We have studied the effects of proteoglycans (PGs) and glycosaminoglycans (GAGs) on the growth and morphology of neurons in culture. PGs from glial cells or Engelbreth-Hohn-Swarm tumor cells (EUS), pure bovine kidney heparan sulfate (HS), shark cartilage type C chondroïtin sulfate (CSc) and bovine mucosa dermatan sulfate (DS) added to embryonic rat neurons strongly enhanced total neurite growth after 48 h in vitro. No trophic effects were seen when PGs treated with a mixture of glycanases were used. PGs, CSc and HS not only enhanced neurite growth but induced the appearance of a majority of neurons with a single long axon whereas, in contrast, DS increased dendrite growth. GAGs bound to the cell surface and were rapidly internalized, a feature that correlated well with the absence of neurotrophicity of GAGs previously immobilized on the culture substratum. Although the mechanisms involved in GAGs neurotrophic effects and in the separate regulation of neuronal polarity by HS and DS were not elucidated, we found that, as opposed to HS, DS was able to enhance neuronal adhesion and spreading and to maintain a high level of expression of microtubule-associated protein 2 (MAP2), a specific dendritic marker. This finding confirms and extends our previous observations on the role of adhesion in the regulation of dendrite growth.

Extracellular matrix (ECM) molecules play a major role in the development of the nervous system. Two well-characterized ECM molecules are laminin and fibronectin, which participate in the attachment, migration and differentiation of peripheral and central neurons, both in vitro and in vivo (for a review see Sanes, 1989). More recently, several studies have indicated that proteoglycans (PGs) secreted in the extracellular space or linked to the neuronal membrane by a true transmembrane segment or a glycolipid anchor participate in neuronal differentiation (Matthew et al., 1985; Cole et al., 1986; Fransson, 1987; Hantaz-Ambroise et al., 1987; Dow et al., 1988; Ruoslahti, 1988; Werz and Scbachner, 1988).

Proteoglycans are composed of a protein core on which long chains of various sulfated sugars are attached, sulfated dermatan, heparan and chondroïtin being the most common found in brain PGs (Margolis and Margolis, 1989; Herndon and Lander, 1990). In spite of their developmentally regulated expression within the mammalian brain (Oohira et al., 1986; Herndon and Lander, 1990), the exact role of these proteoglycans and glycosaminoglycans during development is still unclear. However, several reports indicate that they can regulate neuronal maturation, either directly or in association with other neurotrophic molecules. For example, Muir et al. (1989) reported that a specific proteoglycan could inhibit the neurite growth activity of laminin. Conversely, proteoglycans acting as growth factor receptors (e.g. TGF/i or basic FGF) might have a strong growth-promoting activity (Neufeld et al., 1987; Burgess and Maciag, 1989; Cheifetz and Massagué, 1989; Gordon et al., 1989; Ruoslahti, 1989; Kiefer et al., 1990).

This capacity of proteoglycans and glycosaminoglycans (GAGs) to bind, concentrate and eventually internalize several basic growth factors is finked both to the conformation of the carbohydrate chains and to their high content of negative charges. Thus, it is not surprising that GAGs, in the absence of their protein cores, can themselves be biologically active entities (Verna et al., 1989). However, the exact role and mode of action of proteoglycans and, in particular, the actual function of the complex sugars which, in many cases, constitute the most abundant part of the molecule have not been elucidated.

In previous studies we have used an experimental in vitro model that allows us to study in a quantitative manner the effect of various compounds on the growth of neurites and on the development of neuronal polarity. We have established that the growth of axons and dendrites could be regulated separately (Denis-Donini et al., 1984; Chamak et al., 1987) and that ECM molecules present in astrocyte-conditioned media are instrumental in this regulation (Rousselet et al., 1988, 1990). Moreover, we have demonstrated that the influence of different matrix molecules on neuronal shape and polarity (i.e., the growth of axons or dendrites) varied depending on how they were presented to the cells. In particular, it is clear that substratum-bound and soluble ECM factors induce different patterns of growth and polarity (Chamak and Prochiantz, 1989; Lochter et al., 1991).

Since, in addition to laminin and fibronectin, proteoglycans are also present in astrocyte-conditioned medium, the present studies analysed the potential influence of these molecules on neuronal growth and polarity. We report here that astrocyte-derived proteoglycans and proteoglycans from the EHS tumor stimulate axonal growth in vitro. Moreover, we demonstrate that this influence of PGs on neuronal polarity can be reproduced by the addition in the culture medium of small concentrations of heparan or chondroïtin sulfates and thus does not seem to depend on the presence of the PG core protein. Very interestingly, we find that dermatan sulfate, as opposed to heparan sulfate, has a strong tendency to stimulate dendrite elongation and that the action of these two glycosaminoglycans is associated with their binding to the nerve cells and their subsequent internalization.

Culture media, DMEM and F12 were from Gibco. Penicillin, streptomycin, insulin, transferin, progesterone, putrescine, selenium sodium salt and the different GAGs (bovin kidney heparan sulfate; bovin mucosa dermatan sulfate and shark cartilage type C chondroïtin sulfate) were purchased from Sigma. Protease-free heparinase from Flavobacterium hepari-num, heparitinase and chondroïtin ase ABC from Proteus vulgaris were from Seikagaku Kogyo Co, Ltd (Miles). Antibodies against microtubule-associated protein 2 (MAP2) and Neurofilament-H (NF-H) were kind gifts of Drs A. Fellous and P. Levitt, respectively. The polyclonal antibodies against laminin and high molecular weight core protein of the HSPG from the EHS tumor, as well as the tumor itself, were provided by Dr. M. Vigny. TRITC-bnked second antibodies were from Biosis. Biotinylated antibodies and labelled streptavidin were from Amersham.

Cell culture

All cell culture protocols were as described previously (Rousselet et ai., 1988, 1990). In brief, cells were mechanically dissociated from rat embryonic mesencephalon (ED14), seeded at a density of 25 × 103 cells cm−2 (morphological and imunocytochemical analysis) or 105 cells cm−3 (ELISA and adhesion assays) on plastic tissue culture wells (Nunc) precoated with 1.5 μg ml−1 D,L-polyomithine (Mr 40,000, Sigma) and cultured in chemically defined medium (CDM). Astrocyte or EHS proteoglycans (1 and 4 μg ml−1 respectively) and the various GAGs-(10 μg ml−1) were added 30 minutes after seeding. Mesencephalic astrocytes were prepared by culturing the cells, seeded at the density of 3.5 × 10s cells cm−2, for 3 weeks in the presence of 8% FCS. It is worth noting that, although most of these experiments presented here were done with mesencephalic neurons, similar results were obtained with neurons from cortex, spinal cord or striatum.

Morphological analysis

Cells were cultured for 2 days, fixed with 2.5% glutaraldehyde in PBS pH 7.4 for 30 minutes at room temperature, washed twice with PBS and stained with toluidine blue (0.2% in 1% NajCOj). Stained cells were air dried and examined with an optical microscope (Leitz). For each experiment 50 to 100 neurons were digitalized and analysed with a morphological analysis software (IMSTAR, France). All statistics (Student’s t-test) were done with the help of the Statview II program (Abacus Concepts, Inc.).

Quantification of cell survival

Cells were dissociated and plated as described above. The number of live cells per well at time zero was estimated by trypan blue exclusion under the microscope just before plating and taken as 100%. The percentages of survival after different times in culture were also calculated by the trypan blue exclusion procedure.

Quantification of cell adhesion

Cellular adhesion was quantified according to the method of Dow et al., (1988). Briefly, cells were seeded at 20,000 cells per well and then the multiwell plates were turned up side down. The inverted plates were incubated for 1 hour at 4°C and subsequently 2 hours at 37°C. The cells were then fixed (2.5% glutaraldehyde in PBS), rinsed with water, air dried, stained with 0.1% cristal violet in borate buffer pH 9 (Kueng et al., 1989), washed several times and air dried. Cristal violet bound to the cell nuclei was solubilized in 10% acetic acid and the optical density of the solution in each well determined at 570 nm with an automatic multiwell-plate spectrophotometer.

Immunocytochemistry

Cells seeded on 16 mm diameter glass coverslips coated with 1.5 μg ml−1 polyornithine were cultured for 3 days, fixed for 1 hour at 4°C with paraformaldehyde (4% in PBS), rinsed in PBS-glycine 5 mM pH 7.4, incubated (1 hour 37°C) with anti-MAP2 antibodies (1/400) in PBS plus 10% FCS and 0.01% saponin (buffer A), washed 3 times and further incubated for 1 hour at 37°C with anti-NF-H antibodies (1/50). After 3 washes cells were incubated (1 hour, 37°C) with Texas red-conjugated anti-rabbit Igs (½00), washed 3 times, incubated with biotinylated anti-mouse Igs (½00), washed 3 times, incubated (1 hour, 37°C) with fluorescem-conjugated streptavidin (½00), washed 3 times in PBS and twice in water, mounted in raowiol and observed with a Leitz epifluorescence microscope. All washes and dilutions were done in buffer A.

ELISA on whole cells

The ELISA was performed according to the method of Munn and Cheung, 1989. Briefly, cells were seeded in 96 microwell plates precoated with polyornithine (1.5 μg ml−1), cultured for 2 days, fixed (1 hour, room temperature) with 4% paraformaldehyde (in PBS plus 1 mM Ca, Mg2+), rinsed in PBS-glycine 5 mM pH 7.4, and incubated for 1 hour at 37®C in PBS plus FCS 10%. Anti-MAP2 (1/4000 in PBS plus 10% FCS) antibodies or non-immune rabbit IgGs were added for 1 hour at 37°C. After several washes in PBS plus 0.1% FCS, cells were further incubated with a peroxidase-labelled second antibody (1 hour, 37°C in PBS plus 10% FCS) and washed several times with PBS. Peroxidase activity was revealed with o-phenylenediamine-dihydrochloride and quantified spectro-photometrically at 470 nm. The number of living cells present in sister wells was estimated by direct counting or by the cristal violet method (Kueng el al., 1989).

Proteoglycan purification

Mesencephalic astrocytes cultured for 3 weeks in the presence of 10% FCS were washed several times with DMEM-F12 and cultured for another 2 days in the absence of serum. Conditioned medium adjusted to 6 M urea was adsorbed on a DEAE cellulose column (DE52) and equilibrated in the following buffer: 0.2 M NaCl, 6 M urea, 0.05 M Tris-HCl pH 7.4. The column was washed in the same buffer until no proteins were detected in the effluent and then glial PGs were eluted with 0.7 M NaCl, 6 M urea and 0.05 M Tris-HCl pH 7.4. In some cases conditioning was achieved in the presence of [35S]sulfate salt (50 μ Ci ml’*) in sulfate-frec DMEM-F12. The purity of the preparation was verified by SDS-PAGE analysis (4% acrylamide). Glial PGs were dialysed against DMEM-F12 for 24 hours at 4°C before use. All purification steps were performed in aseptic conditions and in the presence of PMSF (1 mM).

PGs from the EHS tumor were purified according to the method of Hassel et al. (1985). Briefly, tumors propagated in Balb/c mice for 3 weeks were harvested, rinsed in saline extracting medium plus 6 M urea and loaded on a DE 52 anion exchange column equilibrated with 6 M urea, 0.15 M NaCl, 0.04 M EDTA, 8 mM NEM, 2 mM PMSF and 50 mM Tris-HCl pH 6.8. Bound proteins were eluted with different steps of 0.15, 0.30, 0.70, 1 and 1.5 M NaCl in the equilibration buffer. The fractions dialysed against PBS were analyzed by SDS-PAGE, and those containing pure PGs, as checked by electrophoretic profiles were kept frozen at —80°C until use. After slow defrosting, PGs were either filtered with a 0.22 μ m Millipore filter or sterilized with a drop of chloroform.

Homogeneity of the PGs was checked by electrophoresis on SDS-polyacrylamide gels followed either by silver staining (BioRad-kit) or immunoblotting. As indicated in Fig. 1, al) stained material remained in the form of large molecular weight entities unable to penetrate a 6% gel (lane 1), unless part of the sugars were enzymatically degraded by hepariti-nase (lane 2). Immunoblotting with anti-laminin antibodies (lane 3) failed to reveal any contamination by these matrix molecules (the most abundant in EHS tumor).

Fig. 1.

SDS-PAGE and immunoblottings of PGs. Lanes 1 and 2: purified EHS-PGs treated (lane 2) or not (lane 1) with heparitinase, silver staining. Lanes 3 and 4: anti-laminin immunoblotting of PGs (lane 3) and laminin (lane 4). Positions of the relative molecular mass markers arc indicated on the left side of the figure (×10−3).

Fig. 1.

SDS-PAGE and immunoblottings of PGs. Lanes 1 and 2: purified EHS-PGs treated (lane 2) or not (lane 1) with heparitinase, silver staining. Lanes 3 and 4: anti-laminin immunoblotting of PGs (lane 3) and laminin (lane 4). Positions of the relative molecular mass markers arc indicated on the left side of the figure (×10−3).

Enzymatic treatments

Proteoglycans (400 μg ) were incubated for 2 hours at 37°C with heparinase or heparitinase (0.1 U ml−1) or chondroiti-nases (0.05 U ml−1), in 1 ml of DMEM-F12 culture medium supplemented with 25 mM sodium acetate. Proteolytic treatments were achieved overnight at 37aC with trypsine (1 mg ml−1) or proteinase K (1 mg m − 1) and stopped with 10% FCS. Digested PGs were added to the cultures to give the final concentration of 4 μg ml−1. In control experiments, the enzymes were directly added in the culture wells at the appropriate concentrations. The degradation of PGs was monitored by SDS-PAGE.

Electron microscopy

Cells were fixed in glutaraldehyde (3% in PBS) for I hour at room temperature, washed several times with PBS and postfixed in osmium tetroxyde (2% in PBS) for 30 minutes at room temperature. After dehydration they were embedded in Epon, cut, collected on grids, contrasted with uranyl acetate and lead citrate, and viewed with a Jeol 2000 electron microscope (Vuillet et al., 1984; Autillo-Touati et al., 1988). For imtn uno-electron microscopy, cells fixed with 4% paraformaldehyde were incubated with the anti-core protein of the HSPG from the EHS tumor (1/400 in PBS) for 1 hour at 37°C, washed, incubated with peroxidase-linked anti-rabbit IgGs (1/400 in PBS) for another hour and washed several times before addition of diaminobenzydine (0.2 mg m − 1) and H2O2 (0.003%) in Tris 50 mM, pH 7.8 for 5 minutes at room temperature. The cells were then washed thoroughly, postfixed in OsO4, dehydrated, embedded in Epon, cut, collected and viewed.

G AGs biotinylation

GAGs biotinylated with biotin hydrazid (Pierce) according to the manufacturer’s procedure were exhaustively dialysed against 1 M NaCl in PBS and against PBS. Labelling and absence of free biotin hydrazid were checked by PAGE-electrophoresis under the conditions used for DNA analysis (Maniatis), followed by blotting, incubation of the blots with peroxidase-streptavidin and development of the reaction with DAB (0.2 mg ml−I) and H2O2 (0.003%).

Confocal microscopy

Data were obtained with a confocal scanning laser microscope Phoibos 1000 (Sarastro, Stockholm). Excitation was obtained with an Argon ion laser set at 514 nm for TRITC excitation and the emitted light was filtered with an appropriate long pass filter (530 nm). The background noise was reduced and the contrast enhanced by applying a median gaussian filter to the original data. Pseudocolor images coding for fluorescence emission were obtained from a linear look-up table. It decreased from red to yellow, green and blue.

Effects of PGs on neuronal morphology

Mixed proteoglycans (PGs) isolated from astrocyte conditioned medium or from the EHS tumor were added to the cells 30 minutes after seeding and their morphological effects were analysed after 2 days. In the conditions of chemically defined medium (CDM) plus or minus PGs (or GAGs) all cells were neuronal in nature, as demonstrated by their labeling with antibodies directed against the neurofilament triplet or neural specific enolase (not shown, see Rousselet et al., 1988). As shown in Table 1 for EHS PGs (and several GAGs), the addition of these molecules did not affect neuronal survival, which remained constant for 48 hours. In fact at the time of neuronal analysis, more that 90% of the cells attached 3 hours after plating were still alive.

Table 1.

Percentages of surviving neurons after 3, 18, 30 or 48 hours in culture in different conditions

Percentages of surviving neurons after 3, 18, 30 or 48 hours in culture in different conditions
Percentages of surviving neurons after 3, 18, 30 or 48 hours in culture in different conditions

Fig. 2 illustrates the morphological influence of the addition of 4 μg ml−1 EHS proteoglycans, a concentration that did not affect cell viability (Table 1). In control conditions (Fig. 2A), cell bodies are spread with short neurites and numerous cytoplasmic veils. The presence of the proteoglycans induced vigourous neurite growth, most neurons being asymmetric with a single long neurite. In addition, these long neurites tended to fasciculate with one another (Fig. 2B). The percentage of neurons with longest neurite length exceeding 50 pm increased from 10% in the control wells to more than 80% (Fig. 2C) in the presence of PGs. In view of the high neuronal survival (Table 1), it can be assumed that the morphological changes illustrated in Fig. 2 do not reflect the selective survival of specific neuronal subpopulations w’ith distinct morphological traits.

Fig. 2.

Effects of EHS tumor PGs on neuronal morphology. (A) Mesencephalic neurons cultured for 2 days in CDM. Note the spreading of the cell bodies and the large numbers of lamellipodia and veils. (B) Mesencephalic neurons cultured for 2 days in CDM plus PGs (4 μg ml’). Note the round cell bodies and the tendency of the long axon-like neurites to fasciculate. (C) Cumulative quantitative analysis of the length of the longest neurite (L+) in chemically defined medium (CDM) or in CDM plus PGs. This figure pools the results of 3 independent experiments in which 50 neurons per experiment were analysed in each condition.

Fig. 2.

Effects of EHS tumor PGs on neuronal morphology. (A) Mesencephalic neurons cultured for 2 days in CDM. Note the spreading of the cell bodies and the large numbers of lamellipodia and veils. (B) Mesencephalic neurons cultured for 2 days in CDM plus PGs (4 μg ml’). Note the round cell bodies and the tendency of the long axon-like neurites to fasciculate. (C) Cumulative quantitative analysis of the length of the longest neurite (L+) in chemically defined medium (CDM) or in CDM plus PGs. This figure pools the results of 3 independent experiments in which 50 neurons per experiment were analysed in each condition.

We observed similar effects of astrocyte proteoglycans (G-PGs) on neurite growth as indicated in Table 2 (Exp. 1). Indeed, astrocyte and EHS tumor proteoglycans (EHS-PGs) increased total neurite length, but the most striking result was the fivefold increase in the length of the longest neurite. Since PGs extracted from the tumor and from the astrocytes gave identical results, all following experiments were performed with the tumor proteins, which were easier to purify in high quantities.

Table 2.

Morphometric analysis of neurons cultured for 2 days in different conditions

Morphometric analysis of neurons cultured for 2 days in different conditions
Morphometric analysis of neurons cultured for 2 days in different conditions

PGs favor axonal elongation

We were interested in determining whether the long neurites growing in the presence of PGs w’ere in fact axons as suggested by their morphology. In order to examine this directly, cells were fixed and labeled with antibodies specific for either somatodendritic structures (i.e., anti microtubule-associated protein 2, MAP2) or young axons (i.e., the highly phosphorylated isoforms of neurofilament proteins, NF-H) (Pennypacker et al., 1991). These double-staining experiments illustrated in Fig. 3 were achieved after 3 days in vitro. Clearly, the long neurites induced by PGs can be stained only with the axon-specific antibody (Fig. 3D) and not with the anti-MAP2 antibody (Fig. 3C). The absence of MAP2 staining in the long neurites present after 3 days in culture with EHS-PGs confirms the axon-like nature of these neurites and indicates that the axons are sufficiently differentiated not to contain significant amounts of the N1AP2 antigen (Higgins et al., 1988).

Fig. 3.

Double-immunostaining (same field) of mesencephalic neurons cultured with or without PGs. Mesencephalic neurons cultured in CDM (A,B) or in CDM plus PGs (C,D) were reacted after 3 days in culture with anti-neurofilament (axon-specific) (B.D) or anti-MAP2 (de nd rite-specific) antibodies (A,C).

Fig. 3.

Double-immunostaining (same field) of mesencephalic neurons cultured with or without PGs. Mesencephalic neurons cultured in CDM (A,B) or in CDM plus PGs (C,D) were reacted after 3 days in culture with anti-neurofilament (axon-specific) (B.D) or anti-MAP2 (de nd rite-specific) antibodies (A,C).

To examine the localization of added EHS-PGs on neurons in culture, the cells were incubated with an antibody directed against the core protein of the high molecular weight form of the heparan sulfate proteoglycan and the product of the immunoreaction was analysed by electron microscopy. As illustrated in Fig. 4, EHS-PGs bound to the cell membrane and the core protein epitopes recognized by the antibody were not internalised (Fig. 4A). When neurons were in close contact with another cell (Fig. 4B), no staining was observed at the interface of the cells, indicating that the antibody actually recognized added EHS-PGs and did not stain endogenous molecules. In fact, no labeling was seen when EHS-PGs were not previously added to the culture (not. shown). Note that the staining was present at the surfaces of both cell bodies and axons as shown in Fig. 4C.

Fig. 4.

Binding of PGs to mesencephalic neurons as revealed by electron microscopy. Neurons were cultured for 2 days in the presence of EHS-PGs, fixed and labelled with anti-high molecular weight PG core protein antibodies labelled with peroxidase. Cells were processed for electron microscopy as described in Methods. The black precipitate surrounding the cells (arrowheads) is indicative of the presence of the neuron-bound PGs. Note that the PGs are present on the cell body (A) and on the neurites (C). On the contrary, none of them are detectable at the cell contacts (B, little arrows). Bar, 1 μ m.

Fig. 4.

Binding of PGs to mesencephalic neurons as revealed by electron microscopy. Neurons were cultured for 2 days in the presence of EHS-PGs, fixed and labelled with anti-high molecular weight PG core protein antibodies labelled with peroxidase. Cells were processed for electron microscopy as described in Methods. The black precipitate surrounding the cells (arrowheads) is indicative of the presence of the neuron-bound PGs. Note that the PGs are present on the cell body (A) and on the neurites (C). On the contrary, none of them are detectable at the cell contacts (B, little arrows). Bar, 1 μ m.

Effects of purified G AGs on neuronal morphology

PGs are composed of a core protein on which different types of glycosaminoglycans (GAGs) are attached. To test directly the role of GAGs on neuronal morphogenesis, bovine kidney heparan sulfate (HS), shark cartilage chondroïtin sulfate c (CSc) and a bovine mucosa chondroïtin sulfate b (dermatan sulfate, DS) were added to the cultures at a concentration of 10 tg ml“1 which, as demonstrated by preliminary experiments, gave the best effects but did not impair neuronal survival (Table 1). After 2 days, the cells were analysed for their morphologies.

As illustrated in Fig. 5, the three sugars did not exert identical effects on neurite growth. The neuronal morphologies induced by HS (Fig. 5B) and the CSc (Fig. 5D) were almost identical to that observed in the presence of the PGs. Cell bodies were small and rounded, total neurite growth was increased twofold and this increase corresponded to the preferential development of a single axon-like neurite which accounted for more than 75% of total neurite growth (Table 2, Exp. 2). Compared to HS and CSc, DS had an even stronger effect on total neurite growth (threefold increase), but this effect consisted of a strong enhancement of dendrite-like growth, since the axon-like compartment contributed to one third only of the total neuritic arbor (Table 2, Fig. 5C). None of the sugars had any significant effect (compared to CDM) when bound on the polyornithine coating before cell plating, thus indicating that they were active in their soluble form only (Table 2, Exp. 3).

Fig. 5.

Influence of purified GAGs on neuronal morphology. Mesencephalic neurons were cultured for 2 days in CDM (A), HS (B), DS (C) or CSc (D). Note that the three GAGs promote neurite growth, but that neuronal morphology strongly depends on the nature of the GAGs added to the culture.

Fig. 5.

Influence of purified GAGs on neuronal morphology. Mesencephalic neurons were cultured for 2 days in CDM (A), HS (B), DS (C) or CSc (D). Note that the three GAGs promote neurite growth, but that neuronal morphology strongly depends on the nature of the GAGs added to the culture.

To confirm the importance of the sugar moieties on PG-induced neuronal morphogenesis, EHS PGs were treated with heparinase and chondroïtin ase ABC or heparitinase. Control experiments in which the enzymes were added to the cultures showed that heparinase and chondroïtin ase had no effect on cell morphology. The partial cleavage of heparan sulfate polymers and the removal of chondroïtin chains checked by polyacrylamide gel electrophoresis completely abolished the effect of PGs on total neurite growth and on the growth of the axon-like longest neurite as shown in Fig. 6 in the case of heparinase plus . chondroïtin ase ABC. In contrast, treating the PG preparation with either trypsin or proteinase K did not abolish PG-induced neurite growth (not shown) confirming that GAGs by themselves have an interesting effect on neuronal growth and polarity.

Fig. 6.

Effects of sugar removal on PGs activity. PGs were added intact (A) or after degradation with chondrottinase ABC plus heparinase (B). Quantitative estimations of total neurite length and of the length of rhe longest neurite are shown in panel C where the results of 3 independent experiments have been pooled and 150 neurons analysed in control conditions (PGs) or after treatment with the mixture of glycanases (PG-mixt). Symbols relate to the significance of the differences with the results obtained with intact PGs. (s.e.m., Student’s t-test). * P<0.02.

Fig. 6.

Effects of sugar removal on PGs activity. PGs were added intact (A) or after degradation with chondrottinase ABC plus heparinase (B). Quantitative estimations of total neurite length and of the length of rhe longest neurite are shown in panel C where the results of 3 independent experiments have been pooled and 150 neurons analysed in control conditions (PGs) or after treatment with the mixture of glycanases (PG-mixt). Symbols relate to the significance of the differences with the results obtained with intact PGs. (s.e.m., Student’s t-test). * P<0.02.

HS and DS regulate neuronal polarity

In the following sections, we shall only compare the activities of dermatan and heparan sulfate that gave the more distinct morphological differences. However, we observed very few differences between HS and CSc in their abilities to induce axonal growth preferentially. Fig. 7 shows the double immunostaining of neurons cultured for 3 days in the presence of HS (A,B) or DS (C,D). The long neurites present in HS were almost always (98% of the cases) immunodecorated by the axon-specific antibody but showed no or little staining with the anti-MAP2 antibody. In DS, the cells were multipolar with most neurites labelled with the antiMAP? antibody (Fig. 7C). Very surprisingly, although the longest neurites were in majority axons, an important proportion of them (39%) had marked dendrite biochemical characteristics such as the absence of axonal NF-H and rather large quantities of MAP2 antigen (Fig. 7).

Fig. 7.

Influence of purified GAGs on neuronal polarity. Mesencephalic neurons were cultured for 3 days in the presence of HS (A,B) or DS (C.D). Axons and dendrites were identified by double-immunostaining (same field) with the antineurofilament (B,D) and anti-MAP2 (A,C) antibodies.

Fig. 7.

Influence of purified GAGs on neuronal polarity. Mesencephalic neurons were cultured for 3 days in the presence of HS (A,B) or DS (C.D). Axons and dendrites were identified by double-immunostaining (same field) with the antineurofilament (B,D) and anti-MAP2 (A,C) antibodies.

These different effects of the two sugars on the polarity of developing embryonic neurons, combined with the fact that the sugars had to be given in a soluble form (Table 2), led us to compare their cellular distributions. To do so the sugars were biotinylated and added to the cells 30 minutes after plating. The cultures were fixed 1 or 18 hours later and the distribution of HS (Fig. 8A,C) or DS (Fig. 8B,D) revealed with fluorescent streptavidin was observed with a confocal microscope.

Fig. 8.

Confocal microscopy analysis of GAGs distributions. Biotinylated HS (A,C) or DS (B,D) were added to the cell culture for 1 hour (A,B) or 18 hours (C,D). Cells were fixed with paraformaldehyde (4%) and reacted with streptavidin-texas-red. Confocal sections of the neurons shown in this figure correspond to a cut through the raid-height of the cells. Bar, 1 μ m.

Fig. 8.

Confocal microscopy analysis of GAGs distributions. Biotinylated HS (A,C) or DS (B,D) were added to the cell culture for 1 hour (A,B) or 18 hours (C,D). Cells were fixed with paraformaldehyde (4%) and reacted with streptavidin-texas-red. Confocal sections of the neurons shown in this figure correspond to a cut through the raid-height of the cells. Bar, 1 μ m.

One hour after plating, the distributions of the 2 sugars were quite different. DS were rapidly internalized thus making it difficult to observe any labeling of the cell membrane, (Fig. 8B). In contrast, although also rapidly internalized, HS molecules could be observed both inside the cell and at its surface (Fig. 8A). It is interesting to note that the HS distribution at the cell surface is not uniform and indicates some kind of asymmetric organization of the binding sites. Another difference beween the two GAGs is the accumulation of HS in the nucleus, a phenomenon that was not observed consistently in the case of DS (Fig. 8C).

Polarity, adhesion and MAF2 synthesis

In previous reports, we proposed that while axons were able to grow in low adhesion conditions, dendrite growth was only possible in high adhesion conditions (Chamak and Prochiantz, 1989; Rousselet et at., 1990; Prochiantz, 1990). This prompted us to semi-quantify neuronal adhesion 2 hours following plating in CDM or in the presence of PGs, HS or DS. These data were compared with neuronal spreading and with the capacity to synthesize MAP2.

As demonstrated in Fig. 9A, neuronal spreading (apparent surface of the soma) measured 2 days following plating was highest in CDM and reduced in the presence of both GAGs. Although not as efficient as intact PGs (not shown), HS was much more efficient than DS in reducing neuronal spreading. In addition, we found a good correlation between the amounts of MAP2 present in the cells after 2 days in culture and adhesion 2 hours after plating (Fig. 9B,C). Finally, neuronal morphologies of neurons grown for 2 days in the presence of HS or DS were examined in electron microscopy. Fig. 10 illustrates that neurons grown in the presence of HS are rounded and seem to be loosely attached to the substratum (Fig. 10A) whereas in the presence of DS the soma are flattened and present a long and continuous attachment to the culture dish (Fig. 10B). It is noteworthy that the general shape of the nuclei (rounded or flattened) reflected that of the cell bodies.

Fig. 9.

Adhesion and MAP2 expression in ihe presence of PGs or GAGs. (A) Soma surface (jzm2) of neurons cultured in presence or abscence of GAGs. For each condition, 100 isolated neurons were analysed with the 1MSTAR morphometrica) analysis software. (B) Adhesion was measured after 2 hours of cell culture on inverted plates, by the cristal violet staining procedure as described in Methods. Values presented were calculated from three independent experiments. (C) MAP2 expression estimated by a whole-cell ELISA test. Values were calculated from three independent experiments. Symbols relate to the significance of the difference with the values obtained in CDM. * P <0.01

Fig. 9.

Adhesion and MAP2 expression in ihe presence of PGs or GAGs. (A) Soma surface (jzm2) of neurons cultured in presence or abscence of GAGs. For each condition, 100 isolated neurons were analysed with the 1MSTAR morphometrica) analysis software. (B) Adhesion was measured after 2 hours of cell culture on inverted plates, by the cristal violet staining procedure as described in Methods. Values presented were calculated from three independent experiments. (C) MAP2 expression estimated by a whole-cell ELISA test. Values were calculated from three independent experiments. Symbols relate to the significance of the difference with the values obtained in CDM. * P <0.01

Fig. 10.

Electron micrographs of mesencephalic neurons grown in the presence of GAGs. Cells cultured for 2 days on Petriperm dishes in the presence of HS (A) and DS (B) were fixed with glutaraldehyde and osmium tetroxide and processed for electron microscopy as described in Methods. The embedded cells were cut in a plane perpendicular to the culture substratum. Note the ball-shape of the loosely attached cell body of the neurons grown in the presence of HS (contact with the substratum is indicated by arrowheads), as compared to the fiat cell body of neurons cultured in DS. Bar, 1 μ m.

Fig. 10.

Electron micrographs of mesencephalic neurons grown in the presence of GAGs. Cells cultured for 2 days on Petriperm dishes in the presence of HS (A) and DS (B) were fixed with glutaraldehyde and osmium tetroxide and processed for electron microscopy as described in Methods. The embedded cells were cut in a plane perpendicular to the culture substratum. Note the ball-shape of the loosely attached cell body of the neurons grown in the presence of HS (contact with the substratum is indicated by arrowheads), as compared to the fiat cell body of neurons cultured in DS. Bar, 1 μ m.

In this report, we demonstrate that PGs purified either from astrocyte-conditioned medium or from the EHS tumor have a strong influence on neurite growth and, more specifically, on axonal growth. This PG-induced axonal growth is associated with a decrease both in adhesion and in the synthesis of MAP2. The trophic and morphogenetic influence of PGs is abolished when the glycoproteins are enzymatically deglycosylated whereas it is not affected by the hydrolysis of the core protein with trypsin or proteinase K. This importance of the sugar moieties (GAGs) is further confirmed by experiments in which specific sugars, chondroïtin -, dermatan- or heparan-sulfate, were directly added to the cultures. Interestingly enough, it was found that although all GAGs tested strongly promote neurite growth, the type of neurite produced in majority (axon versus dendrites) is highly dependent on the chemical structure of the sulfated carbohydrate chains. This finding illustrated in this report for E14 rat post-mitotic mesencephalic neurons, remains valid for neurons prepared from other brain regions (spinal cord, cortex and striatum, in particular) between E13 and E18.

The culture conditions used in these experiments allow neuronal survival, and typically result in cultures with a cell population more that 99% neuronal. Thus, it is unlikely that the effects of the different PGs and GAGs are mediated through the few non-neuronal cells present in the culture. Furthermore, the low cellular concentration and the very rapid effects of PGs and GAGs (e.g. adhesion was measured 2 hours after seeding) strongly suggest that PGs and GAGs act directly at the level of their target cells. In particular, although we cannot preclude it entirely, it is unlikely that the effects of PGs and GAGs require a long-range diffusion of molecules synthesized by the neurons. Rather, we favor a hypothesis by which these molecules would trigger a chain of intracellular events by acting on receptors or by increasing the efficiency of some autocrine phenomenons.

A crucial point in the interpretation of our results is the possible selective survival, in the different conditions, of specific subpopulations presenting defined morphological traits, e.g. presence of a long axon-like neurite. We consider this possibility very unlikely on the basis of the following considerations. Firstly, cellular survival after 2 days was over 90% whereas more than 80% of the cells presented the same morphology (e.g. length of the longest neurite greater than 50 μm). Secondly, morphological examinations were done after 2 and 6 days yielding identical qualitative and quantitative results although cell survival was lower in the older cultures (not shown). Thirdly, variations in cell adhesion (a critical factor in cell polarity) were measured 2 hours after seeding when all cells were still alive. Finally, in another model of polarity induction through adhesion, we have shown that the effects were fully reversible (Chamak and Prochiantz, 1989). From this, we infer that the effects of PGs and GAGs are instructive and do not reflect a selective mechanism.

Antibodies against MAP2 or against highly phosphorylated isoforms of high molecular weight NF proteins (NF-H) were used to characterize dendrites and axons respectively. MAP2 has been shown to be a good dendritic marker, (Matus et al., 1986; Higgins et al., 1988) and the amounts of MAP2 quantified by an ELISA assay on fixed cells correlated well with the immunological staining. The use of the neurofilament proteins as axonal markers can be more problematic because of the late synthesis of some axon-specific isoforms (Foster et al., 1987). However, in good accordance with the results of Pennypacker et al. (1991) the anti NF-H antibody used in this study allowed us to discriminate between axons and dendrites in 3-day-old cultures, as demonstrated by double immunostaining experiments.

Another point of concern is the purity of the proteoglycan preparations. In particular, these molecules are known to interact strongly with several factors endowed with potent morphogenetic properties such as laminin, fibronectin, basic FGF and TGF β (Ruoslahti, 1988). The EHS proteoglycans used in this study were purified following well established procedures (Hassel et al., 1985) and their analysis on SDS-polyacrylamide gels showed no obvious contaminants. Since we were not interested in purifying distinct subsets of PGs, no CsCI fractionation was achieved, thus the PG mixture we work with is comparable to the one described by others (Kato et al., 1978; Fujiwara et al., 1984). However, the fact that the anion exchange column was equilibrated in 6 M urea diminishes the probability of the presence of contaminating molecules in our preparations. This is in fact well demonstrated by the absence of contaminating laminin, the most abundant matrix molecules in the EHS tumor, clearly illustrated in the Western blot of Fig. 1. Finally, even though the presence of small amounts of highly active contaminating factors can never be entirely precluded, the fact that the morphogenetic effects observed with such proteoglycans were lost after hydrolysis with protease-free sugar-degrading enzymes and could be replicated with purified GAGs eliminates simple expla-nations based on a contamination by any of the factors mentioned above.

The physiological significance of the morphogenetic effects of PGs and GAGs is underlined by the fact that these molecules are synthesized in the nervous system, in particular the brain (Margolis and Margolis, 1989; Herndon and Lander, 1990). In good agreement with published results on the structure of brain-derived PGs (Fransson, 1987; Hoffman and Edelman, 1987; Ratner et al., 1988; Margolis and Margolis, 1989), we verified that astrocytes in culture release PGs in which chon-droitin- and heparan-sulfate are present. The distri-bution of PGs during development, as analysed by several investigators, has shown that these molecules are not only developmentally regulated, but that their distribution coincides with specific pathways either permissive or repulsive for the migration of cells and the elongation of growth cones (Perris et al., 1991).

An important result of our studies is the capability of pure sugars to modify neuronal growth and mor-phology. Such a morphogenetic influence of the GAGs has been observed in another model (Verna et al., 1989). However, to our knowledge, the analysis of how different GAGs can act in a distinct manner on the development of neuronal polarity had not been studied before. Of particular interest are the converse activities of dermatan- and heparan-sulfate, which promote dendrite and axon growth, respectively. The induction of axon growth associated with a decrease in adhesion and in MAP2 synthesis confirms previous results demonstrating that, in contrast to dendrites, axons, because of their high axoplasmic viscosity, are able to grow in low adhesion conditions (Chamak and Pro-chiantz, 1989; Rousselet et al., 1990; Prochiantz, 1990). Although our study is limited to the nervous system, it can be underlined that, in view of the similarities between the mechanisms of polarity establishment and maintainance in several cell types, the results reported here may be of larger physiological significance (Dotti and Simon, 1990).

Our observations on the possible physiological importance of GAGs synthesis and distribution, com-bined with the fact that PGs treated with heparinase, heparitinase and chondroïtin ase ABC lose all their growth-inducing properties raise the question of the respective roles of the core proteins and of the sugar moitiés in PGs physiology. Although confirming the importance of PGs and GAGs in neuronal differen-tiation, the results reported here certainly highlight the importance of the GAGs moiety at the expenses of core proteins. This statement can anyhow be corrected by the fact that our culture conditions (low cell density, CDM), in which control neurons have little growth activity, would not have allowed us to discover an inhibitory action of the core proteins on actively elongating neurites as was reported for NGF-treated PC12 cells (Snow et al., 1990; Oohira et al., 1991).

It is also possible that it is in culture only that the presence of the core protein is of little importance. This might be due to the fact that pure sugar chains are able to bind to the neurons and to mimic the action of intact PGs. It is indeed rather unlikely that, in vivo, protein-free sugar chains can be either secreted into the medium or present at the neuronal surface. Thus, it can be proposed that core proteins act as a means of exposing the sugars at the neuronal surface or of facilitating their secretion into the intercellular space. Moreover it can be speculated that depending on the type of core proteins, PGs could be targeted to specific neuronal compartments, e.g. dendrites or axons. Indeed, Rapraeger and his collaborators have demonstrated a specific targeting of PGs to the apical or the basolateral compartments of the epithelial cell (Rapraeger et al., 1986). This possibility is strengthened by the fact that several PGs are anchored to the cell surface by a glycolipid-link (Hemdon and Lander, 1990) known to act as an apical target signal in polarized cells, neurons included (Lisanti et al., 1989; Dotti et al., 1991). Interestingly enough in this context, Dotti and Simon (1990) have demonstrated the equivalence between axons and the apical compartment of epithelial cells.

The mechanism by which GAGs exert their trophic and polarizing actions must be considered. This question was partially addressed in the present study, in particular in the experiments showing that they can modulate neuronal adhesion and MAP2 expression. More experimental work, however, will be needed to understand the actual physiological role of these molecules. Although we cannot eliminate the possibility that PGs and GAGs are true growth factors acting autonomously after binding specific receptors, it is more likely that these molecules modulate the activity of cell and substratum adhesion molecules as demonstrated in the case of NCAM or laminin (Cole et al., 1986; Muir et al., 1989).

More generally, in view of the high affinity for heparin of several growth factors, such as bFGF or TGFβ, it is possible that the addition of GAGs or PGs increases the trophic influences of such molecules either by augmenting their ability to diffuse (Flaumenhaft et al., 1990) or their binding capacities at the cell surface. Heparin-activated capture of growth factor has been proposed, at least in the case of bFGF, to be required for the further activation of the high affinity tyrosine-kinase-Iinked receptor (Yayon et al., 1991; Rapraeger et al., 1991). In addition, PGs and GAGs might be involved in the specific internalization and intracellular targeting of several factors (Ruoslahti, 1989; Baldin et al., 1990). This latter point is substantiated by our observation that biotinylated GAGs are rapidly internalized in culture. This rapid internalization of GAGs contrasts with the apparent absence of internalization of EHS-PGs. This latter observation has to be taken with caution since the antibody used was directed exclusively against the core protein, leaving open a possible internalization of the GAGs.

Finally, the fact that HS stimulates axonal growth whereas DS allows the growth of all neurites with a strong positive effect on dendrite development sustains the idea that specific domains or even receptors might exist at the cell surface that recognize distinct GAGs sequences. This last point is substantiated by our confocal microscopy experiments which suggest that DS and HS do not behave in the same way when added to neurons in culture. In particular, it can be noted that HS, although internalized and targeted to the nuclei, are found associated with the cell surface in a clearly non-random disposition and seemingly underline an asymmetric organization of the nerve cell even before neurite growth. The possible significance of this distribution and the possible link between the existence of separate domains at the surface of the cell and the development of neuronal polarity is being presently investigated.

We thank Dr. K. Moya for helpful suggestions and for his careful reading of the manuscript. The help of Dr. M. Vigny in the design of some experiments is also acknowledged. Mrs H. Debroas and C. Valenza are acknowledged for their skillful technical assistance. This work was supported by CNRS, FIDIA-France and grants from AFM, DRET (89-200) and MRT (89 C 0701).

Autillo-Touati
,
A.
,
Chamak
,
B.
,
Araud
,
D.
,
Vuillet
,
J.
,
Selte
,
R.
and
Prochiantz
,
A.
(
1988
).
Region-specific neuro-astroglial interactions: ultrastructural study of the in vitro expression of neuronal polarity
.
J. Neurosci. Res
.
19
,
326
342
,
Baldin
,
V.
,
Roman
,
A-M.
,
Bosc-Bleme
,
I.
,
Ai Marie.
F.
and
Bouche
,
G.
(
1990
).
Translocation of bFGF to the nucleus is Gi phase cell cycle specific in bovine aortic endothelial cells
.
EMBO J
.
9
,
15111517
.
Burgess
,
W H.
and
Maciag
,
T.
(
1989
)
The heparin-binding (fibroblast) growth factor family of proteins
.
Ann. Rev. Biochem
.
58
.
575
-
606
.’
Chamak
,
B.
,
Fellous
,
A.
,
Glowinski
,
J.
and
Prochiantz
,
A.
(
1987
).
MAP2 expression and neuritic outgrowth and branching are coregulated through regí on-specific neuroastroglial interactions
.
J. Neurosci
.
7
,
3163
-
3170
.’
Chamak
,
B
, and
Prochiantz
,
A.
(
1989
).
Influence of extracellular matrix proteins on the expression of neuronal polarity
.
Development
106
,
483
491
.
Chelfetz
,
S.
and
Massagué
,
J.
(
1989
).
Transforming growth factor-β (TGF-β) receptor proteoglycan
.
J. Biol. Chem
.
264
,
12025
12028
.
Cole
,
G. J.
,
Loewy
,
A.
and
Glaser
,
L
, (
1986
).
Neuronal cell-cell adhesion depends on interactions of N-CAM with heparin-like molecules
.
Nature
320
,
445
447
.
Denis-Donini
,
S
,,
Glowinski
,
J.
and
Prochiantz
,
A.
(
1984
).
Glial heterogeneity may define the three-dimensional shape of mouse mesencephalic dopaminergic neurones
.
Nature
307
,
641
643
.
Dotli
,
C. G.
,
Parton
,
R. G.
and
Simons
,
K.
(
1991
).
Polarized sorting of glypiated proteins in hippocampal neurons
.
Nature
349
,
158
161
.
Dotti
,
C. G.
and Simons. K
. (
1990
).
Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture
.
Cell
62
,
63
72
.
Dow
,
K. E”
Mlrskl
,
S. E. L.
,
Roder
,
J. C.
and
Rlopelje
,
R. J.
(
1988
).
Neuronal proteoglycans: Biosynthesis and functional interaction with neurons in vitro
.
J. Neurosci
8
,
3278
3289
.
Flaumenhaft
,
R.
,
Moscateui
,
D.
and
Rifkin
,
D. B.
(
1990
).
Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor
.
J. Cell Biol
.
11
,
1651
1659
.
Foster
,
G. A.
,
Dahl
,
D.
and
Lee
,
V. M-Y.
(
1987
).
Temporal and topographic relationships between the phosphorylated and non phosphoryl a ted epitopes of the 200 kDa ncurofilament protein during development in vitro
.
J. Neurosci
.
7
,
2651
2663
.
Fransson
,
L-A.
(
1987
).
Structure and function of cell-associated proteoglycans
.
Trends Biochem. Set
.
12
,
406
411
.
Fujiwara Wiedemann
,
H.
,
Timpl
,
R.
,
Lustig
,
A.
and
Engel
,
J.
(
1984
).
Structure and interactions of heparan sulfate proteoglycans from a mouse tumor basement membrane
.
Eur. J. Biochem
.
143
,
145
157
.
Gordon
,
P. B.
,
Choi
,
H. U.
,
Conn
,
G.
,
Ahmed
,
A.
,
Ehrmann
,
B.
,
Rosenberg
,
L.
and
Hatcher
,
V. B.
(
1989
).
Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor
.
J. Cell Physiol
.
140
,
584
592
.
Hantaz-Ambroise
,
D.
,
Vigny
,
M.
and
Koenig
,
J.
(
1987
).
Heparan sulfate proteoglycan and laminin mediate two different types of neurite outgrowth
.
J. Neurosci
.
7
,
2293
2304
.
Hassel
,
J. R
,,
Leyshon
,
W. C.
,
Ledbetter.
S. R
,
Tyree
,
B.
,
Susuki
,
S.
,
Kato
,
M.
,
Kimata
,
K.
and
Kleinman
,
H. K.
(
1985
).
[solation of two forms of basement membrane proteoglycans
.
J. Biol. Chem
260
,
8098
8105
.
Herndon
,
M. E.
and
Lander
,
A. D.
(
1990
).
A diverse set of developmentally regulated proteoglycans is expressed in the rat central nervous system
.
Neuron
4
,
949
961
.
Higgins
,
D.
,
Waxman
,
A.
and
Banker
,
G.
(
1988
).
The distribution of microtubule-associated protein 2 changes when dendritic growth is induced in rat sympathetic neurons in vitro
.
Neurosci
.
24
,
583
592
.
Hoffman
,
S.
and
Edelman
,
G
, (
1987
).
A proteoglycan with HNK-1 antigenic determinants is a neuron-associated ligand for cytotactin
.
Proc. Natl. Acad. Sci USA
.
84
,
2523
2527
.
Kato
,
M”
Koike
,
Y”
Ito
,
Y”
Susuki
,
S.
and
Klmata
,
K.
(
1987
).
Multiple forms of heparan sulfate proteoglycans in the Engelbreth-Holm-Swarm mouse tumor
,
J. Biol. Chem
.
262
,
7180
7188
.
Kiefer
,
M. C.
,
Stephan
,
J. C.
,
Crawford
,
K
,,
Oklno
,
K.
and
Barr
,
P. J.
(
1990
).
Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that bind basic fibroblast growth factor
.
Proc. Nail. Acad. Sci. USA
.
87
,
6985
6989
.
Kueng
,
W”
Silber
,
S.
and
Eppenberger
,
U.
(
1989
).
Quantification of cells cultured on 96-well plates
.
Anal. Biochem
.
182
,
16
19
.
Lisantl
,
M. P.
,
Carns
,
L. W.
,
Davitz
,
M. A.
and
Rodrigez-Boulan
,
E.
(
1989
).
A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells
.
J. Cell Biol
109
,
21452156
.
Lochter
,
A.
,
Vaughan
,
L
,,
Kaplony
,
A.
,
Prochiantz
,
A.
,
Schachner
,
M.
and
Faissner
,
A.
(
1991
).
Jl/tenascin in subs tract-bou nd and soluble form display contrary effects on neurite outgrowth
.
J. Cell Biol
.
113
,
1159
1171
.
Margolis
,
R. U.
and
Margolis
,
R. K.
(
1989
).
Nervous tissue proteoglycans
.
Dev. Neurosci
.
11
,
276
288
.
Matthew
,
W. D”
Greenspan
,
R. J.
,
Lander
,
A. D.
and
Reichardt
,
L. F.
(
1985
).
Immunopurification and characterization of a neuronal heparan sulfate proteoglycan
.
J. Neurosci
.
5
,
1842
1850
.
Matus
,
A.
,
Bernhardt
,
R.
,
Bodmer
,
R.
and
Alalino
,
D.
(
1986
).
Microtubule-associated protein 2 and tubulin are differently distributed in the dendrites of developing neurons
.
Neurosci
.
17
,
371
389
.
Muir
,
D.
,
Engvall
,
E.
,
Varón
,
S.
and
Manthorpe
,
M.
(
1989
).
Schwannoma cell-derived inhibitor of the neurite-promoting activity of laminin
.
J. Cell Biol
.
109
,
2353
2362
.
Munn
,
D. H.
and
Cheung
,
N-K. V.
(
1989
).
Antibod y-dep ende nt antitumor cytotoxicity by human monocytes cultured with recombinant macrophage colony-stimulating factor
.
J. Exp. Med
.
170
,
511
526
.
Neufeld
,
G.
,
Gospodarowicz
,
D.
,
Dodge
,
L.
and Fuji!, D. K
, (
1987
).
Heparin modulation of the neurotropic effects of acidic and basic fibroblast growth factors and nerve growth factor on PC 12 cells
.
J. Cell. Physiol
.
131
,
131
140
.
Oohlra
,
A.
,
Matsui
,
F.
and
Katoh-Semba
,
R.
(
1991
).
Inhibitory effects of brain chondroïtin sulfate proteoglycans on neurite outgrowth from PC12D cells
.
J. Neurosci
.
11
,
822
827
.
Oohlra
,
A.
,
Matsui
,
F.
,
Matsuda
,
M.
and
Shoji
,
R
(
1986
).
Developmental change in the glycosaminoglycan composition of the rat brain
.
J. Neurochem
.
47
,
588
593
.
Pennypacker
,
K.
,
Fischer
,
I.
and
Levitt
,
P.
(
1991
).
Early in vitro genesis and differentiation of axons and dendrites by hippocampal neurons analyzed quantitatively with ncurofilamcnt-H and microtubule-associated protein 2 antibodies
.
Exp. Neurol
.
III
,
2535
.
Perris
,
R.
,
Krotoski
,
D.
,
La Hier
,
T.
,
Domingo
,
C.
,
Sorrel
,
J. M.
and
Brunner-Fraser
,
M.
(
1991
).
Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development
.
Development
111
,
583
599
.
Prochiantz
,
A.
(
1990
).
Morphogenesis of the nerve cell
.
Comments Dev. Neurobiol
.
1
,
143
155
.
Rapraeger
,
A.
,
Jalkanen
,
M.
and
Bernfield
,
M.
(
1986
).
Cell surfaceproteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells
.
J. Cell Biol
.
103
,
2683
2696
.
Rapraeger
,
A. C.
,
Krufka
,
A.
and
Olwin
,
B. D.
(
1991
).
Requirement of heparan sulfate for bFCF-mediated fibroblast growth and myoblast differentiation
.
Science
252
,
1705
1708
.
Ratner
,
N.
,
Hong
,
D.
,
Lieberman
,
M. A.
,
Bunge
,
R. P.
and
Glaser
,
L.
(
1988
).
Tbe neuronal cell-surface molecule mitogenic for schwann cells is a he parin-binding protein
.
Proc. Natl. Acad. Set. USA
.
85
,
6992
6996
Rousselet
,
A.
Autillo-Touati
,
A.
,
Araud
,
D.
and
Prochiantz
,
A.
(
1990
)
In vitro regulation of neuronal morphogenesis and polarity by astrocyte-de rived factors
.
Dev. Biol
.
137
,
33
45
.
Rousselet
,
A.
,
Fetler
,
L.
,
Chamak
,
B.
and
Prochiantz
,
A.
(
1988
).
Rat mesencephalic neurons in culture exhibit different morphological traits in the presence of media conditioned on mesencephalic or striata) astroglia
.
Dev. Biol
.
129
,
495
504
.
Ruoslahti
,
E.
(
1988
).
Structure and biology of proteoglycans
.
Ann. Rev. Cell Biol
.
4
,
229
255
.
Ruoslahti
,
E.
(
1989
).
Proteoglycans in cel) regulation
.
J Biol. Chem
.
264
,
13369
13372
.
Sanes
,
J. R.
(
1989
).
Extracellular matrix molecules that influence neural development
.
Ann. Rev. Neurosci
.
12
,
491
516
.
Snow
,
D. M
,,
Lemmon
,
V.
,
Carrino
,
D. A
,,
Caplan
,
A. I.
and
Silver
,
J.
(
1990
)
Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro
.
Exp. Neurol
.
109
,
111
130
.
Verna
,
J-M.
,
Flchard
,
A.
and
Saxsod
,
R.
(
1989
).
Influence of glycosaminoglycans on neurite morphology and outgrowth patterns in vitro
.
Int. J. Devi. Neuroset
.
7
,
389
399
.
Vuillet
,
J-
,
Daguet De Montety
,
M-C.
,
Autillo-Touati
,
A.
,
Glowinski
,
J.
,
Prochiantz
,
A.
and
Solté
,
R.
(
1984
).
A combined light and electron microscopic method for the visualization of the same in vitro neuron by autoradiography and serial sections
.
J. Microsc
.
133
,
171
176
.
Werz
,
W
, and
Schachner
,
M.
(
1988
).
Adhesion of neural cells to extracellular matrix constituents. Involvement of glycosaminoglycans and cell adhesion molecules
.
Dev. Brain Res
.
43
,
225
234
.
Yayon
,
A.
,
Klagsbruu
,
M.
,
Esko
,
J. D.
,
Leder
,
P.
and
Ornitz
,
D. M.
(
1991
).
Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor
.
Cell
.
64
,
841
848