Large chondroitin sulfate proteoglycans of developing chick CNS are expressed in cerebral hemisphere neuronal cultures

Decelopmental Brain Research, 73 (1993) 261-272 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00

261

BRESD 51640

Large chondroitin sulfate proteoglycans of developing chick CNS are expressed in cerebral hemisphere neuronal cultures A.K. Hennig c, D. Mangoura a and N.B. S c h w a r t z a,b,c Departments of a Pediatrics, h Biochemistry and Molecular Biology and c Committee on Det,elopmental Biology, Unicersity of Chicago, Chicago, IL 60637 (USA) (Accepted 29 December 1992)

Key words." Chondroitin sulfate proteoglycans; Extracellular matrix; Telencephalon; Chick embryo; Cerebral hemisphere development; Neuronal migration; Neuronal culture

Chondroitin sulfate proteoglycans (CSPG) of the extracellular matrix may play regulatory roles in central nervous system (CNS) development. We have examined the expression of two large CSPGs of the embryonic chick brain, which can be differentiated using the monoclonal antibodies HNK-1 and $103L, in cultures of embryonic day 6 chick cerebral hemisphere neurons. Western blot analysis following immunoprecipitation and endoglycosidase treatment revealed that these cultures produce S103L- and HNK-l-reactive proteoglycans which are biochemically indistinguishable from the CSPGs (previously) identified in homogenized chick embryo brain extracts. The HNK-l-reactive CSPG accumulated in the medium throughout the course of cultures. In contrast, the S103L-reactive CSPG was found in a neuron-associated form during the period of aggregate establishment in culture, as well as in a soluble form secreted into the medium. Immunocytochemical staining of cultures with the SI03L antibody localized reactivity to most neurons during the period of aggregate formation, while neuronal processes and the few flat cells present (presumably neuroblasts and early glia) were negative. Cell selection experiments confirmed that neurofilament-positive cells were the source of the Sl03L-reactive CSPG. The use of differential fixation techniques suggested that the cell-associated S103L reactivity may be intracellular. Because of this pattern of expression and localization, we propose that the developmentally regulated S103L-reactive CSPG may play a role in neuronal migration arrest and organization of neurons into functional aggregates.

INTRODUCTION Brain morphogenesis is a complex, highly ordered process; the underlying mechanisms are as yet poorly understood. The cells of the CNS arise from neurectodermal precursors which line the lumen of the neural tube and migrate outward to final positions in lamina surrounding the neural tube to make up the vesicles of the CNS 3'32"6°. The ~ignals which control the migration, maturation, and organization of these cells into functional nuclei are not known. AS in other developing tissues, the extracellular matrix (ECM) is postulated to play an important role in guiding migration, inducing differentiation and process extension, directing the processes to the correct target areas and initiating appropriate synaptogenesis l°'33'4kSk56'57,62. The components most likely to be responsible for the initial level of this organization are the large proteoglycans and

glycoproteins such as laminin, fibronectin and contactin 33'4v'48,St, all of which are composed of a variety of domains with different structural and functional characteristics, some of which influence cell motility. A network of such macromolecules compartmentalizes the matrix, localizing small, diffusible signal molecules such as growth factors and ions 6'54'55, as well as providing differentiation signals directly via cell surface receptors such as integrins 23. The matrix of the central nervous system is rich in a variety of proteoglycans and their glycosaminoglycan (GAG) components, predominantly chondroitin sulfate, heparan sulfate and hyaluronate 22'36'43. Changes in the quantity and localization of some of these components over the course of CNS development have been reported 18'22'36. In addition, some brain proteoglycans have been shown to influence neuronal locomotion, promote neurite outgrowth in vitro 24, direct axons

Correspondence: N.B. Schwartz, Department of Pediatrics, University of Chicago Hospitals, 5841 S. Maryland Ave., MC 5058, Chicago, IL 60637, USA. Fax: (1) (312) 702-9234.

262 to their targets 7'4j, or modulate the neurite-promoting activity of laminin HBm. Furthermore, neuronal activity has been shown to regulate expression of individual proteoglycans 26'27s'3, and thus to influence the composition of the ECM. These reports indicate that, early in embryogenesis, bidirectional interactions occur between developing neurons and the surrounding matrix, in which proteoglycans may play inductive and regulatory roles. We have previously described the existence and differential expression of two large chondroitin sulfate proteoglycans (CSPG) in embryonic chick brain, which can be distinguished using the monoclonal antibodies S103L and HNK-12L2~. The Sl03L-reactive brain proteoglycan shares portions of the core protein with the large CSPG of chick cartilage zg, the chick homolog of 'aggrecan '~6'28, but carries less chondroitin sulfate and lacks keratan sulfate entirely. The HNK-l-reactive CSPG, which does contain keratan sulfate, has a similar size core protein but does not cross-react with antibodies against the cartilage CSPG core protein. These two brain CSPGs differ also in their expression patterns during embryogenesis. Peak S103L expression in the chick telencephalon 2t coincides with the period of migration and establishment of neuronal nuclei 2-~'6°. In order to circumvent the difficulties in understanding developmental events occurring in intact brain, expression of these CSPGs have been investigated in cultures of CNS neurons derived from day 6 chick embryo (E6) cerebral hemispheres. In these cultures, neuronal proliferation ceases almost completely within the first 24 h after culture initiation, and active organization of neurons into aggregates interconnected by neurite networks takes over, resembling neuronal behavior in ovo 34. Therefore, this seemed an appropriate model system in which to study regulation of S103L-reactive CSPG expression in order to identify the cellular source(s) and to investigate the probable function(s) in cell-to-cell and cell-substrate interactions, aspects that cannot readily be addressed at the tissue/organ level. MATERIALS A N D METHODS Antibodies $103L is a monoclonal rat lgG antibody directed against a well-characterized epitope on the core protein of the large aggregating CSPG of chick cartilage 1537'28. HNK-1 is a monoclonal mouse IgM antibody which recognizes a carbohydrate epitope containing a 3-sulfoglucuronic acid moiety 1,4.11. Hybridomas producing these were propagated in RPMI-1640 medium, and semi-purified antibody prepared from conditioned medium by precipitation with a m m o n i u m sulfate (S103L) or boric acid (HNK-1) 19. Monoclonal anti-keratan sulfate was obtained from ICN Biomedicals and Seikagaku America; monoclonal antibodies against neurofilament-160 and -200 (NF) and glial fibrillary acidic protein (GFAP) and secondary, species-specific antibodies were obtained from Sigma Chemical Company.

(hick embpyos Fertile White Leghorn eggs (Sharp Sales) wcl~_, incubated in ;~ humidifying incubator with automatic rotation (Marsh R~II-X). "lhe first 24 h of incubation was considered day O, and the ag~: ~1 embryos was determined accordingly.

Neuronal cultures Cultures were prepared according to the method of Pettmam~ et al. 45 as modified by Mangoura and Vernadakis 34. Briefly, cerebral hemispheres were removed from 6-day-old chick embryos, cleaned of meningeal membranes, and mechanically dissociated by forcing through a 48-p,m nylon mesh. Dissociated cells were plated on polylysine-coated tissue culture dishes (Falcon) or plastic coverslips (LUX) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Grand Island Biological Company), 1(10 U penicillin and 0.1 mg streptomycin (Sigma) per ml and incubated at 37°C in a humidified 1[)54 CO~ incubator. I mM /3-D-xyloside (p-nitrophenyl-/3-D-xylopyranoside, Koch Light Labora tories) was added to some cultures at initiation s~. Cultures depleted of differentiated neurons were prepared by plating the cell suspension on untreated tissue culture dishes. Non-adherelal cells were removed after a 3-h incubation at 37°C by aspirating the medium and rinsing the culture dish once. After an additional 48-h incubation, adherent cells were released with trypsin and transferred to fresh uncoated dishes. This double selection against neurons resulted in cultures which contained less than I%, neurofilament-positive cells during the initial 2 weeks of culture (Fig. 5). Medium on all cultures was removed and replaced with fresh stock medium every 3 to 4 days. For each set of cultures constituting one experiment, the medium on remaining dishes was changed at the time each subset ot cultures representing a datapoint was harvested.

lmmunocytochemist~ (ICC) Cells cultured on plastic coverslips were washed with PBS containing 1 m M Mg 2+ and Ca 2+, fixed in 4% paraformaldehyde with 0.2% glutaraldehyde in PBS for 1 h (NF and SI03L), with absolute acetone for 10 min at - 2 0 ° C (for GFAP), or with absolute methanol for 10 rain at room temperature. Some samples were subsequently treated with 1% Triton X-100 or 0.1% trypsin for 5 mira then rinsed with PBS, and fixed under different conditions (see Table t). Coverslips were then incubated overnight at 4°C with S103L (1:1,000). anti-NF (1 : 160), or anti-GFAP (1 : 1,600), or with no primary antibody, in diluent consisting of PBS with 2% normal rabbit serum. After additional rinsing, the coverslips were incubated with the second antibody (rabbit anti-rat IgG for S103L or rabbit anti-mouse IgG for NF and G F A P ) at a dilution of 1 : 100 in PBS with 35~ bovine serum albumin (BSA) for 1 h, then with peroxidase-anti-peroxidase complex diluted 1:100 in PBS with 3% BSA for another hour. Coverslips were rinsed again and reacted with 0.05% diaminobenzidine (DAB), 0.01% H 2 0 2 , and 8% NiCI 2 in 50 mM Tris-HC1, pH 7.6, for 4 - 8 rain to visualize antibody reactivity.

Sample preparation Cerebral hemispheres were dissected from embryos at E l l , cleaned of meningeal tissue, and immediately frozen on dry ice for storage at - 7 0 ° C . The samples were thawed in 100 m M T r i s / 5 mM E D T A buffer, pH 7.4, with fresh protease inhibitors, disrupted by sonication, and centrifuged at 12,000× g to remove particulate material. An aliquot of the supernatant was reserved for total protein determination, and the remainder digested with keratanase and chondroitinase prior to immunoprecipitation a n d / o r SDS-PAGE analysis. Cultures were harvested by aspirating the m e d i u m and rinsing the dishes once with 10 m M phosphate-buffered saline (PBS), pH 7.2. The cultured cells were harvested using a rubber policeman, transferred to a centrifuge tube, washed once more with PBS, and the cell pellet was stored at - 2 0 ° C . Culture medium removed prior to harvesting was stored separately. At the time of analysis, protease inhibitors were added to thawing culture medium to final concentrations of 1 m M phenylmethylsulfonyl fluoride (PMSF), 10 m M N-ethyl maleimide (NEM), and 0.36 m M pepstatin, and the medium concen-

263 for 2 - 3 h at 4°C. Samples which had been pre-absorbed fl)r 2 - 3 h with normal rabbit serum-coated Pansorbin were then added and the mixture incubated overnight at 4°C. Precipitates were collected by centrifugation at 2,000× g, washed twice with Slll3L buffer, once with SI03L buffer plus 0.1% SDS and once with 10 m M Tris, pH 7.4, containing 0.1% Nonidet P-4(I, then dissociated by boiling 5 rain in 0.5§/- SDS plus 1% 2-mercaptoethanol.

trated using Centricon 30 concentrators (Amicon Corp.). Cell pellets were resuspended in 100 m M T r i s / 5 m M E D T A buffer, pH 7.4, with fresh protease inhibitors, sonicated, centrifuged to remove particulate material, and total protein determined using the Pierce BCA protein determination kit (Pierce Chemical Co.).

Keratanase and chondroitinase digestion Procedures were derived from previous reports2~42; details of the technique have been published elsewhere. Briefly, samples in 100 m M T r i s / 5 mM E D T A buffer, pH 7.4, with fresh protease inhibitors (1 mM PMSF, 10 m M NEM, and 0.36 m M pepstatin) were treated with 0.5 U chondroitinase ABC or AC a n d / o r 0.25 U keratanase per ml of sample at 37°C for 4 h (all three enzymes were obtained from ICN Biomedicals.)

Western blot analysi.s Cell cultures were separated at harvest into cell-free supernatant ('medium') and washed cell pellet ('cells'). For each time point, material from four 60-mm culture dishes was pooled. The entire cell pellet obtained from each harvest (four 60-ram culture dishes) was used for all data points designated as 'cells'. In contrast. ' m e d i u m ' data points were prepared from the total volume of medium (12 ml) obtained at harvest for CI to C7 cultures (Fig. 3), while 'medium" data points from later E6CH cultures (C7-15, Fig. 4) and neuron-depleted cultures (Fig. 5) were prepared from 3-ml aliquots (the volume of one culture dish). This was done to minimize the amount of reactive material assayed and avoid antigen excess in the immunoprecipitation reaction and assure that the amount of reactive material in the blotted sample was within the range of linearily of the detection method used. Deglycosylated samples or lysed immunoprecipitates were clcctrophoretically separated on 3.5-6~i SDS-polyacrylamide gradient gels according to the method of Laemmli 3¢~. Samples were electroblotted to nitrocellulose at 18 mA in 12 mM Tris, pH 7.4, with 6 m M sodium acetate and [I.3 mM EDTA, for 12-.18 h 2'~. The nitrocellulose sheets were then air-dried and the standard lanes removed for staining with [).1% Amido black in l(/C/, acetic acid/2(l~ methanol. The rest of the sheet was incubated in 6% BSA in 0.125 M borate-

lmm unoprecipita tion Immunoprecipitations were performed essentially as previously described s'34. For S103L s, 100 /zg partially purified antibody was added to each sample in 10 m M Tris, pH 8.0, with 0.5 M NaCI, 0.5% sodium deoxycholate, 0.1 M 6-amino-n-caproic acid, 1% Nonidet P-40, I mM E D T A , 5 mM benzamidine, 1 mM PMSF and 10 mM N E M (S103L buffer). The mixture was incubated for 1 h at room temperature, then 1 h at 4°C. Following addition of 5 /xl of normal rat serum (Sigma) and 440 Izg of rabbit anti-rat lgG (ICN Biomedicals), samples were incubated overnight at 4°C. For HNK-12'~, 10 tzg Pansorbin (Calbiochem) was washed briefly in 15 m M Tris, pH 8.0, with 0.15 M NaCI, 5 m M CHAPS, 0.5% Nonidet P-40, 1 mM PMSF and Ill m M N E M (HNK-I buffer). The pellet was incubated with 100 ~1 of affinity-purified rabbit anti-mouse IgM for 20 rain at 4°C, washed again and incubated with 100 # g partially purified HNK-1

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Fig. 1. S103L reactivity is localized to neuronal cell bodies. Peroxidase-anti-peroxidase immunostaining of E6CH neuronal cultures after 4 days in culture (C4) ( A - C ) or C15 (D). A: diluent containing 2% normal rabbit serum but no primary antibody: neurons did not show any reactivity. B: neurofllament-160 and -200 antibodies. C: S103L; immunoreactivity was seen in neuronal somata (arrows) and axonal hillocks, but not neuronal processes and growth cones (arrowheads), or flat cells (open arrows). D: GFAP; at C14, when foci of GFAP-positive cells appear, no S103L reactivity was present. Bar = 25 p~m for A - C , and 5 4 / z m for D.

264 TABLE I

E6CH cultures: effects of different fixation methods on SI03L staining E6CH cultures maintained for 4 days in culture were fixed with 4% paraformaldehyde in PBS (4% PFA) with or without 0.25~ glutaraldehyde (glut.); or with absolute acetone; or absolute methanol. Some samples were further treated with 1% Triton X-100 or 0.1% trypsin in PBS. Cultures were stained with S103L and peroxidaseanti-peroxidase. Staining was scored as '_+'= some cell aggregates show weak staining, ' + ' = some but not all cells or aggregates are stained, "+ + ' = most aggregates and some individual cells are stained, '+ + + ' = all aggregates and many individual cells are stained, and ' + + + +' = similar cellular distribution but more intense staining than ' + + +'. Asterisk (*) indicates the conditions considered optimal and used for all subsequent studies. Fixation

No treatment 1% Triton

4% PFA + ~ 4%PFA, 0.05%glut. + + 2 4% PFA, 0.2% glut. + 4- 2.5 Acetone NEG Methanol + s

0.1% Trypsin

+ +++ 3 + + + 2.4 4- 4- 4- -r- 6.7.* 4- 4- 4- 4.~, NEG 4- x

Occasional cell clumps stain; cell bodies only. Cell clumps and occasional single cell bodies stain. Most single cell bodies stain; more intense staining than untreated. Destruction of cell processes and some single cells. Occasional slight staining of proximal processes; distal processes and growth cones non-reactive. Somata staining stronger than above. 7 Most proximal processes reactive with occasional staining of entire processes connecting clumps of cells. s Very. weak staining of cell clumps.

2 3 4 5

buffered saline (BBS), pH 8.5, for at least 3 h, followed by a 2-h incubation with agitation in either hybridoma-conditioned medium plus 3% BSA or in anti-keratan sulfate diluted 1:500 in BBS plus 3% BSA. After washing with BBS, blots were incubated for 1-2 h with horseradish peroxidase-conjugated rabbit secondary antibody (anti-rat lgG for S103L, anti-mouse IgM for HNK-1, or anti-mouse IgG for anti-keratan sulfate) diluted 1 : 1,000 in BBS with 3% BSA. After washing again, bound antibody was visualized with 0.03% DAB and 0.0075% H202 in 50 mM Tris, pH 7.6. Bands were scanned in triplicate using a Hoeffer GS 300 transmittance/reflectance densitometer and analyzed by integration using the HSI GS-370 software program. RESULTS

Cellular and subcellular localization o f Sl03L-immunoreactivity in E 6 C H cultures O u r p r e v i o u s investigations o f two large C S P G s in d e v e l o p i n g chick C N S 21'29 r e v e a l e d e x p r e s s i o n p a t t e r n s suggesting t h a t t h e s e m a c r o m o l e c u l e s , p a r t i c u l a r l y the d e v e l o p m e n t a l l y r e g u l a t e d S103L-reactive C S P G , m a y p l a y a role in n e u r o n d i f f e r e n t i a t i o n a n d / o r n e t w o r k o r g a n i z a t i o n . T o investigate this f u r t h e r , e x p r e s s i o n of b o t h C S P G s was e x a m i n e d in n e u r o n a l c u l t u r e s o f c e r e b r a l h e m i s p h e r e n e u r o n s f r o m 6 - d a y - o l d chick e m bryos ( E 6 C H ) , which have p r e v i o u s l y b e e n shown to faithfully r e p r e s e n t the series o f events which o c c u r in vivo for t h e first 2 w e e k s in c u l t u r e 34'35. E 6 C H c u l t u r e s (Fig. 1A) c o n t a i n p r e d o m i n a n t l y ( > 9 5 % ) n e u r o f i l a m e n t - p o s i t i v e n e u r o n s (Fig. 1B), which a g g r e g a t e a n d

e l a b o r a t e n e u r i t i c p r o c e s s e s d u r i n g the first few days in culture. T h e few n e u r o f i l a m e n t - n e g a t i v e flat cells that are p r e s e n t early in c u l t u r e s a r e negative for G F A P d u r i n g the first 2 w e e k s in c u l t u r e 34. A f t e r that time, the fiat cells p r o l i f e r a t e , e v e n t u a l l y covering most of the c u l t u r e vessel surface, a n d foci of G F A P - p o s i t i v e cells begin to a p p e a r by 15 days in c u l t u r e (Fig. ID). D i f f e r e n t fixation a n d p e r m e a b i l i z a t i o n t e c h n i q u e s w e r e t e s t e d to s e e k i n f o r m a t i o n a b o u t the localization of t h e e p i t o p e on n e u r o n s a n d / o r o t h e r cells p r e s e n t in the cultures. A c e t o n e - and m e t h a n o l - f i x e d c u l t u r e s s h o w e d no S103L-like i m m u n o r e a c t i v i t y (likely d u e to d e n a t u r a t i o n o f the e p i t o p e ) . P a r a f o r m a l d e h y d e ( P F A ) fixation b e s t p r e s e r v e d reactivity, a n d g l u t a r a l d e h y d e cross-linking i n t e n s i f i e d the staining (Table I). Since P F A fixation is reversible d u r i n g long a q u e o u s incubations, the a n t i g e n m a y be i n c o m p l e t e l y fixed in the a b s e n c e o f g l u t a r a l d e h y d e , a n d lost d u r i n g s u b s e q u e n t w a s h i n g steps. Post-fixation t r e a t m e n t with T r i t o n X100 o r trypsin e n h a n c e d staining after g l u t a r a l d e h y d e fixation, i n d i c a t i n g t h a t the a n t i g e n was not c o m p l e t e l y accessible to t h e a n t i b o d y a n d suggesting that the antigen r e c o g n i z e d by S103L m a y be localized intracellularly. T h e S103L a n t i b o d y s t a i n e d cells m o r p h o l o g i c a l l y i d e n t i f i a b l e as n e u r o n s (Fig. 1C), a n d which c o r r e s p o n d to n e u r o f i l a m e n t - p o s i t i v e cells in a n t i - N F - s t a i n e d cult u r e s (Fig. 1B). S103L i m m u n o r e a c t i v i t y was localized to the r e g i o n of the cell b o d y with occasional faint staining o f axonal hillocks a n d p r o x i m a l c o a r s e p r o cesses (arrows); fine p r o c e s s e s a n d g r o w t h cones w e r e not s t a i n e d ( a r r o w h e a d s ) . In addition, n e i t h e r flat cells (Figs. 1C a n d 3B, o p e n arrows) n o r the G F A P - r e a c t i v e cells s e e n in o l d e r c u l t u r e s (not shown) were recogn i z e d by this a n t i b o d y . F u r t h e r m o r e , no a c c u m u l a t i o n o f e i t h e r S103L- or A l c i a n b l u e - r e a c t i v e m a t e r i a l in e x t r a c e l l u l a r d e p o s i t s was n o t e d in these cultures, likely d u e to the loss of soluble m a t r i x c o m p o n e n t s d u r i n g the fixation p r o c e s s 36.

Biochemical characterization o f proteoglycans in E6CH cultures D i f f e r e n t i a l e n d o g l y c o s i d a s e d i g e s t i o n s 42 followed by i m m u n o p r e c i p i t a t i o n a n d w e s t e r n blot analysis w e r e p e r f o r m e d on h a r v e s t e d m e d i u m f r o m 5 - d a y - o l d cultures to d e t e r m i n e t h e p r e s e n c e o f p r o t e o g l y c a n s (Fig. 2). E x t r a c t s o f b r a i n d i s s e c t e d at E l l w e r e t r e a t e d in p a r a l l e l for c o m p a r i s o n , a n d stock c u l t u r e m e d i u m was also e x a m i n e d to rule o u t fetal bovine s e r u m o r o t h e r c u l t u r e additives as a source of reactive m a t e r i a l . U n d i g e s t e d s a m p l e s ( U ) s h o w e d no large m o l e c u l a r weight b a n d s a f t e r s e p a r a t i o n on a 3 . 5 - 6 % p o l y a c r y l a m i d e gel d u e to p o l y d i s p e r s i t y a n d failure o f m o l e c u l e s b e a r i n g

265 full-length g l y c o s a m i n o g l y c a n chains to e n t e r t h e gel. A f t e r r e m o v a l o f c h o n d r o i t i n sulfate c h a i n s with c h o n droitinase ABC, culture medium contained both S103L-reactive (Fig. 2A) a n d H N K - l - r e a c t i v e (Fig. 2B) b a n d s with t h e s a m e e l e c t r o p h o r e t i c m o b i l i t y as the b r a i n C S P G s 29. N e i t h e r the S103L-reactive b a n d in E 6 C H c u l t u r e m e d i u m n o r t h a t in b r a i n extract s h o w e d a d d i t i o n a l c h a n g e s in m o b i l i t y a f t e r t r e a t m e n t with k e r a t a n a s e (Fig. 2A: C H - M a n d E l l Brain, l a n e s C vs. KC), i n d i c a t i n g t h e a b s e n c e of k e r a t a n sulfate chains.

A M

I

CH-M

II

E l l BRAIN

]1

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F u r t h e r m o r e , no reactivity with a n t i - K S was seen in any of t h e s e s a m p l e s ( d a t a not shown.) In contrast, the H N K - l - r e a c t i v e b a n d s from b o t h sources s h o w e d a slight d e c r e a s e in m o l e c u l a r size (Fig. 2B: C H - M a n d E l l Brain, l a n e s C vs. KC) indicative of k e r a t a n sulfate removal. T h e p r e s e n c e of k e r a t a n sulfate on t h e H N K 1-reactive C S P G s from c u l t u r e m e d i u m a n d b r a i n extracts was c o n f i r m e d using a m o n o c l o n a l a n t i b o d y specific for k e r a t a n sulfate (Fig. 2C): reactivity seen in H N K - 1 i m m u n o p r e c i p i t a t e s of c h o n d r o i t i n a s e - t r e a t e d (C) s a m p l e s is lost from i m m u n o p r e c i p i t a t e s of s a m p l e s t r e a t e d with k e r a t a n a s e (KC). P r o t e a s e f i n g e r p r i n t i n g by t h e m e t h o d of C l e v e l a n d ~2 f u r t h e r s u p p o r t e d the similarity of the two p r o t e o g l y c a n s in s p e n t c u l t u r e m e d i u m with t h o s e from b r a i n extracts ( d a t a not shown).

Deuelopmental profile of $103L- and HNK-l-proteoglycan expression in E6CH cultures ~

C'

B

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CH-M

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E x p r e s s i o n of t h e two C S P G s over t h e course of the c u l t u r e p e r i o d was i n v e s t i g a t e d by W e s t e r n blot analysis as well as i m m u n o c y t o - c h e m i c a l staining for S103L (Fig. 3), as o p t i m i z e d in p r e v i o u s studies ( c o n d i t i o n s d e s i g n a t e d with an asterisk in T a b l e I). HNK-1 imm u n o c y t o c h e m i s t r y was not p e r f o r m e d since the H N K 1 antibody recognizes a carbohydrate epitope present on n u m e r o u s n e r v o u s system g l y c o p r o t e i n s s2 a n d glycolipids 4. A l t h o u g h the v a r i o u s H N K - l - r e a c t i v e species can be d i s t i n g u i s h e d on w e s t e r n N o t s d u e to their d i f f e r e n t e l e c t r o p h o r e t i c mobilities, i m m u n o c y t o c h e m i cal staining with H N K - I w o u l d not distinguish b e t w e e n t h e m , a n d t h e r e f o r e was not i n c l u d e d in this study. F u r t h e r m o r e , M c G a r r y et al. 39 have r e p o r t e d that 100% o f c u l t u r e d day 7 chick spinal c o r d n e u r o n s react with H N K - 1 by i m m u n o c y t o c h e m i s t r y .

N

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Fig. 2. CSPGs expressed in cultures are indistinguishable from those of intact brain. Aliquots of stock culture medium (M), medium removed from E6CH cultures 5 days after initiation (CH-M), or Tris/EDTA extracts of homogenized Ell brain (Ell Brain) were digested with chondroitinase ABC (C), with chondroitinase and keratanase (KC), or incubated in buffer without enzymes (U), then immunoprecipitated with S103L (panel A) or HNK-1 (panels B and C). Immunoprecipitated material was separated on 3.5-6% SDSPAGE gels and assayed by Western blot using SI03L (panel A), HNK-1 (panel B), or anti-keratan sulfate (anti-KS) (panel C). Migration of laminin A chain standard (440 kDa) is indicated. No bands are visible in undigested samples (U). An S103L-reactive band is seen in all enzyme-treated E6CH medium and brain extract samples (panel A), with no change in electrophoretic mobility after treatment with keratanase. The HNK-l-reactive bands from culture medium and brain show a slight shift in mobility after keratanase digestion (panel B; KC vs. C) as well as reactivity with anti-KS after treatment with chondroitinase alone (panel C; C) which disappears from samples treated with keratanase (KC). No reactivity with any antibody was seen in samples of stock culture medium. Arrows indicate migration of 440 kDa laminin A chain standard.

266 staining increased (Fig. 3B-D). Maximal reactivity was seen on day 5, at which time most ol' the neurons, particularly those in aggregates, were positive (Fig. 3C,D), although staining was still limited primarily to neuronal somata (arrows indicate representative

E6CH cultures contained cells reactive with SI03L by immunocytochemistry as early as 1 day after plating (Fig. 3A). As aggregates form and neurons extend processes during the first five days in culture 34, both the number of S103L-reactive cells and the intensity of

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Fig. 4. Ccll-associated S103L expression is transient in E6CH cultures. S103L reactivity in cultures maintained for longer periods was investigated by Western Not (A). Lysates of cells harvested from four cultures or an aliquot of medium representing the volume present on one culture were assayed as described in the previous figure. In these cultures, cell-associated SI03L reactivity decreased after day 7, although the Sl03L-reactive CSPG continued to accumulate in the culture medium. Immunocytochemistry at day 12 (B) showed no cell-associated S103L reactivity remaining in neuronal somata or processes (arrowheads): flat cells were also non-reactive (open arrow).

strongly reactive cells). At this time, Alcian blue staining for sulfated mucopolysaccharides 4° showed no alcianophilic material present except within a few of the largest aggregates (data not shown). By day 7 (Fig. 3E), S103L staining of cells in aggregates began to decrease (arrowheads indicate non-reactive cell bodies and processes), although reactive cells could still be found in these cultures (arrows). Cultures remained immunoreactive with other antibodies such as anti-neurofilament (results not shown, but see Mangoura et al.34'35). Western blots showed a similar pattern of expression of the S103L-reactive CSPG in association with

rinsed cell pellets (Fig 3F, 'Cells'), with maximal expression occurring at day 5-7. This cell-associated S103L reactivity is lost after 7 days in culture (Fig. 4A, 'Cells'). However, soluble S103L-reactive CSPG continued to accumulate over the course of the cultures (Fig. 4A; 'Medium') despite replacement of medium on the cultures used for later time points. These findings indicate that synthesis of the S103L-reactive CSPG continued throughout the culture period. The absence of reactivity in cell pellets from the later cultures in the presence of high levels of soluble S103L-reactive CSPG in the medium rules out the possibility that cell-associated reactivity results from residual material from medium contaminating the cell pellets. Since S103L reactivity by immunocytochemistry showed the same pattern of expression as the cell-associated S103L-reactive CSPG detected in cell lysates by western blot, the cell-associated form of this CSPG is likely responsible for the neuronal staining. The amount of medium which was assayed differed between the samples in Figs. 3 and 4. In Fig. 3, the entire volume of medium harvested was immunoprecipitated, in order to detect low levels of CSPG in the earlier cultures. In Fig. 4, an aliquot corresponding to the volume of one culture dish was used for immunoprecipitation, in order to avoid antigen excess in the immunoprecipitation reaction, and to keep the amount of reactive material blotted to nitrocellulose within the linear range of detection.

¢3-D-Xyloside decreases large aggregate formation In order to investigate whether disruption of proteoglycan synthesis affected the behavior of cultured neurons, E6CH cultures were established and maintained 4 days in the presence of 1 mM p-nitrophenyl-/3-Dxyloside. This compound disrupts CSPG synthesis by competing with the proteoglycan core protein as a substrate for chondroitin sulfate chain initiation 5~. Cultures maintained 4 days in the presence of /3-i>xyloside showed a decrease in the number of large aggregates (Fig. 5), suggesting that newly synthesized CSPGs

Fig. 3. SI03L reactivity is developmentally regulated in E6CH cultures. Expression of SI03L reactivity in cultures was examined by immunocytochemistry at various time points after culture initiation. A: at 1 day in culture (C1), a few neurons exhibit SI03L immunoreactivity (arrows), while the majority are not reactive (arrowheads); B: at (;3, neurons have elaborated processes and are forming aggregates of neurons which are heavily stained over somata and axonal hillocks; however, their processes (arrowhead) and flat cells (open arrow) remain negative; C,D: at C5, numerous large aggregates are seen and most neurons are found within them or connected via the extensive network of fine and coarse processes. Maximal S103L reactivity is seen at this time, with most neuronal somata (arrows) located both within and outside aggregates showing strong staining which occasionally extends into axon hillocks and proximal processes. E: at C7, neurons in smaller aggregates were positive (arrow), however the number of positive aggregates has greatly declined (arrowheads). Bar = 25 ,~m for A - E . At the same time points, harvested cells and culture medium were examined for Sll)3L-reactive CSPG (F) or HNK-l-reactive CSPG (G) by immunoprecipitation and Western blot. Sample aliquots consisting of washed cell lysate supernatant (Cells) or spent culture medium (Medium) harvested from four 60-mm culture dishes were treated with chondroitinase and keratanase, immunoprecipitated with S103L (F) or HNK-I (G), separated, blotted to nitrocellulose, and stained with the same antibody used for immunoprecipitation. Arrows in panels F and (; indicate bands with the same electrophoretic mobility as the S103L- and HNK-t-reactive CSPGs in brain.

268 are involved in events leading to aggregate establishment.

CSPG expression in neuron-depleted cultures To further establish that the cellular source responsible for production of the S103L-reactive CSPG is the differentiating neurons present in these cultures, we examined $103L reactivity in cultures depleted of neurons which had differentiated to the point of being neurofilament-positive. This was done by plating on untreated plastic dishes, removing non-adherent cells after 3 h, and trypsinizing and passage 48 h later (Fig. 6A,B). These 'neuron-depleted' cultures were negative for S103L reactivity by all methods of examination throughout the first 2 weeks of culture, during the time period addressed in the previous figures (data not shown, but see Fig. 6). In contrast, an HNK-l-reactive band with mobility similar to that of the HNK-l-reactive CSPG (Fig. 6B, arrow) was seen in western blots of medium from these cultures at all times examined. Thus, in the E6CH culture system being described in

CONTROL CULTURES

this report, the presence of differentiated neurons is required for expression of S103L reactivity during the first 2 weeks, during the period in which events in telencephalon histomorphogenesis are reproduced 34'-~5 Neuron-depleted cultures were also examined after longer incubation periods (Fig. 6). Immunocytochemistry for GFAP, NF and S103L reactivity was also performed after 9, 15, and 21 days in culture. After 2 weeks, these cultures were enriched in astrocytes as seen with G F A P staining (Fig. 6C), and contained scarce cells with the morphology of oligodendrocytes (Fig. 6D, arrowhead). In addition, a few cells with neuronal morphology were also apparent (Fig. 6D, arrow). These cells also stained with NF antibodies (data not shown), indicating that the flat cell population remaining after depletion of differentiated neurons contained a stem cell precursor capable of giving rise to neurons. Recent reports 5° indicate the presence in adult mammalian brain of stem cells capable of giving rise to neurons in vitro. The precursor we have found within the flat cell population in E6CH cultures,

[~-D-XYLOSIDE

Fig. 5. fl-D-Xyloside decreases aggregate formation. E6CH cultures incubated in the presence or absence of 1 mM fl-D-xyloside for 4 days were compared with parallel control cultures. Representative phase contrast micrographs of each after 4 days in culture shows the decrease in large aggregates formed in the culture with fl-D-xyloside.

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Fig. 6. Neurons are the only source of the S103L-reactive CSPG in E6CH cultures. Cultures which had been depleted of differentiated neurons at initiation were investigated by Western blot analysis for the presence of SI03L reactivity (A) or HNK-1 reactivity (B). Arrows indicate bands with similar mobility to the respective CSPGs from brain extracts. Cells were also examined morphologically and by immunocytochemistry after 15 days in culture. Numerous loci of GFAP-positive astrocytes were present (C), and few neurons (arrow) and occasional oligos (arrowhead) are also apparent (D). No S103L reactivity was found by immunocytochemistry at any time in these cultures.

which gives rise to neurofilament-positive cells having neuronal morphology after two weeks in culture, may represent an equivalent avian stem cell. Immunocytochemical staining of neuron-depleted cultures with S103L showed no reactivity detectable at any time examined (data not shown). However, soluble S103L-reactive CSPG was detectable by Western blot after 2 weeks in culture, and reactive material appeared in cell lysates at 21 days (Fig. 6A). Since cell-associated reactivity is transient, it is possible that at the times chosen for immunocytochemical examination few neurons were expressing sufficient reactivity to be detected by immunocytochemistry. This association of S103L reactivity with the appearance of neurofilament-positive cells morphologically resembling neurons in cultures depleted of differentiating neurons at initiation, provides additional evidence that neurons in the process of differentiating are the source of the reactivity. We conclude from these experiments that differentiated neurons are the cell type responsible for production of the S103L-reactive CSPG, while the HNK-l-reactive CSPG is primarily produced

by the flat cells in these cultures. Neuronal contribution to H N K - 1 - C S P G expression has not been investigated. DISCUSSION Neuronal cultures derived from E6 chick embryo cerebral hemispheres (E6CH) provide a model for investigating the events that occur during telencephalon development. We have shown that two large CSPGs previously found in the developing chick CNS 2~'29 are expressed in these cultures in patterns which mimic in ovo expression. In this study we report that in E6CH cultures the S103L-reactive CSPG was shown to be produced by differentiated neurons. Sl03L-reactive CSPG was primarily secreted into the medium but additionally retained by the neuronal somata during the period of aggregate establishment in culture, which coincides with cortical center formation in ovo. In the mature telencephalon, neuronal somata are found in spatially and functionally distinct cytoarchitectonic areas or nuclei. During chick embryo develop-

270 ment, each of these nuclei is populated by neurons which arise more or less simultaneously from neuroepithelial stem cells and migrate to the appropriate positions within the enlarging telencephalon wall 2"3'4~'. In contrast to the 'inside-out' pattern reported for development of the mammalian cerebral cortex 37"4~',cerebral hemispheres of the chick are populated in an 'outsidein' pattern6°: waves of migrating neurons from discrete neuroepithelial 'compartments '6j colonize telencephalon regions successively from the outermost (populated by neurons which cease division on E4) to the innermost (populated by neurons ceasing division on E9) mostly radially 25. The neurons comprising each 'wave' appear to aggregate and interact preferentially with others of their own cohort and there is little mixing with different compartments ~'~. The entire complement of telencephalon neurons has arisen by embryonic day 10, and these have populated all major cytoarchitectonic areas by day 16 6°. Differentiation of glial cells begins at embryonic day 10, and their distribution is random throughout the different regions 6°. The onset of S103L-reactive proteoglycan expression in ovo corresponds to the period of active neuronal migration and organization in the chick CNS and ceases by El8 2~. This suggests that this macromolecule may be involved in the late events of neuronal migration. Neurons migrate along glial and neuronal processes via specific ligand interactions 2°'3~46. Previous investigations into the functions of extracellular matrix components have been limited to characterizing isolated macromolecules as permissive or non-permissive substrates for neuronal migration 33'48. Chondroitin sulfate proteoglycans have been shown to inhibit neuronal migration 44 and neuritic outgrowth 7'~'59. Distribution of such substrates could explain how migrating neurons keep to a migration pathway, but additional mechanisms must exist to provide directional cues and selectively stop neurons which have reached their target area, without interfering with the migration of other populations. However, previous investigations into the mechanisms of migration have not revealed how a migrating neuron decides when and where to stop. Pre-commitment to a particular area 37'3~, interaction with other neurons once the neuron has reached its target area 2°, or a combination of the two 2° have all been suggested. Based on the time of expression and the cellular localization pattern, we propose that the S103L-reactive CSPG may be produced by the migrating neurons as a component of the neuronal migration arrest mechanism. The S103L-reactive CSPG was found to be expressed in a cell-associated form during a brief period early in culture, while it also accumulated in soluble

fl)rnr in the medium throughout the culture period. N~> difference between the cell-associated and soluble forms of the Sl03L-reactive CSPG was detectable on western blots, suggesting that the difference in localization might be due to differences in retention of the molecule by the neurons rather than to intrinsic differenees between the two forms of the proteoglycan itself. Neurons may possess a retention mechanism specific for this CSPG in addition to the synthetic and secretion machinery, allowing differential expression at the level of localization as well as the level of synthesis. Another possibility is that a high initial rate of synthesis occurs without a corresponding increase in the rate of secretion, causing an intracellular accumulation of mature product. Developmental changes in subccllular localization of proteoglycans and other related ECM macromolecules in the rat brain have been previously described 36. While these were investigated at a later developmental period than our studies, such findings raise the possibility that rearrangements in localization of individual proteoglycans accompany regulatory functions. Immunocytochemical localization of a migration arrest signal to the cell body would be expected, since newly settled neuronal somata continue to elongate processes and establish synapses. Activation of its expression might result from 'predetermination' to turn on expression at a specific time after cell division ceased. Such predetermination is suggested in the case of the S103L-reactive CSPG by its apparent expression late in those cultures depleted at initiation of differentiated neurons. The same arrest mechanism would be expected to be utilized by migrating neurons throughout the CNS, and would explain the similarity of S103L-reactive CSPG expression patterns in all anatomic areas of the developing brain -~. A similar matrix adhesion/signal mechanism exists in the integrins, which act as receptors for specific ECM molecular domains as well as signal mediators between the matrix and the cytoskeleton 23'5~'53. The use of a large CSPG as an anchoring molecular receptor would in addition change the character of the local ECM, providing information to other cells in the vicinity. S103L reactivity is more often associated with clumps of neurons early in cultures, and is more intense in aggregates of cells than in individual neurons at all times in culture, suggesting a relationship between S103L-reactive CSPG expression and neuronal aggregation. Furthermore, a functional association between CSPG synthesis and aggregate formation has been suggested by the response of E6CH cultures to /3-D-xyloside: the presence of this drug during the period of aggregate formation decreased the number of large aggregates

271

seen after 4 days in culture. The need for an initial arrest signal would be transient; expression of the signal molecule might then be modulated (for example, by a change from retention to release) in order to redirect migration of subsequent cohorts of neurons to prevent interference with the neuronal interconnections being established. The soluble forms of both CSPGs likely represent components which would be localized to the extracellular matrix in vivo. The matrix of developing brain is reported to be highly soluble in aqueous solutions 36. We have shown 29 that greater than 80% of 35SO4labeled material in embryonic day 13 chick brain was extractable with PBS, which includes essentially all of the HNK-1- and S103L-reactive large CSPGs. The solubility of nervous system CSPGs may be a consequence of the absence from the CNS parenchyma of collagen and other fibrous proteins ~°'5~, with which proteoglycans interact 4° to form an insoluble meshwork in other connective tissues. The Alcian blue staining of E6CH cultures indicated the absence of a matrix containing polyanionic material, in contrast to the marked reactivity seen surrounding cultured chondrocytes. Therefore, we view material secreted into the medium in cell cultures as representative of what would be incorporated into an extracellular matrix if the cells were in situ in developing brain. Numerous developmentally regulated nervous tissue proteoglycans have been reported 13"1~'22"29'36"43"4~'~3.Because of their domain structure, these macromolecules could provide a means of coordinating the multiple regulatory mechanisms required in a tissue such as the CNS in which a variety of cell types migrate and differentiate in the same areas over a relatively short period of time. To date, the various roles of nervous tissue proteoglycans have not been delineated experimentally. The studies presented here show that, with respect to the two CSPGs described 2~'29, cerebral hemisphere neuronal cultures accurately reflect conditions in the developing chick brain in ovo, and provide a relevant model for investigating the expression and functions of extracellular matrix components which influence the early events in brain development. Using this model, we have defined the time course of expression, verified the cellular localization, and identified the cell type responsible for synthesis of these proteoglycans. These findings suggest that the developmentally regulated S103L-reactive CSPG may function as an anchoring mechanism to halt neuronal migration during the formation of cortical nuclei. In addition, the differential localization suggests that S103L-CSPG expression may represent a mechanism by which neurons communicate information regarding their position and

state of development. In any case, the developmental expression pattern implicates this CSPG in the process of neuronal migration arrest and final positioning in the developing chick CNS. Acknowledgements. This work was funded by USPHS Grants AR19266, HD-17332, and HD-09402; A.K.H. was supported by Training Grant HD-07136. We thank Kurt Kamper for technical assistance and Glenn Burrell for help in preparing this manuscript.

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