. Nature 326, 833-837. Drago, J., Nurcombe, V., Pearse, M. J., Murphy, M. and Bartlett, P. F. (1991a). Basic fibroblast growth factor upregulates steady-state levels of laminin B1 and B2 mRNA in cultured neuroepithelial cells. Exp. Cell Res. 196, 246-254. Drago, J., Murphy, M., Carroll, S. M., Harvey, R. P. and Bartlett, P. F. (1991b). Fibroblast growth factor-mediated proliferation of central nervous system precursors depends on endogenous production of insulinlike growth factor I. Proc. Natl. Acad. Sci. USA 88, 2199-2203. Dreyer, D., Lagrange, A., Grothe, C. and Unsicker, K. (1989). Basic fibroblast growth factor prevents ontogenetic neuron death in vivo. Neurosci. Lett. 99, 35-38. Drucker, B. J. and Goldfarb, M. (1993). Murine FGF-4 gene expression is spatially restricted within embryonic skeletal muscle and other tissues. Mech. Dev. 40, 155-163. Duband, J.-L., Rocher, S., Chen, W.-T., Yamada, K. M. and Thiery, J. P. (1986). Cell adhesion and migration in the early vertebrate embryo:

955

location and possible role of the putative fibronectin receptor complex. J. Cell Biol. 102, 160-178. Duband, J.-L. and Thiery, J. P. (1987). Distribution of laminin and collagens during avian neural crest development. Development 101, 461478. Duband, J.-L. and Thiery, J. P. (1990). Spatio-temporal distribution of the adherens junction-associated molecules vinculin and talin in the early avian embryos. Cell Diff. Dev. 30, 55-76. Eccleston, P. A. and Silberberg, D. H. (1985). Fibroblast growth factor is a mitogen for oligodendrocytes in vitro. Dev. Brain Res. 21, 315-318. Eckenstein, F. P., Esch, F., Holbert, T., Blacher, R. W. and Nishi, R. (1990). Purification and characterization of a trophic factor for embryonic peripheral neurons: comparison with fibroblast growth factors. Neuron 4, 623-631. Finch, P. W., Rubin, J. S., Miki, T., Ron, D., Aaronson, S. A. (1989). Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 245, 752-755. Gensburger, C., Labourdette, G. and Sensenbrenner, M. (1987). Brain basic fibroblast growth factor stimulates the proliferation of rat neuronal precursor cells in vitro. FEBS Lett. 217, 1-5. Giordano, S., Sherman, L., Lyman, W. and Morrison, R. (1992). Multiple molecular weight forms of basic fibroblast growth factor are developmentally regulated in the central nervous system. Dev. Biol. 152, 193-303. Glenney, J. (1986). Antibody probing of western blots which have been stained with India ink. Anal. Biochem. 156, 315-319. Gospodarowicz, D. (1990). Fibroblast growth factor and its involvement in developmental processes. Curr. Top. Dev. Biol.24, 57-93. Gospodarowicz, D., Neufeld, G. and Schweigerer, L. (1986). Molecular and biological characterization of fibroblast growth factor, an angiogenic factor which also controls the proliferation and differentiation of mesoderm and neuroectoderm derived cells. Cell Diff. Dev. 19, 1-17. Grothe, C., Wewetzer, K., Lagrange, A. and Unsicker, K. (1991). Effects of basic fibroblast growth factor on survival and choline acetyltransferase development of spinal cord neurons. Dev. Brain Res. 62, 257-261. Halaban, R., Kwon, B. S., Ghosh, S., P. Delli-Bovi and Baird, A. (1988). bFGF as an autocrine growth factor for human melanomas. Oncogene Res. 3, 177-186. Hall, D. E., Neugebauer, K. M. and Reichardt, L. F. (1987). Embryonic neural retina cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT). J. Cell Biol. 104, 623-634. Hamburger, V. and Hamilton, H. (1951). A series of normal stages in the development of the chick embryo. J. Morph. 88, 49-92. Hancock, K. and Tsang, V. C. W. (1983). India ink staining of proteins on nitrocellulose paper. Anal. Biochem. 133, 157-162. Hayashi, Y., Haimovich, B., Reszka, A., Boettiger, D. and Horwitz, A. (1990). Expression and function of chicken integrin β1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells. J. Cell Biol. 110, 175-184. Heuer, J. G., von Bartheld, C. S., Kinoshita, Y., Evers, P. C. and Bothwell, M. (1990). Alternating phases of FGF receptor and NGF receptor expression in the developing chicken nervous system. Neuron 5, 283-296. Homma, S., Yaginuma, H. and Oppenheim, R. W. (1990). Cell death during the earliest stages of spinal cord development in the chick embryo. Soc. Neurosci. Abst. 16, 836. Hynes, R. O. (1992). Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25. Jacobson, M. (1991). Developmental Neurobiology, 3rd ed., Chapter 2.2. New York: Plenum. Johnson, D. E. and Williams, L. T. (1993). Structural and functional diversity in the FGF receptor multigene family. Adv. Cancer Res. 60, 141. Juliano, R. L. and Haskill, S. (1993). Signal transduction from the extracellular matrix. J. Cell Biol. 120, 577-585. Kalcheim, C. and Neufeld, G. (1990). Expression of basic fibroblast growth factor in the nervous system of early avian embryos. Development 109, 203-215. Krotoski, D. M., Domingo, C. and Bronner-Fraser, M. (1986). Distribution of a putative cell surface receptor or fibronectin and laminin in the avian embryos. J. Cell Biol. 103, 1061-1071. Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

956


Luna, E. J. and Hitt, A. L. (1992). Cytoskeleton-plasma membrane interactions. Science 258, 955-964. Marics, I., Adelaide, J., Raybaud, F., Mattei, M., Coulier, F., Planche, J., Lapeyriere, O. and Birnbaum, D. (1989). Characterization of the hst-related FGF-6 gene, a new member of the fibroblast growth factor gene family. Oncogene 4, 335-340. Mignatti, P., Morimoto, T. and Rifkin, D. B. (1991). Basic fibroblast growth factor released by single, isolated cells stimulates their migration in an autocrine manner. Proc. Natl. Acad. Sci. USA 88, 11007-11011. Mitrani, E., Gruenbaum, Y., Shohat, H. and Ziv, T. (1990). Fibroblast growth factor during mesoderm induction in the early chick embryo. Development 109, 387-393. Mohammadi, M., Dionne, C. A., Li, W., Li., N., Spivak, T., Honegger, A. M., Jaye, M. and Schlessinger, J. (1992). Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenensis. Nature 358, 681-684. Morrison, R. S. and De Vellis, J. (1981). Growth of purified astrocytes in a chemically defined medium. Proc. Natl. Acad. Sci. USA 78, 7205-7209. Morrison, R. S., Sharma, A., De Vellis, J. and Bradshaw, R. A. (1986). Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture. Proc. Natl. Acad. Sci. USA 83, 7537-7541. Murphy, M., Drago, J. and Bartlett, P. F. (1990). Fibroblast growth factor stimulates the proliferation and differentiation of neural precursor cells in vitro. J. Neurosci. Res. 25, 463-475. Neff, N. T., Lowrey, C., Decker, C., Tovar, A., Damsky, C., Buck, C. and Horwitz, A. F. (1982). A monoclonal antibody detaches embryonic skeletal muscle from extracellular matrices. J. Cell Biol. 95, 654-666. Neufeld, G., Mitchell, R., Ponte, P. and Gospodarowics, D. (1988). Expression of human basic fibroblast growth factor cDNA in baby hamster kidney-derived cells results in autonomous cell growth. J. Cell Biol. 106, 1385-1394. O’Neill, C., Jordan, P., Riddle, P. and Ireland, G. (1990). Narrow linear strips of adhesive substratum are powerful inducers of both growth and total focal contact area. J. Cell Sci. 95, 577-586. Orr-Urtreger, A., Givol, D., Yayon, A., Yarden, Y. and Lonai, P. (1991). Developmental expression of two murine fibroblast growth factor receptors flg and bek. Development 113, 1419-1434. Peters, K. G., Chen, W. G. and Williams, L. T. (1992a). Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development 114, 233-243. Peters, K. G., Marie, J., Wilson, E., Ives, H. E., Escobedo, J., Del Rosario, M., Mirda, D. and Williams, L. (1992b). Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis. Nature 358, 678-681. Peters, K. G., Ornitz, D., Werner, S. and Williams, L. (1993). Unique expression pattern of the FGF receptor 3 gene during mouse organogenensis. Dev. Biol. 155, 423-430. Reh, T. A., Nagy, T. and Gretton, H. (1987). Retinal pigmented epithelial cells induced to transdifferentiate to neurons by laminin. Nature 330, 6871. Reh, T. A. and Radke, K. (1988). A role of the extracellular matrix in retinal neurogenesis in vitro. Dev. Biol. 129, 283-293. Reichardt, L. F. and Tomaselli, K. J. (1991). Extracellular matrix molecules and their receptors: Functions in neural development. Ann. Rev. Neurosci. 14, 531-70. Reid, H. H., Wilks, A. F. and Bernard, O. (1990). Two forms of the basic fibroblast growth factor receptor-like mRNA are expressed in the developing mouse brain. Proc. Natl. Acad. Sci. USA 87, 1596-1600. Represa, J., León, Y., Miner, C. and Giraldez, F. (1991). The int-2 protooncogene is responsible for induction of the inner ear. Nature 353, 561563. Saga, S., Hamaguchi, M., Hoshino, M. and Kojima, K. (1985). Expression of meta-vinculin associated with differentiation of chicken embryonal muscle cells. Exp. Cell Res. 156, 45-56. Sanes, J. R. (1989). Extracellular matrix molecules that influence neural development. Ann. Rev. Neurosci. 12, 491-516.

Schnürch, H. and Risau, W. (1991). Differentiating and mature neurons express the acidic fibroblast growth factor gene during chick neural development. Development 111, 1143-1154. Schubert, D. and Kimura, H. (1991). Substratum-growth factor collaborations are required for the mitogenic activities of activin and FGF on embryonal carcinoma cells. J. Cell Biol. 114, 841-846. Seed, J., Olwin, B. B. and Hauschka, S. D. (1988). Fibroblast growth factor levels in the whole embryo and limb bud during chick development. Dev. Biol. 128, 50-57. Slack, J. M. W. (1990). Growth factors as inducing agents in early Xenopus development. J. Cell Sci. Suppl. 13, 119-130. Taira, M., Yoshida, T., Miyagawa, K., Sakamoto, H., Terada, M. and Sugimura, T. (1987). cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity. Proc. Natl. Acad. Sci. USA 84, 2980-2984. Tanaka, A., Miyamoto, K., Minamino, N., Takeda, M., Sato, B., Matsuo, H., and Matsumoto, K. (1992). Cloning and characterization of an androgen-induced growth factor essential for the androgen-dependent growth of mouse mammary carcinoma cells. Proc. Natl. Acad. Sci. USA 89, 8928-8932. Togari, A., Dickens, G., Kuzuya, H. and Guroff, G. (1985). The effect of fibroblast growth factor on PC12 cells. J. Neurosci. 5, 307-316. Unsicker, K., Reichert-Preibsch, H., Schmidt, R., Pettmann, B., Labourdette, G. and Sensenbrenner, M. (1987). Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons. Proc. Natl. Acad. Sci. USA 84, 5459-5463. Wagner, J. A. (1991). The fibroblast growth factors: An emerging family of neural growth factors. Curr. Top. Microbiol. Immunol. 165, 95-118. Wagner, J. A. and D’Amore, P. A. (1986). Neurite outgrowth induced by an endothelial cell mitogen isolated from retina. J. Cell Biol. 103, 13631367. Walicke, P. A. (1988). Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions. J. Neurosci. 8, 2618-2627. Walicke, P., Cowan, W. M., Ueno, N., Baird, A. and Guillemin, R. (1986). Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. Natl. Acad. Sci. USA 83, 3012-3016. Wanaka, A., Milbrandt, J. and Johnson, Jr., E. M. (1991). Expression of FGF receptor gene in rat development. Development 111, 455-468. Westermann, R., Grothe, C. and Unsicker, K. (1990). Basic fibroblast growth factor (bFGF), a multifunctional growth factor for neuroectodermal cells. J. Cell Sci. Suppl. 13, 97-117. Wilkinson, D. G. (1990). Segmental gene expression in the developing mouse hindbrain. Semin. Dev. Biol. 1, 127-134. Windle, W. F. and Austin, M. F. (1936). Neurofibrillar development in the central nervous system of chick embryos up to 5 days’ incubation. J. Comp. Neurol. 63, 431-463. Yayon, A. and Klagsbrun, M. (1990). Autocrine transformation by chimeric signal peptide-basic fibroblast growth factor: Reversal by suramin. Proc. Natl. Acad. Sci. USA 87, 5346-5350. Yoshida, T., Miyagawa, K., Odagiri, H., Sakamoto, H., Little, P. F. R., Terada, M. and Sugimura, T. (1987). Genomic sequence of hst, a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2 encoded protein. Proc. Natl. Acad. Sci. USA 84, 7305-7309. Zachary, I. and Rozengurt, E. (1992). Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell 71, 891-894. Zhan, X., Bates, B., Hu, X. and Goldfarb, M. (1988). The human FGF-5 oncogene encodes a novel protein related to fibroblast growth factors. Mol. Cell. Biol. 8, 3487-3495.

(Accepted 3 August 1993)