A central nervous system keratan sulfate proteoglycan: localization to boundaries in the neonatal rat brain

163

Del,elopmental Brain Research, 75 (1993) 163-173 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00

B R E S D 51687

A central nervous system keratan sulfate proteoglycan: localization to boundaries in the neonatal rat brain Eldon E. Geisert Jr. and Deborah

J. B i d a n s e t

Department ~>fCeU Biology% Neurobiolo,~' Research Center, UnirersiO, of Alabama at Birmingham, UAB Station, Birmingham, AL 35394-0019 (USA) (Accepted 4 May 1993)

K~Lvwords: Development; Keratan sulfate; Proteoglycan; Astrocyte; Cortex; Thalamus; Axonal growth

During the development of the central nervous system (CNS), adhesive molecules promote the formation of axonal pathways and appropriate neuronal connections by facilitating cellular interactions. In addition to the interactions that bring neurons together, recent evidence suggests inhibition of neuronal interactions also plays a role by restricting axons to their appropriate pathways and forming boundaries betwccn functional units of the developing CNS. The present study describes the distribution of a recently identified large keratan sulfate proteoglycan. A B A K A N , in the postnatal day 14 (P14) and adult rat brain. In the adult brain A B A K A N appears to be relatively evenly distributed throughout the CNS, while at P14 this proteoglycan is found at high concentrations between different functional units of the neonatal brain. For example, A B A K A N appears to separate different cortical areas and mark the boundaries between thalamic nuclei. In vitro assays demonstrate that this keratan sulfate proteoglycan is a potent inhibitor of neurite growth. The distribution of A B A K A N at P14 and the effects of this kcratan sulfate proteoglycan on neurite growth suggest that A B A K A N functions as a molecular barrier to axonal growth in the developing rat brain.

INTRODUCTION

The formation of appropriate connectivity patterns in the developing CNS requires the guidance of growing axons and the termination of growth once the appropriate target is reached. It is becoming increasingly clear that the cellular interactions associated with these processes involve several adhesion molecules ~2"~9 that promote growth and a separate group of molecules that inhibit cellular a d h e s i o n 7'32"33'46"4~'51. The balance between these growth promoting and growth inhibiting factors appears to play a key role in the formation of axonal pathways during development and may also be involved in the prevention of axonal growth in the adult CNS. Several recent studies have focused on the role of proteoglycans in the inhibition of axonal growth ~''~'3~'4~. The proteoglycans are a structurally diverse group of proteins found throughout the body, including the brain. They share one structural feature, the presence of one or more glycosaminoglycan chains that are covalently linked to a core protein. Many of the functional

properties of proteoglycans are attributable to their highly anionic and hydrophilic carbohydrate side chains. Thus, when fully hydrated, the glycosaminoglycan side chains can occupy a considerable volume, creating sufficient steric hindrance to interfere with cellular adhesion 3~'-~s. In addition to tile general properties of proteoglycans, more specific interactions are believed to be effected through the core protcm . . . . . . . as well as the glycosaminoglycan side c h a i n s ~'~'~5. The diversity seen in both core protein and g[ycosaminoglycan composition suggests that proteoglyeans can have a variety of functions that are related to interactions of the different core proteins as well as the contributions of the glycosaminoglycan side c h a i n s 1~'22"~4"~1)'4~. In a previous study, an adult brain, astrocyte associated keratan sulfate proteoglycan, ABAKAN, was identified using the monoclonal antibody TED15 2L. The epitope recognized by TED15 is within the keratan sulfate side chain of this proteoglycan and appears to be expressed within the postnatal brain. This keratan sulfate glycosaminoglycan was found in avian and mammalian brain and not within the nervous sys-

Correspondence." E.E. Geisert Jr., Department of A n a t o m y and Neurobiology, University of Tennessee, Health Science Center, S75 Monroe Ave., Memphis, TN 38163, USA.

164

tern of lower vertebrates. This distribution led us to hypothesize that the proteoglycan may be involved in the inhibition of neurite growth. In this paper we describe the spatial and temporal expression of ABAKAN in the developing rat brain. In addition, we demonstrate that ABAKAN inhibits neurite outgrowth and neuronal attachment in vitro. M A T E R I A L S A N D METHODS Monoclonal antibodies'. The monoclonal antibody TED15 was produced from a fusion in which two mice were immunized with m e m b r a n e proteins from cultured rat glial cells, with a final boost of whole rat brain white matter. The characterization of the IgM T E D I 5 was reported previously 21. TED1, a monoclonal antibody directed against glial fibrillary acidic protein (GFAP) and the antibody 13-38, which recognizes the extracellular domain of N-CAM, were also produced and characterized in our laboratory z°'21. A mouse monoclonal antibody (IgM) of irrelevant specificity was purchased from ICN (Irvine, CA) and used as a control in the immunohistochemical staining. lmmunoblot method. The general methods used in the immunoblot analysis of the proteoglycan were previously described 1'). Protein samples were obtained from embryonic day 17 (El7) rat brain, postnatal day 1 (P1) rat brain, P14 rat brain and from adult rat brain. The animals were deeply anesthetized with sodium pentobarbital (100 m g / k g ) and decapitated. The brains were homogenized in cold phosphate buffered saline (pH 7.4) and the concentration of protein in the sample was determined by the Lowry method 36. The protein concentrations of the samples were balanced and the samples were placed in Laemmli's buffer 34. Equal a m o u n t s of proteins were loaded and run on a 3 to 15% gradient gel by SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose paper. These blots were then blocked in borate buffer (pH 8.4) containing 5% non-fat dry milk and probed with the TED15 antibody. After rinsing in borate buffer, the blots were incubated in horseradish peroxidase (HRP)-labeled goat anti-mouse IgM secondary antibody (Southern Biotechnology), rinsed extensively and reacted with 0.05% diaminobenzidine and 0.01% hydrogen peroxide. lrnmunohistochemistry. Four adult and four P14 Sprague-Dawley rats (Charles River Labs) were deeply anesthetized with sodium pentobarbital (100 m g / k g of body weight) and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed from the skulls, cryoprotected in 30% sucrose and sectioned (40 ktm) on a freezing microtome. A one-in-ten series of sections from each animal was immunostained with monoclonal antibody TED15 in borate buffered saline (pH 8.4) overnight at 4°C. Following three rinses, the sections were placed in H R P labeled goat anti-mouse lgM (Southern Biotechnotogy) for 2 h, rinsed in phosphate buffer (pH 7.4) and reacted with diaminobenzidine and hydrogen peroxide. Adjacent sections were stained for G F A P using either the monoclonal antibody TED121 (Geisert et al., 1992) or a polyclonal antiserum directed against G F A P (Accurate Chemical). Enzymatic digestions. To characterize the antigen recognized by TED15, enzymatic digestions were carried out on both frozen sections of adult rat brain and A B A K A N purified as stated in Geisert et al. 21. Sections were placed in a solution of keratanase (0.1 U / m l ; Seikagaku America, Inc., Rockville, MD) in 0.05 M Tris-HCl (pH 7.5) for 4 h at 37°C. Following the enzymatic digestions the sections were rinsed in Tris buffer, transferred to borate buffered saline and processed by indirect immunohistochemical methods. Control sections were treated in a similar m a n n e r with the exception that keratanase was not added to the Tris buffer. Digestions of purified A B A K A N were also carried out using the enzymes endo-/3-galactosidase (Boehringer Mannheim), purified sulfatase (Sigma-Type V1 from Aerobacter aerogenes) and keratanase following the protocols of the different suppliers. All three digests were analyzed by immunoblot analysis using TED15.

7issue culture assays. Two different assay systems were used t,~ examine the interaction of neurons with A B A K A N . In the first system, neurons were plated on glass coverslips that were spotted with proteoglycan or laminin in a m a n n e r similar to that described by Muir et al. 3s. The second approach was to develop a neuron binding assay using microtiter wells coated with different substrates. Neurons and astrocytes were cultured as previously described~. Briefly, neurons were cultured from E l 7 rat cortices following the basic procedures outlined by Bartlett and Banker 2. The neurons were seeded into the microtiter wells at 2.104 cells/well or onto glass coverslips at a density of 5- I1)3 c e l l s / c m z in HB101 medium (Hana Biologics). Astrocytes were cultured from neonatal (P2-PS) rat pup cortices in a m a n n e r similar to that described by McCarthy and de Vellis 37 and were maintained in BME containing 11)% fetal calf serum. A variety of different methods were tested to define mixtures that would serve as an appropriate base ti~r the molecular coatings to be used in the analysis of neuron attachment and neurite extension: poly-L-lysine (PLL) only; nitrocellulose dissolved in methanol: PLL followed by nitrocellulose; and nitrocellulose followed by PLL. The best overall substrate was a coating of nitrocellulose followed by PLL. When this base coating was used on either glass coverslips or in 96-well plates, the coverslips and wells bound the proteoglycan and laminin almost as well as a base of nitrocellulose alone. Furthermore, this base was also capable ~f binding frec keratan sulfate chains. The binding of this initial coating was confirmed by ELISA and by antibody staining of the coated coverslips. The ability of the initial coating to bind a second coating consisting of keratan sulfate, laminin or A B A K A N was also examined by ELISA and antibody staining of the coated coverslips. It was shown that within a single well or on a glass coverslip the nitrocellulose/PLL base is capable of binding the initial substrate (laminin, keratan sulfate or ABAKAN), and then accepting a second coating with either of the remaining two substrates. Before performing the biological assays, it was necessary to know the a m o u n t of added proteoglycan that was actually bound to the various substrates. To determine this, purified A B A K A N was iodinated using lodogen (Pierce Immunotechnology, Rockford, IL) according to manufacturers instructions and added (5.10 a c p m / w e l l ) to microtiter wells previously coated with the nitrocellulose/PLL base coat, the base coated with taminin or 0.05% heat-denatured BSA. After a 90-rain incubation at room temperature, the wells were rinsed and both bound and u n b o u n d counts were determined. The a m o u n t of 12Sl-labeled A B A K A N bound to these surfaces was quantified and expressed as the percent bound of the total added (bound + u n b o u n d counts). The percentage of t2SI-labeled A B A K A N bound to the nitrocellulose/PLL base was 55.7_+ 6.7%, to the base coated with laminin was 24.4_+ 1.7% and to the heat-denatured BSA was 6.6 + 0.5%. Thus, approximately 25% of the A B A K A N added in our biological assays remained bound to laminin substrate. To examine neurite growth on different substrates, 18 m m glass coverslips were coated with 125 ~1 of nitrocellulose dissolved in methanol as described previously ~0,4,~ The coverslips were allowed to dry and then were coated with 0.5 ml of a 1 m g / m l solution of poly-L-lysine (PLL-56 kDa-Sigma, St. Louis, MO, No.P2636). The following day, laminin (a gift from Dr. Dale Abrahamson, Dept. of Cell Biology, University of Alabama at Birmingham) diluted to 20 t z g / m l in d d H 2 0 , was coated at 0.5 ml/coverslip and allowed to dry. The laminin coated coverslips were then inverted and placed on capillary tubes spaced 3 m m apart. Using capillary action 1 /~g protein (either laminin or ABAKAN), in 10 p.l of (/.02 M phosphate buffered saline (PBS), was applied to each capillary tube, allowing 4 stripes/coverslip. Alternatively, laminin-coated coverslips were spotted with 1 ~ g protein (laminin, A B A K A N or A B A K A N plus TED15) in 2 p.I 0.02 M PBS containing rhodamine B isothiocyanate (Sigma, St. Louis, MO). The coverslips were placed into a 12-well tissue culture plate and three small paraffin feet were placed on the edges of the coverslip. The wells were blocked with 0.(/5% heat-denatured BSA and rinsed 2 times with 0 . 9 ~ saline. Neurons were then seeded onto the coverslips and allowed to attach t~)r 3 h at 37°C. The coverslips were then inverted over feeder layers of astrocytes in HB101, with the small paraffin feet suspending the neurons approximately 2 mm above the astrocyte layer. After 48 h, the neurons were

165

m

examined and photographed. To study neuron attachment, 96-well plates were coated in a similar manner. RESULTS A B A K A N was identified using the monoclonal antibody T E D I5. When immunoblots of protein solubilized from rat brain were probed with TED15, a diffuse band with a M~ greater than 200.103 in size was recognized, suggesting that the antigen was a high m o l e c u l a r weight glycoprotein. W h e n purified A B A K A N was digested with keratanase and probed with TED15, the antigen was no longer recognized 2~, indicating that the TED15 epitope is within the keratan sulfate glycosaminoglycan side chain. In the present study, A B A K A N was digested with endo-/3-galactosidase and sulfatase to provide an independent means of identifying the epitope recognized by TED15. Immunoblot analyses of these two samples also revealed the absence of T E D I 5 reactivity indicating that both endo-/3-galactosidase, which digests poly-N-acetyllactosamine and sulfatase, which removes sulfate ester groups, could remove portions of the TED15 epitope. Thus, A B A K A N does contain keratan sulfate glycosaminoglycan side chains and the TED15 antibody recognizes the sulfated polylactosamine (keratan sulfate) epitope of this proteoglycan.

Deeelopmental expression To define the expression of A B A K A N during the development of the CNS, samples of total protein from embryonic and postnatal rat brains were analyzed by immunoblot methods (Fig. 1). A darkly labeled broad band above the highest molecular weight marker (M~ = 200.103) was observed on the lanes containing proteins from the adult rat brain (Fig. 1E). The proteoglycan recognized by TED15 was not detected in protein samples from the embryonic brain (Fig. 1B). In protein samples from postnatal rat brain, a gradual increase in the levels of the proteoglycan was observed during the first two postnatal weeks. At P1 the levels of detectable A B A K A N were very low (Fig. 1C) and by P14 (Fig. 1D) the relative amount of this proteoglycan approached adult levels (Fig. 1E). Distribution in the adult rat brain In the adult rat brain, TED15 labels most of the structures at approximately the same intensity. T h e r e is a distinct decrease of labeling in heavily myelinated regions of the brain (Fig. 2) and spinal cord. In gray matter, the pattern of labeling appears to be diffuse at low magnifications; however, when regions of gray matter are examined at high magnifications, a reticulated

i~!iii~i!ii~: 220m- iiiii~iiiii~i!~

100m,-

43~

ABE

DE

Fig. I. Immunoblots probed with the monochmal antibody TED15 (B-E), demonstrate the upregulation of ABAKAN during CNS development in the rat. TED15 recognizes a high molecular weight proteoglycan on immunoblots of adult rat brain (E). Lane A is a Coomassie blue stained gel of P14 rat brain proteins. At El7 there is no detectable ABAKAN on the immunoblots (B). The levels of ABAKAN increase slightly by P1 (C) and by PI4 (D) are near adult levels (E). The same amount of protein was loaded on all lanes. Relative molecular weights are indicated to the left ( × 10~).

pattern of immunoreactivity is observed (Fig. 2D). Within the adult white matter a pattern of staining is observed that appears to be due to labeling of astrocytes between the myelin sheaths. This pattern of immunoreactivity is specific to the TED15 antibody, for it was not observed in control sections reacted with the secondary antibody only or in sections that were reacted with a non-specific mouse IgM. Minor differences in the immunoreactivity patterns were observed in different regions of the adult rat brain. In general, the intensity of labeling observed in the neocortex was similar across all cortical areas and layers, with only a few slight variations. In the cingulate cortex, layer 1 had a slight increase in the intensity of immunoreactivity and the reticulated pattern was more closely spaced (Fig. 2A). In the somatosensory cortex, a relatively even intensity of labeling within the barrel fields (Fig. 2B) was observed. The intensity of the immunoreaction product varied the most within the hippocampus (Fig. 2C). The layers of neuronal cell bodies were relatively unlabeled, with only a few thin labeled processes passing between the densely packed neurons. In the dentate gyrus there was an even labeling throughout the molecular layer. The granular cell layer was unlabeled with the exception of fine immunoreactive processes passing through. A dramatic increase in the intensity of labeling was observed immediately below the granular cell layer within the hilum (Fig. 2D). Within the thalamus there was an

166

Fig. 2. The distribution of ABAKAN in the adult is demonstrated in four photomicrographs of adult rat brain sections stained with TEDI5. In A, the staining pattern within midline cortex (Ctx) and corpus callosum (CC) is illustrated. Notice that an even labeling of gray matter is present and that the white matter tracts contain less immunoreaction product. In sections of the somatosensory cortex (B) an even labeling is observed throughout the cortex with only a slight modulation of the intensity of immunoreaction product in the barrel fields. The caudate/putamen (CP) contains an even labeling of reaction product within the gray matter and the white matter of the internal capsule is relatively unlabeled. In C, the pattern of immunoreactivity within the hippocampus (Hip) and thalamus (Th) is illustrated. The greatest degree of variation in the intensity of labeling is seen in the hippocampus with the neuronal layers containing little immunoreaction product and variations of the reaction product regionally. At higher magnification (D) this variation in the intensity of labeling is clear. The granule cell layer (G) has very little immunoreaction product, the molecular layer ventrally is lightly labeled and the hilum contains high levels of immunolabeling. Panels A, B and C are at the same magnification and the scale bar in C represents 1 mm.

167 even labeling of most thalamic nuclei with an obvious

body. W h e n s i m i l a r s e c t i o n s w e r e r e a c t e d w i t h m o n o -

d e c r e a s e in i m m u n o r e a c t i v i t y o b s e r v e d in r e g i o n s t h a t

clonal antibodies directed against either GFAP

a r e h e a v i l y m y e l i n a t e d (Fig. 2C).

CAM,

To confirm that the TED15

i m m u n o r e a c t i v i t y ob-

s e r v e d in t h e a d u l t b r a i n was d u e to t h e p r e s e n c e o f a keratan with

sulfate proteoglycan,

keratanase.

Sections

sections were

were

then

was

no

difference

in t h e

o r N-

intensity

of

immunoreactivity between keratanase treated sections and control sections.

digested

probed

with

TED15, TED1 (anti-GFAP) and 13-38 (anti N-CAM). No TED15

there

i m m u n o r e a c t i v i t y was o b s e r v e d in s e c t i o n s

Distribution in the neonatal rat brain Since ABAKAN

the

immunoblot

was u p r e g u l a t e d

analysis

revealed

that

in t h e n e o n a t a l rat b r a i n

treated with keratanase, while control sections treated

(Fig. 1), t h e d i s t r i b u t i o n o f A B A K A N

in a s i m i l a r m a n n e r w i t h o u t t h e a d d i t i o n o f k e r a t a n a s e

P14 rat b r a i n by i m m u n o h i s t o c h e m i c a l

demonstrated

p a t t e r n o f i m m u n o r e a c t i v i t y was s u r p r i s i n g l y d i f f e r e n t

an i n t e n s e s t a i n i n g w i t h t h e T E D 1 5 anti-

was e x a m i n e d in methods. The

Fig. 3. The immunoreaction patterns observed at P14 are illustrated in four photomicrographs. In A, the TED15 immunoreaction patterns observed in the midline cortex (Ctx) and corpus callosum (CC) are illustrated. Notice that the cortex is not evenly labeled and that the corpus callosum contains more reaction product than is observed in the surrounding gray matter. B is a photomicrograph of a section through the somatosensory cortex and the caudate/putamen (CP). Within layer IV the characteristic patterns of the developing barrel fields is observed. The TED15 immunolabeling pattern observed in a horizontal section through the entorhinal cortex is illustrated in C. Notice that the regions of intense TEDI5 immunoreactivity appear to mark the boundaries between: the subiculum (S) and the presubiculum (Pr); the presubiculum and the parasubiculum (Pa), the parasubiculum and the entorhinal cortex (Ent) and, the entorhinal cortex and the neocortex. In addition, some of the interfaces between cellular layers in the entorhinal cortex contain high levels of TED15 immunoreaction product. D is a higher magnification of the dentate gyrus shown in C. Notice that the pattern of labeling in the granule cell layer (G), molecular layer to the left and hilum is similar to that observed in the adult. A and B are at the same magnification and the scale bar in B represents 1 ram. The scale bar in C represents 1 ram.

168 from that observed in the adult brain. The most obvious difference was that some regions that were destined to become myelinated fiber tracts were heavily labeled (Figs. 3,4) at P14 as compared to the white matter in the adult brain. The subcortical pathways and corpus callosum were heavily labeled. The fornix and alveus of the hippocampus were also heavily labeled (Fig. 4). In general, all of the major pathways destined to become heavily myelinated contained high

levels of TED15 immunoreactivity. Within these heavily labeled regions, a reticulated pattern of immunoreactivity was observed. This pattern was consistent with the directions of the astrocytic processes which run parallel to axons in these regions. That is to say, if the pathway was cut parallel to the axons within it, then the pattern had a linear striation that mimicked the trajectory of the axons, while if a pathway was cut such that the axons would be running in and out of the

Fig. 4. Some of the labeling patterns observed when sections of P14 rat thalamus are stained with TED15 are illustrated in a photomicrograph of a horizontal section. High levels of TED15 immunoreactivity are observed at the boundaries between many of the thalamic nuclei. The third ventricle on the midline of the thalamus is labeled by the 3-arrow and rostral is at the top of the figure. The abbreviations are as follows: medial dorsal nucleus (MD); anteroventral nucleus (AV); anterodorsal nucleus (AD); stria medullaris (SM); thalamic reticular nucleus (R); ventroposterior nucleus (VP); posterior nucleus (P); ventral lateral geniculate nucleus (VLG); lateral geniculate nucleus (LG); medial geniculate nucleus (MG); and pretectal area (PT). The scale bar represents 1 mm.

169 plane of section, the immunoreaction pattern was a series of dots. The second difference observed in the immunoreactivity pattern at P14 relative to that seen in the adult was within the cerebral cortex. At P14, patches of immunoreactivity were observed throughout the cortex, appearing to define different cortical areas. Within these individual areas the immunoreactivity appeared to define functional subdivisions of the cortex. Specific patterns of TED15 immunoreactivity were observed within different cortical regions. For example, within the somatosensory cortex a distinct pattern of labeling was observed in layer IV of the barrel fields. Regions devoid of immunostaining were surrounded by regions that contained high levels of TED15 immunoreaction product (Fig. 3B). A reticulated pattern of labeling was observed in the heavily labeled regions along with a distinct lack of intracellular neuronal staining. It appeared as if the extracellular regions surrounding the cortical neurons were filled with a diffuse pattern of immunoreactivity. Another region that demonstrated a distinct pattern of TED15 immunoreactivity was the entorhinal cortex (Fig. 3C). Although the general level of labeling was lower in the entorhinal cortex relative to the somatosensory cortex, A B A K A N nevertheless

was distributed in a distinct pattern that reflects the anatomical divisions of the region. The pattern of labeling observed in the hippocampus and dentate gyrus was similar to that observed in the adult rat brain; however, alterations in the patterns of immunoreactivity were observed in the transition from the subiculum to the presubiculum along with a very distinct narrow strip of labeling found at the border region between the presubiculum and the parasubiculum. In addition, TEDl5-immunoreactive regions appeared to surround the parasubiculum, dividing it from the entorhinal cortex. The entorhinal cortex demonstrated a complex pattern of labeling. There was a clear border between the entorhinal cortex and the lateral neocortex, similar to that observed in the parasubicular/entorhinal cortex boundary. Within the entorhinal cortex even the cellular layers appeared to be separated by the immunoreaction product (Fig. 3C). When adjacent sections were stained for GFAP, there was an even labeling of astrocytes throughout the area with no indications of cellular border regions (data not shown). In the adult brain, this distinct pattern of TED15 immunoreactivity at the boundaries between these cortical areas and between the cortical layers within the entorhinal cortex was not observed. As with

Fig. 5. The GFAP (A) and TED15 (B) labeling within the thalamus (Fig. 4) is illustrated at a higher magnification. The Immunopositive region, indicated by the asterisks, is one of the border regions between thalamic nuclei. The arrows indicate individual astrocytes that are immunoreactive for GFAP (A) or ABAKAN(B). Based on their morphological features the TED15 positive cells appear to be a specific subset of astrocytes with ABAKANon their external surface. A and B are at the same magnificationwith the scale bar in B representing 100 p.m.

170

other cortical areas in the adult rat brain, there was a generalized staining of the cortical gray matter with no indication of cellular or regional boundaries. In the thalamus, TED15 immunoreactivity was observed outlining many of the thalamic nuclei (Fig. 4). The thalamic nuclei were identified by examining adjacent sections stained by the Nissl method. At the rostral pole of the thalamus, the anterior nuclear group was surrounded by the immunoreactive lamina. Within the anterior group the different nuclei were also subdivided by T E D I 5 positive areas. In Fig. 4, the boundary between the anterodorsal and anteroventrat thalamic nuclei is clearly visible. The thalamic reticular nucleus is outlined by TED15 immunoreaction product and the region between the medial and lateral subdivisions of the thalamic reticular nucleus also appears to be labeled. Continuing along the lateral aspect of the thalamus, the ventral lateral geniculate nucleus contains a significant amount of label. The lateral geniculate nucleus found immediately caudal to the ventral lateral geniculate nucleus was also completely surrounded by T E D I 5 immunoreactivity. These patterns of labeling were also observed within the mesencephalon. For example, the pretectal area was clearly outlined by immunoreaction product (Fig. 4). At a higher magnification, individual TED15 positive cells were observed (Fig. 5B). In adjacent sections stained for GFAP, cells with a morphology similar to those labeled with TED15 were observed (Fig. 5A). In vitro assays on neurite growth

To determine if A B A K A N is capable of altering the interactions of growing neurites with their environment, cultured E l 7 rat cortical neurons were plated onto glass coverslips coated with laminin and spotted with either purified ABAKAN, purified A B A K A N that was p r e i n c u b a t e d with the a n t i b o d y T E D 1 5 ( A B A K A N / T E D 1 5 ) or laminin. The neurons were allowed to attach to the coverslips that were then inverted over an astrocyte feeder layer. When the cultures were examined 48 h later, there were clear differences in the abilities of the different spots to support neurite outgrowth and neuronal attachment. In control cultures where laminin was dotted onto laminin-coated coverslips, there was virtually no difference in neurite outgrowth on the spotted laminin as compared to the surrounding laminin coating. The pattern of neurite outgrowth observed on spots of purified ABAKAN pretreated with the antibody TED15 was also similar to that observed on the surrounding laminin coated substrate (Fig 6B). When the coverslips spotted with the purified A B A K A N were examined, neurons did not bind to the ABAKAN

Fig. 6. The growth of neurons on coverslips coated with laminin and spotted with A B A K A N (A) or with a mixture of A B A K A N and T E D I 5 (B) is illustrated in two phase-contrast photomicrographs Arrows denote the boundary between the experimental substratc ( A B A K A N or A B A K A N preincubated with TED15) in the upper portion of each photomicrograph and the laminin coating on the lower portion. In A, neurons can be observed attaching to and extending neurites over the laminin coated substrate. Neurons did not attach to the A B A K A N coated spots in the upper half of the photomicrograph. Furthermore, neurites from neurons on adjacent laminin coated substrate do not grow into the A B A K A N coated region. In B, neurons are seen to attach to the A B A K A N / T E D 1 5 coated spot and neurites from the spot and from the adjacent region coated with laminin grow freely between the two substrates. A and B are at the same magnification and the scale bar in B represents 100 ~m.

coated regions and neurites from neurons on the surrounding laminin coated substrate appeared to avoid A B A K A N spots (Fig. 6A). Thus, it appeared that A B A K A N blocked the binding of neurons to the substrate and that A B A K A N prevented growing neurites from invading proteoglycan-containing regions. Since ABAKAN appeared to alter the ability of neurons to bind to laminin, a binding assay was developed and used to quantify the effects of ABAKAN on neuronal binding. Microtiter wells were prepared as described in the Methods Section and then coated with laminin, followed by a second coating with either laminin or different concentrations of ABAKAN. Neurons were placed in the wells and allowed to bind for 90 min, after which the wells were rinsed with 0.02 M

171 PBS. Each well was examined on an inverted microscope and the number of c e l l s / m m 2 was quantified. The number of cells bound was expressed as number of cells b o u n d / m m 2 (mean _+ s.d.). The neuronal binding to laminin was 9 3 4 + 136 cells per mm 2. When ABAKAN was coated onto the well surface at 1 p.g per well, there was a dramatic decrease in the ability of neurons to bind to the substrate with only 318_+ 70 cells bound per mm 2. A further decrease in neuronal binding was observed when 10/xg A B A K A N was added per well, with 119 +_ 26 cells bound per mm 2. Thus, the presence of 1 Ixg of ABAKAN on a favorable substrate inhibited neuronal binding by 66% and 10 /xg A B A K A N inhibited binding by 87%. The decrease in binding is statistically significant at the 0.05 level using the M a n n - W h i t n e y U-test. Taken together, these data demonstrate that A B A K A N is a potential inhibitor of both neuronal attachment and neurite extension. DISCUSSION The present study examines the distribution and potential role of a recently identified keratan sulfate proteoglycan, ABAKAN, that is found in the adult mammalian brain and appears to be associated with astrocytes. This proteoglycan is identified using a previously characterized monoclonal antibody, TED15 21. The epitope recognized by TED15 is not only sensitive to keratanase, but is also sensitive to both endo-/3galactosidase and sulfatase. These data indicate that ABAKAN carries a sulfated polylactosamine (keratan sulfate) carbohydrate side chain and that a sulfate ester is part of the epitope recognized by TED15. This proteoglycan is not readily recognized by the keratan sulfate-specific monoclonal antibody 5D46. Since the binding of both 5D4 and TED15 is dependent upon the presence of a sulfate ester group, the selective recognition of keratan sulfate proteoglycans in the CNS by TED15 and not by 5D4 must be due to a difference in the degree or pattern of sulfation. Within the CNS a number of different proteoglycans are present that are temporally and spatially regulated during development 10,11,23,27,28,31,40,48.56 One proposed role of CNS proteoglycans is the regulation of axonal growth, with some proteoglycans facilitating axonal growth and other proteoglycans inhibiting it. Complexes of laminin and heparan sulfate proteoglycan ~4'44 stimulate neurite outgrowth from sensory neurons and hippocampal neurons, suggesting that the heparan sulfate proteoglycan facilitates neurite outgrowth. Chondroitin sulfate proteoglycans are implicated in both the promotion of axonal elongation 29 and in the inhibition of axonal growth and cell attachment 38'41'49"5°. Recently, a series

of studies have shown that a keratan sulfate proteoglycan 111 found in the midline barrier structure of the developing chick spinal cord 13,47,48,54 inhibits the growth of axons on laminin and N-CAM 1°. In the present study we demonstrate that ABAKAN is capable of blocking neurite growth on laminin and that it will also inhibit the binding of neurons to laminin. These observations are in basic agreement with previous s t u d i e s 1°'3~'49"5° which demonstrate that proteoglycans can block neurite growth on laminin or N-CAM. In previous studies, enzymes such as keratanase, chondroitinase ABC and heparitinase1°'3~'49 or monoclonal antibodies specific for the glycosaminoglycan side chains t° were used to reduce or mask the inhibitory effect of proteoglycans on neurite growth. There are several different ways to interpret these data. The enzymatic digestion of the glycosaminoglycan side chains not only removes portions of the carbohydrate side chains, decreasing the steric hindrance of the glycoprotein, but may also change the conformation of the remaining glycoprotein. These changes at the molecular level potentially affect the interactions of the proteoglycan with both substrates in the assay system and cells used to test the in vitro environment. The second approach, adding an antibody directed against the glycosaminoglycan side chain, theoretically alters the effects of the carbohydrate side chain by masking it with another protein. In this study, the carbohydrate side chains were masked by preincubating A B A K A N with the monoclonal antibody TED15 prior to the addition of neurons to the culture system. The results suggest that steric hindrance is not the sole mechanism responsible for the inhibition of neurite growth. Instead, some molecular aspect of the glycosaminoglycan side chains must be in part responsible for the blocking of neurite outgrowth on laminin. Proteoglycans demonstrating this inhibition of process growth appear to be a molecular component of functional barriers which separate different regions of the developing CNS 1°'13'47'48'54. In the neonatal rat brain, ABAKAN is found at boundary regions that separate functional units. In these boundary regions of the developing CNS, a variety of extracellular molecules are present: collagens and large glycoproteins like fibronectin, laminin and J1/tenascin s'~'15. The temporal and spatial distribution of these extracellular matrix proteins correlates with spatial restrictions in axonal growth. For example, the association of cytotactin (tenascin) with glial and neuronal plasma membranes during the development of the mouse somatosensory barrel fields suggests that this molecule plays a role in cortical pattern formation 53. This association of cytotactin and other substrate-adhesion molecules with

172 proteoglycans indicates that these glycoproteins may also play a role in t h e d e v e l o p m e n t o f t h e b r a i n 5"12'17' 42,45,52. B a s e d on the d i s t r i b u t i o n o f A B A K A N within the d e v e l o p i n g b r a i n a n d the i n h i b i t i o n of n e u r i t e g r o w t h o n A B A K A N in culture, A B A K A N a p p e a r s to be a p r o m i n e n t c o m p o n e n t o f t h e s e b o u n d a r i e s not only in t h e s o m a t o s e n s o r y c o r t e x b u t also in o t h e r b r a i n r e g i o n s such as the e n t o r h i n a l cortex a n d the t h a l a m u s . This p r o t e o g l y c a n is ideally s i t u a t e d to play several roles in the d e v e l o p i n g brain: (1) A B A K A N m a y be involved in the s e g r e g a t i o n of n e u r o n s into distinct f u n c t i o n i n g units or nuclei; (2) this p r o t e o g l y can c o u l d limit t h e g r o w t h o f d e n d r i t e s or axons to a specific l o c a t i o n within t h e brain; (3) the p r o t e o g l y c a n c o u l d force axons within p a t h w a y s to leave t h e p a t h w a y to i n v a d e a d j a c e n t A B A K A N n e g a t i v e regions; a n d 4) t h e u p r e g u l a t i o n o f this p r o t e o g l y c a n late in d e v e l o p m e n t m a y d e c r e a s e t h e ability of cells to form new connections. Several lines o f e v i d e n c e suggest t h a t A B A K A N is a s s o c i a t e d with a s e l e c t e d p o p u l a t i o n of a s t r o c y t e s in t h e m a t u r e a n d t h e d e v e l o p i n g brain. In t h e rat r e t i n a a n d o p t i c n e r v e A B A K A N is only f o u n d a s s o c i a t e d with the a s t r o c y t e s o f t h e optic nerve 21. T h e r e was no l a b e l i n g within the r e t i n a l layers, i n d i c a t i n g that the n e u r o n s as well as t h e r e t i n a l glial cells ( a s t r o c y t e s a n d Mfiller cells) do not express t h e e p i t o p e r e c o g n i z e d by T E D 1 5 . In t h e o p t i c nerve, A B A K A N was d i s t r i b u t e d in a lattice-like s t a i n i n g p a t t e r n t h a t was very similar to the p a t t e r n o b s e r v e d in the s a m e section c o u n t e r s t a i n e d for G F A P 2~. W i t h i n t h e t h a l a m u s o f the develo p i n g rat brain, individual cells a r e i m m u n o r e a c t i v e for T E D 1 5 . T h e s e cells a r e m o r p h o l o g i c a l l y similar to astrocytes in a d j a c e n t sections t h a t a r e l a b e l e d with antib o d i e s d i r e c t e d a g a i n s t G F A P . In the e n t o r h i n a l cortex it a p p e a r s as if G F A P - p o s i t i v e a s t r o c y t e s at the b o r d e r r e g i o n s w e r e positive for A B A K A N while o t h e r G F A P - p o s i t i v e a s t r o c y t e s away from b o r d e r regions did not c o n t a i n high levels o f A B A K A N . Thus, A B A K A N is n o t a g e n e r a l i z e d m a r k e r for m a t u r e a s t r o c y t e s like G F A P . The patterns of TED15 immunoreactivity change d r a m a t i c a l l y d u r i n g t h e d e v e l o p m e n t o f the brain. In the early p o s t n a t a l brain, T E D 1 5 i m m u n o r e a c t i v i t y is high in s o m e axonal p a t h w a y s (Figs. 3 a n d 4). This l a b e l i n g o f t h e axonal p a t h w a y s a p p e a r s to b e associa t e d with a s t r o c y t e s in the white m a t t e r a n d possibly with axons in e a r l y p h a s e s in m y e l i n a t i o n . H o w e v e r , in t h e a d u l t CNS, t h e s e p a t h w a y s d i s p l a y relatively little T E D 1 5 i m m u n o r e a c t i v i t y (Fig. 2). W h e n t h e levels o f A B A K A N w e r e e x a m i n e d by i m m u n o b l o t analysis, t h e a m o u n t o f A B A K A N in a d u l t rat white m a t t e r was similar to t h a t o b s e r v e d in s a m p l e s of a d u l t r a t gray

m a t t e r ( B i d a n s e t , p e r s o n a l observation). T h e s e d a t a suggest that the lack o f T E D 1 5 i m m u n o r e a c t i v i t y in heavily m y e l i n a t e d tracts is d u e to a m a s k i n g of T E D 1 5 e p i t o p e by s o m e c o m p o n e n t o f the a d u l t white m a t t e r . In conclusion, A B A K A N is a k e r a t a n sulfate p r o t e o glycan t h a t inhibits a t t a c h m e n t a n d growth of c u l t u r e d n e u r o n s . T h e d i s t r i b u t i o n of A B A K A N is c o n s i s t e n t with t h e hypothesis that A B A K A N is one of the m o l e c u l e s that c o n t r i b u t e s to t h e f o r m a t i o n of m o l e c u lar b o u n d a r i e s t h a t restrict axonal growth within the d e v e l o p i n g CNS. T h e fact t h a t A B A K A N is f o u n d at high levels t h r o u g h o u t the a d u l t C N S suggests that it m a y function to stabilize cellular i n t e r a c t i o n s by dec r e a s i n g the ability of n e u r o n to form new c o n n e c t i o n s a n d this m a y in p a r t c o n t r i b u t e to the lack of axonal r e g e n e r a t i o n in the a d u l t CNS. We would like to thank Truman Grayson, Michelle McCafferty and Allison Stewart for their technical assistance and Drs. J. Michael Wyss and John A. Robson for their constructive comments on this manuscript. We would like to thank Dr. John Baker for his ongoing assistance with this project. This project was supported by the Whitehall Foundation, Inc.

Acknowledgements.

REFERENCES 1 Akeson, R. and Warren, S.L., PC12 adhesion and neurite formation on selected substrates are inhibited by some glycosaminoglycans and a fibronectin involved tetrapeptide, Exp. Cell Res., 162 (1986) 347-362. 2 Bartlett, W.P. and Banker, G.A., An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture, J. Neurosci., 4 (1984) 1944-1953. 3 Bidanset, D.J., Guidry, C., Rosenberg, L.C., Choi, H.U., Timpl, R. and Hook, M., Binding of the proteoglycan decorin to collagen type VI, J. Biol. Chem., 267 (1992) 5250-5256. 4 Bidanset, D.J., LeBaron, R., Rosenberg, L., Murphy-Ultrich, J.E. and Hook, M., Regulation of cell substrate adhesion: effects of small galactosaminoglycan-containing proteoglycans, J. Cell Biol., 118 (1992) 1523-1531. 5 Bixby, J.L. and Jhabvala, P., Extracellular matrix molecules and cell adhesion molecules induce neurites through different mechanisms, J. Cell Biol., 111 (1990) 2525-2532. 6 Caterson, B., Christner, J.E. and Baker, J.R., Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate, J. Biol. Chem., 258 (1983) 8848-8854. 7 Caroni, P. and Schwab, M.E., Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter, Neuron, 1 (1988) 85-96. 8 Chiquet, M. and Fambrough, D.M., Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis, J. Cell Biol., 98 (1984) 1926-1936. 9 Chiquet-Ehrismann, R., Mackie, E.J., Pearson, C.A. and Sakakura, T., Tenascin; an extracellular matrix protein involved in tissue interactions during fetal development and oncogenesis, Cell, 47 (1986) 131-139. 10 Cole, G.J. and McCabe, C.F., Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth, Neuron, 7 (1991) 1007-1018. 11 Crossin, K.L., Hoffman, S., Seong-Seng, T. and Edelman, G.M., Cytotactin and its proteoglycan ligand mark structural and functional boundaries in somatosensory cortex of the early postnatal mouse, Dev. Biol., 136 (1989) 381-392. 12 Crossin, K.L., Prieto, A.L., Hoffman, S., Jones, F.S., and Friedlander, D.R., Expression of adhesion molecules and the establishment of boundaries during embryonic and neural development, Exp. Neurol., 109 (1990) 6-18.

173 13 Davies, J.A., Cook, G.M.W., Stern, C.D. and Keynes, R.J., Isolation from chick somites of a glycoprotein fraction that causes the collapse of dorsal root ganglion growth cones, Neuron, 4 (1990) 11-20.

14 Dow, K.E. and Riopelle, R.J., Specific effects of ethanol on neurite-promoting proteoglycans of neuronal origin, Brain Res., 508 (1990) 40-45. 15 Erickson, H.P. and Iglesias, J.L., A six-armed oligomer isolated from cell surface fibronectin preparations, Nature 311 (1984) 267-269. 16 Gallagher, J.T., The extended family of proteoglycans: social residents of the pericellular zone, Curr. Opin. Cell Biol., 1 (1989) 1201-1218. 17 Gatchalian, C.L., Schachner, M. and Sanes, J.R., Fibroblasts that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules Tenascin (Jl), N-CAM, fibronectin and a heparan sulfate proteoglycan, J. Cell Biol., 108 (1989) 1873 1890. 18 Geisert Jr., E.E., Johnson, H.G. and Binder, L.I., Expression of microtubule-associated protein 2 by reactive astrocytes, Proc. Natl. Acad. Sci. USA, 87 (1990) 3967-3971. 19 Geisert Jr., E.E. and Stewart, A.M., Changing interactions between astrocytes and neurons during CNS maturation, Dec. Biol., 143 11991) 335-345. 20 Geisert Jr.. E.E., Murphy, T.P., Irwin, M.H. and Larjava, H., A novel cell adhesion molecule, G-CAM, found on cultured rat gila, Neurosci. Lett., 133 (19911 262-266. 21 Geisert Jr., E.E., Williams, R.C. and Bidanset, D.J. A CNS specific proteoglycan associated with astrocytes in rat optic nerve, Brain Res., 571 (19921 165 168. 22 Goetinck, P.F., Proteoglycans in development, Curr. Topics De~,. Biol., 25 11991) 111-131. 23 Gowda, D.C., Margolis, R.U. and Margolis, R.K., Presence of the HNK-I epitope on poly (N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain, Biochem., 28 (1989) 4468-4474. 24 Hardingham, T.E. and Fosang, A,J., Proteoglycans: many forms and many functions, FASEB J., 6 (1992) 861-870. 25 Hassell, J.R., Kimura, J.H. and Hascall, V.C., Proteoglycan core protein families, Annu. Rec. Biochem., 55 (1986) 539-567. 26 Heinegard, D., Franzen, A., Hedbom, E. and Sommarin, Y., Common structures of the core proteins of interstitial proteoglycans, Ciba Found. Symp., 124 (1986) 69-88. 27 Herndon, M.E. and Lander, A.D., A diverse set of developmentally regulated proteoglycans is expressed in the rat central nervous system, Neuron, 4 (1990) 949-961. 28 Hoffman, S., Crossin, K.L. and Edelman, G.M., Molecular forms, binding functions and developmental expression patterns of cytotactin and cytotaetin-binding proteoglycan, an interactive pair of extracellular matrix molecules, J. Cell Biol., 106 (1988) 519-532. 29 lijima, N., Oohira, A., Mori, T., Kitabatake, K. and Kohsaka, S., Core protein of ehondroitin sulfate proteoglycan promotes neurite outgrowth from cultured neocorfical neurons, J. Neurochem., 56 11991) 706-7/18. 30 Jackson, R.L., Busch, S.J. and Cardin, A.D., Glycosaminoglycans: molecular properties, protein interactions and roles in physiological processes, PhysioZ Ret,., 2 (1991) 481-539. 31 Johnson-Greene, P.C., Dow, K.E. and Riopelle, R.J., Characterization of glycosaminoglycans produced by primary astrocytes in vitro, Glia, 4 (1991) 314-321. 32 Kapfhammer, J.P. and Raper, J.A., Interactions between growth cones and neurites growing from different neural tissues in culture, J. Neurosci., 7 (1987) 1595-1600. 33 Kapfhammer, J.P. and Raper, J.A., Collapse of growth cone structure on contact with specific neurites in culture, J. Neurosci., 7 (1987) 201-212. 34 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 68//-685. 35 Lindahl, U., Thunberg, L., Bhckstr6m, G., Riesenfeld, J., Nordling, K. and Bj6rk, 1., Extension and structural variability of the antithrombin-binding sequence in heparin, J. BioL Chem., 259 (1984) 12368 12376.

36 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 37 McCarthy, K.D. and de Vellis, J., Preparation of separate astroglial and oligodendrial cell cultures from rat cerebral tissue, J. Cell Biol., 85 (1980) 890-902. 38 Muir, D., Engvall, E., Varon, S. and Manthorpe, M. Schwannoma cell-derived inhibitor of the neurite-promoting activity of laminin, J. Cell Biol., 109 (1989) 2353-2362. 39 Neugebauer, K.M., Tomaselli, K.J., Lilien, J. and Reichart, L.F., N-cadherin, NCAM and integrins promote retinal neurite outgrowth on astrocytes in vitro, J. Cell Biol., 1117(1988) 1177-1187. 40 Oohira, A., Matsui, F. and Katoh-Semba, R., Inhibitory effects of brain chondroitin sulfate proteoglycans on neurite outgrowth from PCI2D cells, J. Neurosci., 11 (19881 822-827. 41 Perris, R. and Johansson, S., Amphibian neural crest cell migration on purified extracellular matrix components: a chondroitin sulfate proteoglycan inhibits locomotion of fibronectin substrates, J. Cell Biol., 105 (1987) 2511 2521. 42 Rauvala, H., Pihlaskari, R., Laitinen, J. and Merenmies, J., Extracellular adhesive molecules in neurite growth, Biosci. Rep., 9 (1989) 1-12. 43 Ruoslahti, E., Structure and biology of proteoglycans, Annu. Rec. Cell Biol., 4 (1988) 229 255. 44 Sandrock, A.W. and Matthews, W.D., Identification of a peripheral nerve neurite growth-promoting activity by development and use of an in vitro bioassay, Proc. Natl. Acud. Sci. USA 84 (1987) 6934-6938. 45 Sanes, J.R., Schachner, M. and Covault. J., Expression of several adhesion molecules (N-CAM, LI, Jl, NILE, Uvomorulin, Laminin, Fibronectin and a Heparan Sulfate Proteoglycan) in embryonic, adult and denervated adult skeletal muscle, J. Cell Biol., 102 (1986) 42/)-431. 46 Schwab, M.E. and Caroni, P., Oligodendrocytes and CNS myelin are nonpermissive substrates for neurite growth and fibroblast spreading in vitro, .L Neurosci., 8 (1988) 2381-2393. 47 Silver, J., Postin, M. and Rutishauser, U.. Axon pathway boundaries in the developing brain. 1. Cellular and Molecular determinants that separate the optic and olfactory projections, J. Neurosci., 7 (1987) 2264-2272. 48 Snow, D.M., Steindler, D.A. and Silver, J.. Molecular and cellular characterization of the glial roof plate of the spinal chord and the optic tectum: a possible role for proteoglycans in the development of an axon barrier, Det'. Biol., 138 (19901 359-376. 49 Snow, D.M., Lemmon, V., Carrino, D.A., Caplan, A.l. and Silver, J., Sulfated proteoglycans in astroglial barriers inhibit neurile outgrowth in vitro, Exp. Neurok, 109 (19911) 111-1311. 50 Snow, D.M., Watanabe, M., Letourneau, P.C. and Silver, J., A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth, Det'elopment, I 13 (1991) 1473 1485. 51 Stahl, B., Muller, B., Von Boxberg, Y., Cox, E.C. and Bonhoeffer, F., Biochemical characterization of a putative axonal guidance molecule of the chick visual system, Neuron, 5 (1990) 735 743. 52 Steindler, D.A. and Cooper, N.G.F., Glial and glycoconjugate boundaries during postnatal development of the central nervous system, Det,. Brain Res., 36 (1987) 27-38. 53 Steindler, D.A., Cooper, N.G.F., Faissner, A. and Schachner, M., Boundaries defined by adhesion molecules during development of cerebral cortex: the J1/tenascin glycoprotein in the mouse somatosensory cortical barrel field, Det'. Biol., 131 (1989) 243-260. 54 Tosney, K., Ceils and cell-interactions that guide motor axons in the developing chick embryo, Bioessays, 13 (1991) 17-23. 55 Turnbull, J.E., Fernig, D.G., Ke, Y., Wilkinson, M.C., and Gallagher, J.T., Identification of the basic fibroblasl growth factor binding sequence in fibroblast heparan sulfate, J. Biol. Chem., 267 (1992) 111337-11/341. 56 Zaremba, S., Naegele, J.R.. Barnstable, C.J. and Hockfield, S., Neuronal subsets express multiple high-molecular-weight cellsurface glycoconjugates defined by monoclonal antibodies Cat-301 and VCI.I, J. Neurosci., I11 (I990) 2985-2995.