Heparan Sulfate Proteoglycans Are Ligands for Receptor Protein Tyrosine Phosphatase {sigma}

RPTP ${sigma}$ is a cell adhesion molecule-like receptor protein tyrosinephosphatase involved in nervous system development. Its avianorthologue, known as cPTP ${sigma}$ or CRYP ${alpha}$ , promotes intraretinal axongrowth and controls the morphology of growth cones. The molecularmechanisms underlying the functions of cPTP ${sigma}$ are still to bedetermined, since neither its physiological ligand(s) nor itssubstrates have been described. Nevertheless, a major classof ligand(s) is present in the retinal basal lamina and glialendfeet, the potent native growth substrate for retinal axons.We demonstrate here that cPTP ${sigma}$ is a heparin-binding protein andthat its basal lamina ligands include the heparan sulfate proteoglycans(HSPGs) agrin and collagen XVIII. These molecules interact withhigh affinity with cPTP ${sigma}$ in vitro, and this binding is totallydependent upon their heparan sulfate chains. Using molecularmodelling and site-directed mutagenesis, a binding site forheparin and heparan sulfate was identified in the first immunoglobulin-likedomain of cPTP ${sigma}$ . HSPGs are therefore a novel class of heterotypicligand for cPTP ${sigma}$ , suggesting that cPTP ${sigma}$ signaling in axons andgrowth cones is directly responsive to matrix-associated cues.

INTRODUCTION

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Introduction

The complex pattern of neural connectivity established duringnervous system development relies on the ability of the axon'smotile tip, the growth cone, to receive, transduce, and integratemultiple environmental signals. Protein phosphorylation on tyrosineresidues plays a key role in these processes (21, 29). Two majorfamilies of enzymes, the protein tyrosine kinases and the proteintyrosine phosphatases (PTPs), control cellular phosphotyrosinelevels. These enzymes are found in both cytoplasmic and transmembrane(receptor-like) forms, and the biochemical interactions betweenthem lead to a diversity of cellular behaviors (25, 76).

Receptor protein tyrosine phosphatases (RPTPs) have recentlyjoined the list of molecules involved in neural developmentand in particular in axon growth and guidance (reviewed in references6, 68, 72, and 79). Type 2 RPTPs, containing cell adhesion molecule(CAM)-like extracellular regions, may be particularly well equippedto trigger signals involving cell-cell or cell-extracellularmatrix contacts (68). Recent experiments with Drosophila havedemonstrated the involvement of the RPTPs DLAR and DPTP69D inmotor (19, 20, 50), retinal (27, 57), and midline (73) axonguidance. In leech, a LAR gene-related RPTP (HmLAR2) is implicatedin Comb cell behavior, specifically in process outgrowth andmutual avoidance by sibling growth cones (2, 28). Several vertebrateRPTPs have been shown to promote neurite outgrowth in cell culture,including cPTP ${sigma}$ (51), RPTP ${kappa}$ (23), RPTPµ (10), and RPTP ${delta}$ (82).Moreover, it has recently been shown that RPTP ${delta}$ also has a potentialguidance function, at least in vitro (74).

In mice, gene deficiencies in type 2 RPTPs lead to various abnormalities.LAR deficiency leads to a reduction in size of basal forebraincholinergic neurons, diminished hippocampal innervation, anddefects in other tissues, such as the mammary gland (63, 78,86). RPTP ${delta}$ deficiency leads to impaired learning and enhancedhippocampal long-term potentiation (77). The most extreme defectsare seen in RPTP ${sigma}$ -deficient mice, which show poor fecundity,hypomyelination of peripheral nerves, ataxias, and abnormalitiesin development of the hypothalamus and pituitary (24, 80). Althoughthe developmental mechanism of these defects is not known, itis of interest that the avian orthologue of RPTP ${sigma}$ , cPTP ${sigma}$ (69,85), regulates axon outgrowth of embryonic neurons (51).

Despite this accumulation of functional data, much less is knownabout the extracellular cues that trigger signal transductionthough RPTPs. Several RPTPs interact homophilically in trans,including RPTPµ (8), RPTP ${kappa}$ (62), and RPTP ${delta}$ (82), but theeffects of such homophilic interactions on enzyme function areas yet unclear. The laminin-nidogen complex has been shown tobe a heterotypic ligand for a nonneural isoform of LAR (59),while type 5 RPTP ${zeta}$ can bind to several molecules, including contactinand tenascin and the cytokines midkine and pleiotrophin (60).Significantly, pleiotrophin has also recently been shown tosuppress the catalytic activity of RPTP ${zeta}$ (53).

cPTP ${sigma}$ , originally described as CRYP ${alpha}$ (69), is a type 2 RPTP expressedas two major isoforms: cPTP ${sigma}$ 1 (CRYP ${alpha}$ 1) has three immunoglobulin-like(Ig) domains and four fibronectin type III (FNIII) domains inits extracellular region, while cPTP ${sigma}$ 2 (CRYP ${alpha}$ 2) has four extraFNIII domains. Both isoforms are strongly expressed in the chickenembryo nervous system, in particular within retinal and tectalaxons and on their growth cones (70, 71). Moreover cPTP ${sigma}$ 1 promotesintraretinal axon growth in vitro and controls growth cone morphologyvia the maintenance of lamellipodia (51). One or more ligandsfor cPTP ${sigma}$ are localized in the retinal and tectal basal lamina(BL) and on the glial endfeet of these tissues (33, 51), butthe identity of these ligands has remained elusive. To understandhow cPTP ${sigma}$ may function at the molecular and cellular levels,we sought to identify the molecular nature of these ligands.Here we show that cPTP ${sigma}$ is a heparin binding protein and thatextracellular matrix heparan sulfate proteoglycans (HSPGs),in particular agrin and collagen XVIII, are binding partnersfor cPTP ${sigma}$ in vitro. Receptor affinity probe assays on tissuesections reveal that the binding of cPTP ${sigma}$ ectodomains to HSPGsis absolutely dependent on the presence of heparan sulfate (HS)side chains. Site-directed mutagenesis in a putative heparinand HS (heparin/HS) binding site in cPTP ${sigma}$ completely abolishedthis interaction. These data, together with the overlappingexpression patterns of cPTP ${sigma}$ , agrin, and collagen XVIII in thedeveloping chick retina, suggest that the reported interactionsare physiologically relevant and that HSPGs could be a majorligand class for cPTP ${sigma}$ .

MATERIALS AND METHODS

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Materials and Methods

Expression constructs, fusion protein production, and site-directed mutagenesis. The pBG plasmid was constructed by subcloning the HindIII-XbaIfragment coding for human placental alkaline phosphatase (AP)from APTag2 (13) into pcDNA3.1 (Invitrogen). A HindIII fragmentcoding for the extracellular region of cPTP ${sigma}$ 1 (amino acids 1to 721; GenBank accession number L32780) was subcloned intopBG, in frame with AP, resulting in pBG ${sigma}$ 1-AP. pBG ${sigma}$ 1-AP was transfectedinto 293T cells (grown in Dulbecco's modified Eagle medium,10% fetal calf serum, 1% penicillin-streptomycin mixture; sigma)using Superfect (Qiagen). Conditioned medium containing thesecreted cPTP ${sigma}$ 1-AP fusion protein was collected after 6 to 7days, sterile filtered, buffered to pH 7.4 with 20 mM HEPES,and stored at 4°C. The fusion protein was quantified bymeasuring the AP activity as described previously (17).

The p ${sigma}$ 1VST expression vector was constructed by subcloning theHindIII fragment coding for the cPTP ${sigma}$ 1 extracellular region intothe pCS3N-VST shuttle vector (A. R. Aricescu and A. W. Stoker,unpublished data), which contains a HindIII site, a linker codingfor the vesicular stomatitis virus (VSV) peptide tag, a spacersequence, and an AclI site, all flanked by two NotI sites. p ${sigma}$ 1VSTwas used to express the secreted cPTP ${sigma}$ 1-VSV fusion protein in293T cells as described above, or for higher expression levels,the cPTP ${sigma}$ 1-VSV coding fragment was transferred into the RCAS(A)avian retrovirus (46) as follows: p ${sigma}$ 1VST was linearized withAclI and ligated into the ClaI opened RCAS(A); the shuttle vectorbackbone was then removed via NotI digestion and self-ligation,resulting in the RCAS- ${sigma}$ 1VST retrovirus. This construct was transfectedinto line 0 chicken embryo fibroblasts (grown in Dulbecco'smodified Eagle medium plus 10% fetal calf serum plus 2% chickserum) using Superfect. After six passages the conditioned mediumcontaining cPTP ${sigma}$ 1-VSV was collected, sterile filtered, and bufferedto pH 7.4 with 20 mM HEPES.

The pFN3 ${Delta}$ -AP expression plasmid, containing a cPTP ${sigma}$ 1 extracellularconstruct lacking the membrane-proximal FNIII-like domain, fusedto the amino terminus of AP, was a gift from John Chilton. Thisplasmid was used as a template for site-directed mutagenesisaccording to Stratagene's QuickChange mutagenesis kit protocol.Primer sequences can be obtained from A. R. Aricescu. All mutantconstructs were verified by DNA sequencing and transfected into293T cells, and conditioned media were collected as describedabove.

Solid-phase binding assays. Heparin-albumin (Sigma), agrin (purified as described in reference36), and collagen XVIII (purified as described in reference38) were immobilized on 96-well microtiter plates at a concentrationof 2 µg/ml (heparin-albumin) or 5 µg/ml (agrin andcollagen XVIII) for 2 h at room temperature. Remaining bindingsites were saturated by overnight incubation at 4°C in phosphate-bufferedsaline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄,1.5 mM KH₂PO₄ [pH 7.4]) containing 3% bovine serum albumin (BSA).Wells were incubated for 3 h at room temperature with conditionedmedia from 293T cultures, containing AP fusion proteins. Afterfour washes in PBS and one wash in SEAP buffer (0.5 mM MgCl₂,1 M diethanolamine [pH 9.8]), the bound AP activity was determinedby adding 200 µl SEAP buffer containing 10 mM p-nitrophenylphosphate. Progress curves were recorded for 1 h at room temperature,at 405 nm, using a Dynex MRX microplate reader, and the initialrates were determined using the SigmaPlot 4.01 software (SPSS,Inc.). Each data point represents the average of triplicatewells ± the standard deviation. Nonspecific binding wasdetermined from cPTP ${sigma}$ 1-AP bound to BSA (immobilized at a concentrationof 3 mg/ml) and was subtracted from each data point.

The data were plotted for Scatchard analysis in order to getinitial estimates of the binding parameters. cPTP ${sigma}$ 1-AP was foundto bind HSPGs with very high affinities, and at low concentrations,more than 10% of the initial AP activity was bound. Therefore,in order to obtain more accurate K_d values, we fitted the saturationcurve by nonlinear regression to the following equation, whichtakes into account significant ligand depletion, with negligiblenonspecific binding (75): b*_tot² - b*_tot[ß(K_d + L*_tot)+ r_tot] + ß L*_totr_tot = 0, where b*_tot is the totalamount of bound ligand (cPTP ${sigma}$ 1-AP) expressed in optical densityunits (OD) per minute, ß is the specific AP activityexpressed in OD per minute per molar concentration units, K_dis the equilibrium dissociation constant, L*_tot is the totalligand (cPTP ${sigma}$ 1-AP) concentration, and r_tot is the maximal specificbinding expressed in OD per minute. The specific activity usedfor cPTP ${sigma}$ 1-AP fusion protein was 1.12 OD/min/nM.

In vivo viral injection. White Leghorn eggs were obtained from Needle Farm (Herts) andincubated at 38°C to the desired stage. The RCAS- ${sigma}$ 1VST vectorwas used to generate stocks of viral inoculi as described previously(54). Virus (0.1 µl) was injected into the optic vesicleof stage 11 embryos ( $~$ 33 h) (40) using a PM1000 microinjector(MicroData Instrument Inc.). E6 embryos were processed for immunohistochemistryor in situ hybridization.

Immunohistochemistry and in situ hybridization. E6 or E10 chicken embryo heads were fixed in 4% paraformaldehydein PBS, cryoprotected in 30% sucrose in PBS, and frozen in OCTcompound (TissueTek). Cryosections (10 to 12 µm) weremounted on 3-aminopropyltriethoxysilane-coated glass slides.For immunohistochemistry, sections were blocked with 1% BSA-0.25%Triton X-100 in PBS for 15 min at room temperature. Primaryantibodies used were IG2 (anti-cPTP ${sigma}$ rabbit polyclonal) (70)diluted at 1:500; P5D4 (anti-VSV glycoprotein monoclonal; Sigma)at 1:500; 6D2 (anti-agrin monoclonal) (35) at 1:20, and 6C4(anti-collagen XVIII monoclonal) (38) at 1:20, all in 3% BSA-0.25%Triton X-100 in PBS. After 1 h of incubation at room temperature,the sections were washed three times in 0.1% BSA-0.05% TritonX-100 in PBS and secondary antibodies were added for 1 h atroom temperature: goat anti-rabbit peroxidase-conjugated (Promega),diluted at 1:100; rabbit anti-mouse peroxidase-conjugated (Promega),at 1:100; and goat anti-mouse fluorescein isothiocyanate-conjugated(Jackson Labs), at 1:200. After three final washes the peroxidasereactions were performed using the DAB substrate kit (VectorLaboratories); the fluorescein isothiocyanate-labeled sectionswere mounted in Fluorosave (Calbiochem), and all sections wereanalyzed using an Axiophot fluorescence microscope (Zeiss).

For in situ hybridization, the PRD4A viral probe (58) was used,as described previously (52).

Receptor affinity probe (RAP) in situ. The in situ localization of cPTP ${sigma}$ ligands on tissue sectionsand flat-mounted retinal basal laminae was performed using thecPTP ${sigma}$ 1-AP conditioned medium, as previously described (33, 51).In modified RAP assays, the cPTP ${sigma}$ 1-AP probe was preincubatedwith 100 µg of heparin (Sigma), bovine kidney HS (Sigma),or chondroitin sulfate (Calbiochem)/ml for 1 h at room temperaturebefore addition to the tissue samples. Alternatively, the tissuesamples were pretreated for 2 h at 37°C with 0.5 U of heparinaseIII (Sigma) in 50 µl of PBS, pH 7.4, containing 0.1% BSAor with 0.1 U of chondroitinase ABC (Sigma) in 50 µl ofTris-acetate buffer, pH 8. The protease inhibitor 4-(2-aminoethyl)-benzenesulfonylfluoride was added to both reactions at a final concentrationof 2 mM.

Immunoblotting and blot-overlay assays. An HSPG-enriched fraction (HfV) from E9 chicken embryo vitreousbodies homogenized in Ca²⁺-free and Mg²⁺-free Hanks' balancedsalt solution, pH 7.3 (CMF), was partially purified by ion-exchangechromatography as follows. The vitreous homogenate was supplementedwith 1:100 protease inhibitors cocktail with no metal chelators(Sigma) and centrifuged at 12,000 x g for 30 min at 4°Cto remove debris. The supernatant was applied on a Q-SepharoseFast Flow column (Amersham Pharmacia Biotech) and washed with0.5 M NaCl in CMF, and the HfV fraction was eluted with 1.5M NaCl in CMF. Eluted fractions were concentrated on a CentriconYM-30 (Amicon), and the buffer was changed to CMF. Aliquotsof the HfV, 100 ng of immunopurified agrin, and collagen XVIII(as described in references 36 and 38) were preincubated for2 h at 37°C with either 0.5 U of heparinase III, 0.1 U ofchondroitinase ABC, or 0.1 U of collagenase (Sigma type VII)in PBS, pH 7.4, containing 2 mM 4-(2-aminoethyl)-benzenesulfonylfluoride.

For immunoblotting, the reactions were mixed with equal volumesof Laemmli sample loading buffer (62 mM Tris [pH 6.8], 2% sodiumdodecyl sulfate (SDS), 10% glycerol, 5% ß-mercaptoethanol,0.001% bromophenol blue), boiled, and separated by SDS-6% polyacrylamidegel electrophoresis. Proteins were transferred onto Hybond-CExtra nitrocellulose membranes (Amersham Pharmacia Biotech),blocked overnight at 4°C in 5% nonfat milk in PBS, and probedfor 1 h at room temperature with antiagrin (6D2; 1:20) or anticollagenXVIII (6C4; 1:20) monoclonal antibodies. The secondary antibodyused was rabbit anti-mouse horseradish peroxidase (Dako) dilutedat 1:2,000. Chemiluminescence detection was performed usingthe ECL Western blotting kit (Amersham Pharmacia Biotech).

For blot overlay assays, the samples were processed as describedabove except that following overnight blocking, the membraneswere incubated for 3 h at room temperature with cPTP ${sigma}$ 1-VSV conditionedmedium supplemented with 0.5% Igepal CA-630 (Sigma). The membraneswere then washed three times with PBS containing 0.5% IgepalCA-630, and bound cPTP ${sigma}$ 1-VSV was detected using the anti-VSVmonoclonal antibody (P5D4; 1:1,000) as described above.

Homology modelling of cPTP ${sigma}$ domains. Individual domains of the extracellular region of cPTP ${sigma}$ weredefined using the Pfam server (http://www.sanger.ac.uk/Pfam[5]). The corresponding sequences were submitted to the SWISS-MODELv3.5 protein modelling server (http://www.expasy.ch/swissmod/SWISS-MODEL.html[31]) for analysis. Suitable modelling templates were identifiedin the ExNRL-3D database using SWISS-MODEL Blast. The sequencealignments obtained were analyzed, and the most appropriateExPDB entries were selected as templates. All the models generatedwere quality checked by the WHAT IF verification routines (WHAT-CHECK[45]) and the Biotech protein validation suite (http://biotech.embl-heidelberg.de:8400/).Energy minimization was done with the GROMOS implementationof Swiss-PdbViewer v3.6b2 (30) (http://www.expasy.ch/spdbv/).

RESULTS

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Results

cPTP ${sigma}$ binds to the retinal BL in vivo and in vitro. Our previous results established that the BL and glial endfeetof the chicken embryo retina, optic stalk, and chiasm containa prominent ligand for the cPTP ${sigma}$ 1 ectodomain (33). This was detectedin vitro using the cPTP ${sigma}$ 1-AP fusion protein in a RAP in situtechnique (26).To test whether a related binding pattern occursin vivo, we constructed the RCAS- ${alpha}$ 1VST retrovirus that encodesa secretable cPTP ${sigma}$ 1 ectodomain fused with the VSV peptide tag(cPTP ${sigma}$ 1-VSV). cPTP ${sigma}$ 1-VSV was readily detected as it accumulatedin conditioned medium from infected cells in culture (Fig. 1A).RCAS- ${alpha}$ 1VST virus was injected into the optic vesicle of chickembryos at stage 11 ( $~$ 33 h), and by embryonic day 6 (E6) theretrovirus had infected the whole retina (Fig. 1B). The cPTP ${sigma}$ 1-VSVprotein, secreted by infected retinal cells in situ, accumulatedspecifically in the ligand-rich BL region (Fig. 1D), providinggood evidence in vivo that at least one physiological ligandfor cPTP ${sigma}$ is located in this region.

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FIG. 1. Binding of cPTP ${sigma}$ to the E6 chick retinal basal lamina in vivo. (A) Time course of cPTP ${sigma}$ 1-VSV production by chicken embryo fibroblasts infected with RCAS- ${alpha}$ 1VST retrovirus. Samples of culture medium conditioned for 1 (lane 2), 2 (lane 3) or 3 (lane 4) days after transfection were analyzed by immunoblotting using antibody specific for the VSV epitope. Lane 1 contains conditioned medium from untransfected cells. (B and D) RCAS- ${alpha}$ 1VSV retrovirus-infected retinas at E6, probed with a digoxigenin-labeled viral probe by in situ hybridization (B) or with VSV antibody (D). Noninfected tissues were probed by in situ hybridization (C) or with VSV antibody (E). Arrowheads indicate the retinal BL. pe, pigmented epithelium. Scale bar, 0.1 mm.

cPTP ${sigma}$ is a heparin-binding protein. We screened for reagents that could modulate RAP in situ bindingof the cPTP ${sigma}$ ectodomain to the retinal BL. Increasing concentrationsof sodium chloride abolished this interaction, whereas the nonionicdetergent Igepal CA-630 had no effect even at high concentrations.We hypothesized therefore that the cPTP ${sigma}$ -ligand interaction waspredominantly ionic rather than hydrophobic and that it mayinvolve charged glycosaminoglycan chains.

Using a secreted cPTP ${sigma}$ 1 ectodomain fused with AP at its C terminus(cPTP ${sigma}$ 1-AP) (33), we found that in solid-phase binding assayscPTP ${sigma}$ 1-AP bound to heparin-albumin at subnanomolar concentrations(Fig. 2A). Using a nonlinear regression analysis, a K_d valueof 0.32 ± 0.05 nM was calculated, demonstrating thatcPTP ${sigma}$ binds heparin with very high affinity. Control constructscontaining AP alone or AP fused with the ectodomain of RPTPµdid not bind to heparin-albumin in similar assays (data notshown). In addition, the nonspecific binding of cPTP ${sigma}$ 1-AP onwells coated just with albumin was negligible (data not shown).Therefore, we propose that the cPTP ${sigma}$ 1-AP construct binds heparinvia the extracellular region of cPTP ${sigma}$ .

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FIG. 2. Saturation curves and Scatchard plots for the binding of cPTP ${sigma}$ to heparin, agrin, and collagen XVIII. Microtiter plates were coated with heparin-albumin (A), agrin (B), or collagen XVIII (C) and incubated with a range of cPTP ${sigma}$ 1-AP concentrations. Saturation curves were fitted by nonlinear regression analysis as described in Materials and Methods. Each value represents the mean ± standard deviation of three measurements.

cPTP ${sigma}$ binds to retinal HSPGs. We next investigated whether ligand interactions detected inRAP assays (33) are mediated by HS chains and whether heparinand soluble HS can compete in binding. Preincubation of cPTP ${sigma}$ 1-APconditioned medium with heparin completely blocked the BL signal(Fig. 3B). HS also interfered with binding but did not completelyblock it (Fig. 3C); this may be due to the type of HS we used(bovine kidney), since it is known that significant variationsin the HS structure (the degree of sulfation, for example) existbetween different tissues. No effect was observed when cPTP ${sigma}$ 1-APwas preincubated with the same amount of chondroitin sulfate(Fig. 3D), itself a highly charged polyanionic molecule.

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FIG. 3. Binding of cPTP ${sigma}$ to the E6 chick retinal BL is mediated by HS chains. Results of receptor affinity probe assays using the extracellular region of cPTP ${sigma}$ 1 fused to alkaline phosphatase are shown. Retina sections were untreated (A to D) or pretreated with heparinase III (E) or chondroitinase ABC (F). cPTP ${sigma}$ 1-AP conditioned medium was used alone (A) or preincubated with heparin (B), HS (C), or chondroitin sulfate (D). The BL staining indicates cPTP ${sigma}$ 1-AP binding. Arrowheads indicate the retinal BL. pe, pigmented epithelium. Scale bar, 0.1 mm.

To establish whether heparin and HS modulate the binding becausethey bound cPTP ${sigma}$ 1-AP directly, or because they simply maskedthe binding site(s) on the BL ligand, the sections were pretreatedwith heparinase III or chondroitinase ABC. Heparinase III completelyremoved the BL signal (Fig. 3E), while chondroitinase ABC hadno effect (Fig. 3F). Therefore, we conclude that the cPTP ${sigma}$ 1-APbinding observed on the retinal BL is mediated by HS chainsand (since free heparin is not present in the retina) that theligand(s) is an HSPG(s).

cPTP ${sigma}$ binds to the extracellular matrix HSPGs agrin and collagen XVIII. The major HSPGs present in the retinal BL are agrin and collagenXVIII (39). Perlecan can also be detected at very low levels.Agrin and collagen XVIII immunopurified from the vitreous body(36, 38) were tested as candidate ligands for cPTP ${sigma}$ in solid-phasebinding assays. cPTP ${sigma}$ 1-AP bound at picomolar concentrations toboth agrin and collagen XVIII (Fig. 2B and C). The equilibriumdissociation constants measured were 0.18 ± 0.01 nM foragrin and 0.21 ± 0.02 nM for collagen XVIII, demonstratingthat cPTP ${sigma}$ binds these HSPGs with very high affinity. This experimentshows that native agrin and collagen XVIII behave as potentialligands for cPTP ${sigma}$ , at least in vitro.

These interactions were investigated in a further assay to determinewhether cPTP ${sigma}$ -proteoglycan interactions do occur mainly via theHS chain, as predicted above. An HSPG-enriched (HfV) fractionfrom E9 chicken embryo vitreous bodies was prepared by ion-exchangechromatography and, together with pure agrin and collagen XVIII,was tested on a blot overlay assay. The HfV fraction containedboth agrin and collagen XVIII (Fig. 4A). Agrin, a large proteoglycanwith an apparent molecular mass of 500 kDa, appeared in theHfV fraction and was not digested by treatment with collagenase(lane c) or chondroitinase ABC (lane d). However, treatmentwith heparinase III (lane b) reduced agrin's size to 250 kDa,corresponding to its core protein. Similarly, the molecularmass of collagen XVIII was reduced from 300 kDa to the 180-kDacore protein (lane f). Chondroitinase ABC treatment had no effect(lane h), whereas collagenase removed any trace of collagenXVIII (lane g). When the same HfV samples were probed with thecPTP ${sigma}$ 1-VSV conditioned medium in the blot overlay assay, twobands, corresponding in size to agrin and collagen XVIII, wereobserved in the untreated (lane i) and chondroitinase ABC-treated(lane l) samples. Heparinase III digestion (lane j) completelyremoved any signal on the overlay, showing that cPTP ${sigma}$ bindingrequires the presence of HS chains. This agrees with the RAPin situ results described above. Finally, collagenase preincubation(lane k) specifically removed the lower-molecular-weight band,corresponding to collagen XVIII.

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FIG. 4. cPTP ${sigma}$ binds agrin and collagen XVIII via their HS chains. An HSPG-enriched fraction from chicken embryo vitreous bodies (A), purified agrin (B), and purified collagen XVIII (C) samples, separated by SDS-6% polyacrylamide gel electrophoresis, were transferred to nitrocellulose and probed with antibodies specific for agrin or collagen XVIII or were incubated with cPTP ${sigma}$ 1-VSV conditioned medium. cPTP ${sigma}$ 1-VSV was detected using antibody to VSV. Samples were either not treated with enzymes (lanes a, e, and i) or predigested with heparinase III (lanes b, f, and j), collagenase (lanes c, g, and k), or chondroitinase ABC (lanes d, h, and l). Arrows in panel A indicate the two bands corresponding in molecular mass to agrin (upper arrow) and collagen XVIII (lower arrow) observed in the blot overlay assay.

The same patterns were obtained when purified agrin (Fig. 4B)or purified collagen XVIII (Fig. 4C) samples were enzyme treatedand probed in immunoblots or overlay assays. The collagen XVIIIblots (Fig. 4C) are more smeared than the fresh HfV samples,presumably due to partial degradation during the purificationsteps. Altogether, these data suggest that agrin and collagenXVIII can act as binding partners for cPTP ${sigma}$ , irrespective ofthe cPTP ${sigma}$ fusion tag used (AP or VSV), and that these interactionsoccur necessarily via their HS chains.

Overlapping expression of cPTP ${sigma}$ , agrin, and collagen XVIII in the developing chick retina. cPTP ${sigma}$ , agrin, and collagen XVIII are expressed in the embryonicnervous system in a developmentally regulated manner (36, 38,52, 71). We examined whether the expression patterns of cPTP ${sigma}$ and the proposed ligands are juxtaposed, as would be expectedif they were to interact in vivo. At E6 cPTP ${sigma}$ is localized inretinal axons in the optic fiber layer (OFL) in close contactwith the BL (Fig. 5A). Agrin (Fig. 5C) and collagen XVIII (Fig.5E) are localized at E6 mainly in the BL, which forms part ofthe complex substrate on which the retinal ganglion cells (RGCs)extend their axons. At E10, when the retinal plexiform layershave formed, cPTP ${sigma}$ is localized mainly in the OFL and in theinner plexiform layer (IPL) (Fig. 5B). Agrin, produced likecPTP ${sigma}$ by the RGCs, is present in the OFL and IPL but also accumulatesin large amounts in the BL (Fig. 5D). Collagen XVIII, however,produced by the ciliary body (39), is present only on the retinalinner surface, representing the BL (Fig. 5F).

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FIG. 5. Expression patterns of cPTP ${sigma}$ , agrin, and collagen XVIII in the developing chick retina. E6 (A, C, and E) and E10 (B, D, and F) chick retina sections were probed with antibodies to cPTP ${sigma}$ (A and B), agrin (C and D), and collagen XVIII (E and F). At E6, cPTP ${sigma}$ is strongly expressed in the retinal axons (outer fiber layer [OFL]), while both agrin and collagen XVIII show strong expression in the juxtaposed BL. At E10, cPTP ${sigma}$ shows strong expression, mainly in neurite layers: OFL and IPL (inner plexiform layer). Agrin expression overlaps in fiber layers and is also a major constituent of the BL along with collagen XVIII. RGC, retinal ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; PE, pigmented epithelium. White arrowheads indicate the BL staining. Scale bar, 0.1 mm.

The expression patterns described above suggest that cPTP ${sigma}$ wouldindeed be able to interact with both agrin and collagen XVIIIduring eye development.

The first Ig domain of cPTP ${sigma}$ contains a putative heparin-binding site. Basic amino acid clusters located on the surface of extracellulardomains of proteins are likely to bind polyanionic glycosaminoglycanchains (for a review, see reference 42). Using the SWISS-MODEL(v3.5) automated protein modelling server (31), we obtainedthree-dimensional (3D) models for all the Ig domains and threeout of four FNIII domains of cPTP ${sigma}$ 1 (except the FNIII-4 domain,which had less than 30% identity to any 3D structure in theProtein Data Bank [PDB]). Several positively charged clusterswere observed on the molecular surfaces (computed using theSwiss-PdbViewer v3.6b2 program [30]), the most prominent beingin the Ig-1 domain (Fig. 6A). This cluster consists mainly ofa ß-hairpin formed by strands ßC and ßC'(Fig. 6B) and contains the sequence R⁶²VTWNKKGKKVNSQR⁷⁶, plusthe side chains of Arg96 and Arg99, from a loop between theßE and ßF strands. The Ig-1 domain homologymodel was generated using telokin as a template (39.3% identity;PDB codes 1FHG and 1TLK), a typical Ig superfamily I-set structure.Twenty-four out of the 35 key structural residues as definedin reference 4 are identical, representing all the major secondarystructures and most of the peripheral regions (Fig. 6C).

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FIG. 6. Structural model of the Ig-1 domain of cPTP ${sigma}$ and sequence alignment with the Ig domains of telokin, NCAM, and FGFR1. (A) Electrostatic surface representation of the Ig-1 domain of cPTP ${sigma}$ ; blue and red represent positive and negative electrostatic potential, respectively. The large positive-potential patch represents the putative heparin binding site. Basic residues are labeled in yellow and numbered according to Stoker (69). (B) Ribbon view of the predicted folding with the same orientation as in panel A. N and C denote the amino and carboxyl termini. The ß strands are labeled, from A to G, according to the telokin fold. (C) Structure-based sequence alignment between telokin (the template used for modelling) and the heparin-binding Ig domains of cPTP ${sigma}$ , NCAM, and FGFR1. The corresponding sequences of the mouse and human orthologues of cPTP ${sigma}$ (mPTP ${sigma}$ and hPTP ${sigma}$ ) are included. The heparin-binding sites are highlighted in blue (proposed site for cPTP ${sigma}$ ). The telokin key structural residues and their conserved equivalents in cPTP ${sigma}$ are highlighted in green. The ß strands are underlined. The secondary structure definitions were reported in the following sources: for telokin, reference 44; for NCAM-Ig2, reference 47; and for FGFR1-Ig2, reference 61. This figure was made using the Swiss-PdbViewer v3.6b2 (30).

The cluster described above in the cPTP ${sigma}$ Ig-1 domain containsa BBxBB motif suggested to interact optimally with HS chains(where B represents Arg or Lys) (42). Other proteins containingheparin/HS binding sites located in I-set Ig domains have beendescribed, including the classical examples: neural cell adhesionmolecule (NCAM) and fibroblast growth factor (FGF) receptors(FGFRs). High-resolution 3D structures of both NCAM (1EPF, X-raystructure) (47) and FGFR1 (1CVS, X-ray structure) (61) haverecently been published, and these were aligned with the cPTP ${sigma}$ Ig-1 model using Swiss-PdbViewer (Fig. 6C). The proposed heparin-bindingsite of cPTP ${sigma}$ is perfectly conserved in its mouse and human orthologuesand aligns well with the heparin-binding site found in NCAM(14). However, the location of the FGFR1 heparin-binding sitedoes not seem to overlap, suggesting a different molecular architectureof the FGFR1-heparin complex.

The heparin-binding site of cPTP ${sigma}$ is essential for binding to the retinal BL. To test the accuracy of the molecular modelling prediction,we mutated the charged amino acids in the putative heparin-bindingsite of the Ig-1 domain and, as controls, other basic clusterspresent in cPTP ${sigma}$ Ig domains 2 and 3. Several basic clusters arealso present in the membrane-proximal FNIII-4 domain. However,a cPTP ${sigma}$ 1 extracellular construct lacking this FNIII-4 domain,termed FN3 ${Delta}$ -AP, bound in a similar way to full-length cPTP ${sigma}$ 1 inRAP in situ assays (see Fig. 8A and B). Therefore, FNIII-4 doesnot contain the BL HSPG binding site. The FN3 ${Delta}$ -AP construct wasused as a backbone for incorporation of further mutations, asshown in Fig. 7A. Mutations M1, M2, M3, and M4 are all directedto the charged amino acids from the putative heparin/HS bindingsite in Ig-1. Mutation M5 targets a basic cluster in Ig-2, andM6 targets a similar cluster in the loop connecting Ig-2 andIg-3. All constructs were transfected into 293T cells, and thecells produced essentially equal amounts of fusion protein inthe conditioned media (data not shown). All the mutations directedat the large basic cluster in Ig-1 (Fig. 6A) showed impairedheparin binding in solid-phase assays (Fig. 7B). Binding ofM5 and M6 constructs was not significantly affected. The severelyreduced binding properties of the M1 to M4 mutants precludedthe measurement of K_d values.

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FIG. 8. The heparin/HS binding site in domain Ig-1 is essential for retinal BL binding. Receptor affinity probe assays were performed using cPTP ${sigma}$ 1-AP (A), FN3 ${Delta}$ -AP (B), M1 (C), M2 (D), M3 (E), M4 (F), M5 (G), and M6 (H) conditioned media, respectively. The BL staining indicates fusion protein binding. Arrowheads indicate the retinal BL. pe, pigmented epithelium. Scale bar, 0.1 mm.

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FIG. 7. Identification of the cPTP ${sigma}$ heparin-binding site by site-directed mutagenesis. (A) Basic residues in the heparin-binding site in domain Ig-1 (mutations M1, M2, M3, and M4), another cluster in Ig-2 (mutation M5), or in the loop connecting Ig-2 and Ig-3 (mutation M6) were replaced with alanine. wt corresponds to the original cPTP ${sigma}$ sequence. (B) The mutated proteins were tested in solid-phase binding assays for binding to heparin-BSA. Bars represent means ± standard errors of three determinations.

The mutated constructs were tested on E6 chick retina sectionsby RAP in situ. The BL binding pattern observed correlated withthe solid-phase heparin binding profile (Fig. 8). Therefore,we conclude that the heparin/HS binding site was correctly predictedby molecular modelling. This site, which is highly conservedin the orthologues of cPTP ${sigma}$ (Fig. 6C), is essential for the bindingof cPTP ${sigma}$ 1 ectodomains to the BL HSPGs.

Müller glia endfeet contain HSPG and non-HSPG binding sites for cPTP ${sigma}$ . Radial Müller glial cells are the only nonneuronal celltype in the chicken retina. RAP in situ experiments demonstratethat in addition to the BL ligand(s), cPTP ${sigma}$ binds strongly toradial glia endfeet in contact with the BL. This interactioncontributes to the role of cPTP ${sigma}$ in neurite growth and in maintenanceof growth cone lamellipodia (33, 51). Here we have assessedwhether or not the endfeet binding sites are also HSPGs.

Unfixed preparations of retinal basal laminae, with glial endfeetattached, were used for RAP in situ experiments. Both heparinaddition and heparinase III treatment diminished cPTP ${sigma}$ 1-AP bindingon glial endfeet significantly (Fig. 9B and C). Unlike resultsin fixed cryosections, however (Fig. 3B), this interaction wasnot blocked completely. In addition, several cPTP ${sigma}$ 1 mutant constructsexhibited reduced binding to the glial endfeet (Fig. 9H, I,and J), but again there was residual cPTP ${sigma}$ binding. Neither chondroitinsulfate nor chondroitinase ABC pretreatment could significantlyimpair the interaction between cPTP ${sigma}$ and its ligands expressedon glial endfeet (Fig. 9D and E). These observations suggestthat the glial endfeet, a highly differentiated area of theMüller glia, express both an HSPG and a non-HSPG classof cPTP ${sigma}$ ligands.

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FIG. 9. Müller glia endfeet express two classes of cPTP ${sigma}$ ligands. Retinal basal laminae with glial endfeet attached were flat mounted and probed by RAP in situ with conditioned media containing cPTP ${sigma}$ 1-AP (A to E), FN3 ${Delta}$ -AP (F), M1 (G), M2 (H), M3 (I), M4 (J), M5 (K), and M6 (L). Heparin addition (B) or heparinase III pretreatment (C) reduces binding compared to control treatments (A and F) but does not abolish it. cPTP ${sigma}$ mutants with impaired heparin-binding properties also show reduced binding on glial endfeet (H to J). Chondroitin sulfate addition (D) or chondroitinase ABC pretreatment (E) does not affect cPTP ${sigma}$ binding significantly. Arrows indicate ringlike endfeet. Scale bar, 10 µm.

DISCUSSION

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Discussion

Cell adhesion molecule-like RPTPs are emerging as a major classof molecules involved in regulating neural development, includingaxon growth and guidance, synaptic function, and nerve repair(41, 78, 84; also reviewed in references 6, 55, 72, and 79).Currently, little is understood regarding the molecular mechanismsof RPTP signaling, one reason being that in only a few casesare their interaction partners known. Where homophilic bindinghas been demonstrated between RPTPs, for example with RPTPµ(8), RPTP ${kappa}$ (62), or RPTP ${delta}$ (82), it is not understood how, or evenif, such binding directly influences RPTP signaling. Where heterotypicligands have been proposed, it is clearer that ligand bindingcan influence phosphatase activity, either positively (66) ornegatively (53). To advance our understanding it will be necessaryto define many more ligand classes for the RPTPs.

cPTP ${sigma}$ has recently been implicated in promoting intraretinalaxon growth and controlling the growth cone morphology of RGCs(51). Putative, heterotypic ligands for cPTP ${sigma}$ have been localizedon Müller glia endfeet and RGC neurites and in the retinalBL (33, 51). The retinal BL (inner limiting membrane) is a 50-to 70-nm-thick extracellular matrix sheet separating the retinafrom the vitreous body. Its major constituents include laminins,nidogen, collagen IV, HSPGs (mainly agrin and collagen XVIII),and a chondroitin sulfate proteoglycan (39). The retinal BLis an excellent substrate for axon growth in vitro. In vivo,during the intraretinal axon outgrowth (E3 to E7 in chick),axons grow initially on the BL and a layer formed by radialglia endfeet (34). Accordingly, enzymatic disruption of theBL severely perturbs retinal architecture and axonal outgrowth(37). Our solid-phase binding assays have not detected interactionsbetween cPTP ${sigma}$ and laminin 1, laminin 2, fibronectin, or collagenIV (McKinnell and Stoker, unpublished data). However, cPTP ${sigma}$ bindsheparin with high affinity as well as the HS chains of HSPGsagrin and collagen XVIII. In addition, using molecular modelling,we have identified a heparin/HS binding site in the first Igdomain of cPTP ${sigma}$ , which is necessary for these interactions. Inour previous work we showed that both the Ig and FNIII domainsof cPTP ${sigma}$ are required for ligand binding, at least under RAPin situ conditions (33). Therefore, it appears that the HS bindingsite in the first Ig domain described here is essential butnot sufficient for the interaction with HSPGs. The FNIII domainsmay be required either to achieve an appropriate functionalconformation or to interact with other ligands in a putativecPTP ${sigma}$ signaling complex.

HSPGs are well established as modulators of neurite outgrowth.Previous studies have reported that HSPGs enhance the growth-promotingabilities of laminin and NCAM (reviewed in reference 7) andof basic FGF (12). Heparinase III treatment of cell-free basallaminae greatly inhibited the growth rate and distribution densityof RGC axons (12) and induced aberrant growth of axons in culturedinsect wing appendages (83). In Xenopus, heparitinase removalof HS chains during optic tract formation retards retinal axonelongation; later HS removal, after axons have extended to thetract, elicits a tectal bypass phenotype (81). Moreover, additionof exogenous HS chains to the developing Xenopus optic pathwayseverely disrupted target recognition (81). These effects havebeen attributed, at least in part, to the disruption of FGFsignaling. Our present data suggest that further signaling receptorsthat bind to HSPGs, including cPTP ${sigma}$ , may also be directly affectedin these assays.

The HSPGs agrin and collagen XVIII are the first heterotypicbinding partners described for cPTP ${sigma}$ . These interactions cannot only bring further insight to our understanding of the molecularmechanisms behind currently known cPTP ${sigma}$ functions but also suggestnovel ones. Agrin, for example, is abundant in axonal pathwaysof the chick central nervous system, such as the optic nerveand the tecto-bulbar pathway (36). The temporal coincidenceof its expression with axonal outgrowth suggests a growth-promotingrole for agrin, as has also been proposed for cPTP ${sigma}$ (51). Surprisingly,though, purified agrin displays an inhibitory activity for neuriteoutgrowth in vitro (36). However, a large variety of moleculesbind in vivo to agrin via its HS chains, protein core, or both,including laminin-1, thrombospondin, fibronectin, FGF2, merosin(laminin-2), and pleiotrophin (15). All of these are in turnpotent substrates for neurite extension, and their binding toHSPGs is important for modulating this activity. A functionalinteraction between agrin and cPTP ${sigma}$ may or may not involve suchmolecules as well. Furthermore, agrin is expressed in severalisoforms. Alternative promoter and first (N-terminal) exon usagedefines a long (LN) and short (SN) isoform (11, 56). LN is secretedand incorporated into the BL, while SN remains attached on theneuronal surface. The purified agrin used as a substrate foraxon growth experiments in vitro was LN, whereas the antibodywe have used in this study does not discriminate between thetwo isoforms (36). It is possible, therefore, that the agrininvolved in neurite outgrowth modulation is of the SN type.Both isoforms mentioned above have the potential to interactwith cPTP ${sigma}$ either as a ligand (LN agrin) or as a coreceptor onthe neuronal surface (SN agrin). Which one of these interactionshas functional consequences is still to be determined.

The best-characterized function for agrin remains its abilityto induce acetylcholine receptor clustering at neuromuscularjunctions (43). Transgenic animals have provided convincingevidence for the essential role of LN agrin in triggering synapticdifferentiation (11; reviewed in reference 32). cPTP ${sigma}$ 1 is expressedon motor neurons and also binds to sites on muscles in RAP insitu experiments (J. Chilton, F. Haj, and A. Stoker, unpublisheddata). This interaction appears to be HS independent, however,and its relationship, if any, to agrin has yet to be determined.

Despite the large amount of data supporting the role of agrinat neuromuscular junctions, less is known about its roles inthe brain, where it is a major HSPG. Recent reports (16, 22)have revealed a potential role in Alzheimer's disease. The ß-amyloidpeptide (Aß) accumulates as aggregates within senileplaques and cerebrovascular deposits. Aß binds tothe HS chains of agrin in vitro, leading to the accelerationof Aß fibril formation (16). Agrin also localizesto Aß deposits in brains affected by Alzheimer's disease.Interestingly, senile plaques promote neurite outgrowth andusually contain numerous dystrophic neurites, events that maybe dependent on FGF2, which can associate with agrin's HS chains(18). It would be interesting to see whether PTP ${sigma}$ plays any rolein this process. In the avian retina, agrin is found mainlyin the BL and retinal plexiform layers (from E8 onwards) (35,48), consistent with a possible role in interneuronal synapseformation (49). Since cPTP ${sigma}$ is present at high levels in theIPL (71) and, as reported here, binds to agrin, this raisesthe possibility that cPTP ${sigma}$ might be involved in retinal synaptogenesis.There is evidence already that the highly related RPTP ${delta}$ may beinvolved in modulating synaptic plasticity (77).

Another BL ligand for cPTP ${sigma}$ is collagen XVIII. Collagen XVIIIhas recently emerged as a modulator of cell migration and axonguidance (1), and a mutation in the human COL18A1 gene causesKnobloch syndrome, a disease involving vitreoretinal degeneration(65). A 38-kDa C-terminal fragment termed NC1 appears to beresponsible for stimulating cell and axon migration (1). Althoughthis fragment has heparin-binding ability, its neuronal receptorhas not been identified. We found that binding of cPTP ${sigma}$ to collagenXVIII is dependent on the integrity of the latter's HS chains,at least in blot-overlay assays, although we cannot rule outdirect protein-protein interactions. NC1 contains a putativeO-glycosylation site, although it is not clear yet whether itis glycosylated in vivo. It will be of interest to see if RPTP ${sigma}$ also acts as a receptor for NC1/endostatin, considering theinvolvement of both proteins in neurite growth and guidance.

The HS chains of agrin and collagen XVIII may directly regulatethe phosphatase activity of cPTP ${sigma}$ as is seen with ligands interactingwith other RPTPs (53, 66). Whether or not such regulation occursthrough receptor dimerization, which occurs with certain RPTPs(reviewed in reference 3), remains to be seen. Given that HSchains are highly variable and complex, it may be unlikely thatthere is a singular HS structure that acts as a specific ligandfor cPTP ${sigma}$ . Instead, one likely way in which HSPGs could interactwith cPTP ${sigma}$ is by acting as a facilitator of either receptor oligomerizationor the binding of further, cognate ligands. An example of thisis seen with the cobinding of FGF and HS to the FGF receptor,producing a supramolecular complex with full signaling potential(64). If HSPG-cPTP ${sigma}$ interactions represent part of a multiproteinsignaling complex necessary for regulating the RPTP, then thecharacterization of additional components of this complex willbe essential. It is also feasible that binding of HS to cPTP ${sigma}$ represents an adhesive function rather than a signaling function,and this will require further investigation.

We provide evidence that glial endfeet contain at least twobinding partners for cPTP ${sigma}$ . Previous work of members of our groupdemonstrated that Müller glia endfeet contain ligands forcPTP ${sigma}$ and that interactions with these ligands sustain neuriteoutgrowth (51). Here we have shown that endfeet contain HS-dependentbinding sites for cPTP ${sigma}$ . It is possible that interactions betweenthese sites and cPTP ${sigma}$ lead at least in part to the axonal phenotypeobserved earlier (51). However, we also report that endfeetcontain non-HS binding sites for cPTP ${sigma}$ , which remain to be characterized.These binding sites were not evident on cultured Müllerglia, where cPTP ${sigma}$ binding was entirely sensitive to heparinasetreatment and heparin competition (Aricescu, unpublished data).The non-HS binding sites on endfeet are also apparently removedor masked by methanol fixation, both in flat-mounted basal laminaeand in tissue sections. Glial endfeet, in both the retina andtectum, are highly differentiated structures (9, 67). Tectalendfeet, for example, specifically accumulate ephrin B1 (9),and axon outgrowth experiments in vitro demonstrate that Müllerglia are highly polarized (67). As previously suggested (51),the endfeet contain a rich mixture of positive and negativecues for neurite outgrowth, and it is therefore possible thatcPTP ${sigma}$ is interacting, perhaps simultaneously, with at least twoligand types on endfeet, with the net effect of promoting neuriteelongation.

In conclusion, we demonstrate that cPTP ${sigma}$ has a novel, heparinbinding activity and that its ability to bind to the retinalBL is mediated by HS chains of extracellular matrix HSPGs. Thesematrix HSPGs are identified as agrin and collagen XVIII andare the first heterotypic ligands identified for cPTP ${sigma}$ . TheseHSPGs may be ligands in their own right or may act as partnersin presenting other ligand proteins to cPTP ${sigma}$ . Further investigationswill clarify the direct effect of HSPGs on cPTP ${sigma}$ function, helpingus to understand how extracellular ligands regulate RPTP functionand how such interactions in turn influence axon development.

ACKNOWLEDGMENTS

We thank John Flanagan for providing us with the plasmid AP-Tag2,Bruce Morgan for the PRD4A viral probe and the pFN3 ${Delta}$ -AP plasmid,Stéphane Swillens for advice regarding the nonlinearregression analysis, and Caroline Paternotte for help with DNAsequencing. We also thank Michael Hurley for critical commentson the manuscript.

This work was supported by a University College London GraduateResearch Scholarship (A.A.), the Medical Research Council (I.M.),and grants from the University of London Central Research Fund(A.S.) and the National Institutes of Health (NS33981-02) (W.H.).

FOOTNOTES

* Corresponding author. Mailing address: Neural Development Unit, Institute of Child Health, 30 Guilford St., London WC1N 1EH, United Kingdom. Phone: 44-20-79052244. Fax: 44-20-78314366. E-mail: a.stoker{at}ich.ucl.ac.uk.

REFERENCES

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