expression of multiple chondroitin/dermatan sulfotransferases in the neurogenic regions of the...

Expression of Multiple Chondroitin/Dermatan Sulfotransferases inthe Neurogenic Regions of the Embryonic and Adult CentralNervous System Implies That Complex Chondroitin Sulfates Have aRole in Neural Stem Cell Maintenance

KAORU AKITA,a ALEXANDER VON HOLST,a YOKO FURUKAWA,b TADAHISA MIKAMI,b KAZUYUKI SUGAHARA,b,c

ANDREAS FAISSNERa

aDepartment of Cell Morphology and Molecular Neurobiology, Ruhr-University Bochum, Bochum, Germany;bDepartment of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe, Japan; cGraduate School ofLife Science, Hokkaido University, Frontier Research Center for Post-Genomic Science and Technology, Kita-ku,Sapporo, Japan

Key Words. Chondroitin sulfate proteoglycan • Dermatan sulfotransferase • Neural stem cell niche • Neurosphere • Sulfation

ABSTRACT

Chondroitin/dermatan sulfotransferases (C/D-STs) underliethe synthesis of diverse sulfated structures in chondroitin/dermatan sulfate (CS/DS) chains. Recent reports have sug-gested that particular sulfated structures on CS/DS poly-mers are involved in the regulation of neural stem cellproliferation. Here, we examined the gene expression profileof C/D-STs in the neurogenic regions of embryonic andadult mouse central nervous system. Using reverse tran-scription-polymerase chain reaction analysis, all presentlyknown C/D-STs were detected in the dorsal and ventraltelencephalon of the embryonic day 13 (E13) mouse embryo,with the exception of chondroitin 4-O-sulfotransferase(C4ST)-3. In situ hybridization for C4ST-1, dermatan 4-O-sulfotransferase-1, chondroitin 6-O-sulfotransferase (C6ST)-1and -2, and uronosyl 2-O-sulfotransferase revealed a cellularexpression of these sulfotransferase genes in the embryonicgerminal zones of the forebrain. The expression of multiple

C/D-STs is maintained on cells residing in the adult neuralstem cell niche. Neural stem cells cultured as neurospheresmaintained the expression of these enzymes. Consistent withthe gene expression pattern of C/D-STs, disaccharide analysisrevealed that neurospheres and E13 mouse brain cells synthe-sized CS/DS chains containing monosulfated, but also signifi-cant amounts of disulfated, disaccharide units. Functionally,the inhibition of sulfation with sodium chlorate resulted in asignificant, dose-dependent decrease in neurosphere numberthat could not be rescued by the addition of individual purifiedglycosaminoglycan (GAG) chains, including heparin. Thesefindings argue against a simple charge-based mechanism ofGAG chains in neural stem cell maintenance. The synergisticactivities of C/D-STs might allow for the adaptive modificationof CS/DS proteoglycans with diversely sulfated CS/DS chainsin the extracellular microenvironment that surrounds neuralstem cells. STEM CELLS 2008;26:798–809

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Various biological roles of proteoglycans (PGs) have beenreported in the developing and mature central nervous system(CNS). Their functions are partially mediated by the glycos-aminoglycan chains that are covalently bound to the coreprotein. Recently, evidence has revealed that sulfation ofglycosaminoglycans is spatiotemporally regulated in thebrain [1– 4]. The variation of this modification allows for aconsiderable structural diversity of glycosaminoglycans,which may constitute the basis for diverse biological roles. Itis established that heparan sulfate (HS) chains modulate theactivities of various growth and morphogenetic factors [5, 6].Among them, fibroblast growth factor (FGF) and fibroblastgrowth factor receptor (FGFR) signaling represents one of

the crucial effectors for the neural stem cell population. Forexample, FGF-2 not only promotes cell proliferation of em-bryonic cortical stem cells [7] but also controls their differ-entiation into neuronal and glial lineages [8]. Developmentalstage-specific structural differences of HS chains that serveas temporal regulators to form the active FGF-FGFR signal-ing complex have been described in various progenitor cellpopulations, including the neuroepithelial layers of the brain[2]. Therefore, it has been suggested that the expression ofselected heparan sulfotransferases is temporally regulated inneural precursor cells [9]. HS chains that are expressed onembryonic day 9 (E9) to E11 mouse neural precursor cellsexhibit differential binding affinities for FGF-1 and -2 [10].Notably, the binding of cytokines and growth factors is notlimited to HS-type glycosaminoglycans. Oversulfated chon-droitin/dermatan sulfate (CS/DS) chains from different

Correspondence: Prof. Andreas Faissner, M.D., Department of Cell Morphology and Molecular Neurobiology, Faculty of Biology,Ruhr-University Bochum, NDEF 05/594, Universitatsstrasse 150, D-44780 Bochum, Germany. Telephone: 49-234-3223851; Fax: 49-234-3214313; e-mail: [email protected] Received June 12, 2007; accepted for publication November 27, 2007; firstpublished online in STEM CELLS EXPRESS December 13, 2007. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2007-0448

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sources also interact with several heparin-binding growthfactors with nanomolar affinities [11–14].

Previously, we have established that the monoclonal antibody473HD is directed against phosphacan/DSD-1-PG/6B4-PG [15–17], a splice variant of receptor protein tyrosine phosphatase(RPTP)-� that has been purified from postnatal rodent CNS [17].Subsequent analysis of the 473HD epitope (or DSD-1 epitope) hasshown that the antibody recognizes a unique CS/DS glycosamino-glycan structure. The complex carbohydrate comprises GlcUA-GalNAc(4S), GlcUA(2S)-GalNAc(6S), and GlcUA-GalNAc(4S)/GalNAc(6S)-IdoUA/IdoUA(2S), where 2S, 4S and 6S represent2-O-, 4-O-, and 6-O-sulfate, respectively [18, 19]. We recentlyreported that CS/DS-PGs bearing this unique sulfated structure areexpressed in the periventricular germinal zones of the developingmouse CNS and in the subventricular zone of the adult mouse CNS[20], where multipotent stem cells reside [21]. The expression ofsome CS/DS-PG core proteins has been detected in the embryonicneural stem cell niche and in neurospheres [22, 23]. Furthermore,consistent with the expression of the 473HD epitope, variousmono- and disulfated disaccharide units have also been identifiedby the compositional analysis of CS/DS chains purified from theembryonic mammalian CNS [11, 13, 23–25]. Interestingly, cellsurface expression of the 473HD epitope is prominently observedon the neural stem cell population, and the addition of the mono-clonal antibody (mAb) 473HD into neurosphere culture mediumdecreases the number of neurospheres [20]. Using the neurosphereculture system, Ida et al. [23] have recently reported that particularsulfatedstructuresonCS/DSchainssuchastheIdoUA(2-O-sulfate)�1-3GalNAc(4-O-sulfate) (CS-B), GlcUA(2-O-sulfate)�1-3GalNAc(6-O-sulfate) (CS-D), and GlcUA�1-3GalNAc(4, 6-O-disulfate)(CS-E) units possess the potential to promote FGF-2-mediated cellproliferation of rat embryonic neural stem/precursor cells. Thesefindings suggested that the sulfation profile on CS/DS chains is oneof the crucial factors regulating cell proliferation of neural stemcells in the CNS.

Sulfate groups are transferred from 3�-phosphoadenosine5�-phosphosulfate to the specific acceptor sites in CS/DS chainsby chondroitin/dermatan sulfotransferases (C/D-STs) that arelocated in the Golgi apparatus [26–28]. As illustrated in Figure1A, these enzymes are classified into the following fourgroups: chondroitin/dermatan 4-O-sulfotransferase (C4ST/D4ST), chondroitin 6-O-sulfotransferase (C6ST), uronosyl 2-O-sulfotransferase (UA2OST), and N-acetylgalactosamine 4-sul-fate 6-O-sulfotransferase (GalNAc4S-6ST). Three C4STisoforms [29–32], two C6ST isoforms [33, 34], D4ST-1 [35],UA2OST [36], and GalNAc4S-6ST [37] have been identified inmammals. It has been reported that gene expression levels ofsome enzymes correlate with the amount of sulfated productsthat corresponded to each enzymatic activity [1, 25], whichholds the promise that studies of gene expression of C/D-STswill yield more detailed insights about the sulfation profiles inmixed CS/DS chains.

To begin to elucidate how the sulfation on CS/DS chains isregulated in the neural stem cell niche, we first studied the geneexpression patterns of a range of C/D-STs in the neurogenicregions of the embryonic and adult CNS. The mRNAs of thesesulfotransferases were also examined in neurospheres obtainedfrom E13 mouse telencephalon. Furthermore, we analyzed thestructural characteristics of the CS/DS carbohydrate chains pre-pared from the conditioned neurosphere culture medium andfrom E13 mouse brain. Our results suggest that expression ofmultiple C/D-STs is maintained in the cells residing in theneurogenic regions of embryonic and adult CNS, rather thandisplaying temporal regulation of the expression of these en-zymes. Furthermore, we show that the addition of sodium chlor-ate inhibited the expression of the 473HD epitope on neuro-sphere-forming cells concomitant with a decrease in the number

of neurospheres, implying that defined sulfated molecules suchas PGs are closely involved in cell proliferation and mainte-nance of neural stem/progenitor cells.

MATERIALS AND METHODS

Animals and AntibodiesNMRI mice with timed pregnancies were obtained from CharlesRiver Laboratories (Wilmington, MA, http://www.criver.com), and

Figure 1. Expression of C/D-ST mRNAs in the developing CNS. (A):Schematic structure of sulfated disaccharides in the chondroitin sulfate(CS)/dermatan sulfate chains. The repeating CS disaccharide units consist-ing of glucuronic acid (GlcUA) (white hexagons) and N-acetylgalac-tosamine (GalNAc) (light gray hexagons) are depicted. These CS-disaccha-ride units are modified by four different sulfotransferases: C4ST, C6ST,UA2OST, and GalNAc4S–6ST, as indicated in the scheme. The activity ofthe chondroitin sulfotransferases leads to the addition of sulfate groups atdefined positions (black circles), which results in the generation of specifiedCS units as shown in the figure (underlined). In case GlcUA is converted toiduronic acid (IdoUA) (dark gray hexagons) by its C-5 epimerization, theenzyme D4ST preferentially adds a sulfate group at the C4 position ofGalNAc, which is adjacent to IdoUA. The detailed substrate specificities foreach enzyme are further explained in the text and the selected references.(B): Analysis of chondroitin/dermatan sulfotransferase expression in theembryonic day 13 (E13) mouse telencephalon by reverse transcription-PCR. cDNA was synthesized using total RNA purified from E13 C-cortexand E13 G-eminence. PCR was performed with a serial number of cycles(20, 24, 28, 32, and 36). The amplified products were visualized byelectrophoresis on a 1.5% agarose gel containing ethidium bromide. Notethat with the exception of C4ST-3, mRNAs of all C/D-STs cloned so farwere detected in these tissues. Abbreviations: C4ST, chondroitin 4-O-sulfotransferase; C6ST, chondroitin 6-O-sulfotransferase; C-cortex, cere-bral cortex; D4ST, dermatan 4-O-sulfotransferase; GalNAc4S–6ST, N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase; G-eminence,ganglionic eminence; PCR, polymerase chain reaction; UA2OST,uronosyl 2-O-sulfotransferase.

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the ages of the embryos were verified according to Bard et al. [38].The antibodies 473HD and KAF13 and their specificities have beendescribed [17, 20]. All commercial antibodies were obtained fromChemicon (Temecula, CA, http://www.chemicon.com), Roche Di-agnostics (Basel, Switzerland, http://www.roche-applied-science.com), or Dianova (Hamburg, Germany, http://www.dianova.de) asindicated in supplemental online data 4.

Primary Neurosphere CulturePrimary neurospheres were grown from E13 forebrain cell suspen-sions as described previously [20, 39]. The cultures were supple-mented with or without epidermal growth factor (EGF) (20 ng/ml;PeproTech, London, http://www.peprotech.com), FGF-2 (20 ng/ml;Peprotech), and heparin (0.5 U/ml; Sigma) as indicated. We usedclonal density assays [40] (200 cells per cm2) to assess the effect ofsodium chlorate on neurosphere formation by adding 5 or 30 mMchlorate in the presence of EGF, FGF-2, or both. For rescue exper-iments, GlcUA�1–3GalNAc(4-O-sulfate) (CS-A), CS-B,GlcUA�1–3GalNAc(6-O-sulfate) (CS-C), CS-D, and CS-E (allfrom Seikagaku, Tokyo, http://www.seikagaku.co.jp/english) wereadded at 10 �g/ml in the continued presence of 30 mM sodiumchlorate. After 5 days of cultivation, the total number of neuro-spheres under control and treatment conditions was microscopicallydetermined.

Reverse Transcription-Polymerase Chain Reactionand Semiquantitative AnalysisBriefly, total RNA was isolated and reverse transcribed as previ-ously described [39]. The polymerase chain reactions (PCRs) wereperformed in the linear range with primer sequences and PCRconditions as detailed in supplemental online data 3 and 4. Allamplicons were cloned and verified by sequencing. For semiquan-titative analysis, the density of the amplified products was measured(NIH ImageJ version 1.36) and plotted as ratio of the �-actin band.

In Situ HybridizationThe probes for mRNA of the sulfotransferases C4ST-1, D4ST,C6ST-1 and -2, and UA2OST, as well as RPTP-�, were obtained byreverse transcription (RT)-PCR. Digoxigenin (DIG)-labeled anti-sense and sense riboprobes were generated according to the manu-facturer’s instructions (DIG RNA Labeling Kit; Roche Diagnos-tics). Cryosections were hybridized with the riboprobes at 50°Covernight. After washing and blocking, the sections were incubatedwith an alkaline phosphatase-conjugated anti-DIG antibody(1:2,000) overnight at 4°C. The probes were visualized using NitroBlue Tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, and colordevelopment was stopped at variable time points to obtain reason-able signal-to-noise ratios.

Immunoblot AnalysisDetergent extracts of samples were obtained, separated, blotted, anddeveloped as described previously [39]. For immunoprecipitation, 4ml of KAF13 was added to neurosphere lysates or conditionedmedium. After incubation at 4°C overnight, 10 �l of protein A-Sepharose was added and incubated at 4°C for 2 hours. Immuno-precipitates were spun down and further treated as the proteinlysates (details given in supplemental online data 4).

Immunostaining of Neurosphere SectionsNeurosphere sections were prepared as described for the in situhybridizations, and the immunohistochemical stainings were per-formed as described previously [20].

Disaccharide Analysis of CS/DS Chains fromEmbryonic Brain and Conditioned NeurosphereCulture MediaDisaccharide analysis of CS/DS chains from E13 mouse brain wasperformed as described previously [11]. Using the same analyticalmethod, disaccharide compositions of CS/DS chains from condi-tioned neurosphere culture media were also analyzed.

Other MethodsThe protein concentration was determined using BCA protein assaykit (Bio-Rad) with bovine serum albumin as the standard. Student’st test was used for statistical analysis of the experiments. p � .05was taken as the minimal level of significance. A detailed descrip-tion of all methods can be found in supplemental online data 4.

RESULTS

Gene Expression Patterns of C/D-STs in theNeurogenic Regions of the Embryonic and AdultMouse BrainTo examine which C/D-STs are expressed in the neurogenicregions at embryonic stages, we performed RT-PCR using totalRNA purified from E13 mouse cerebral cortex and ganglioniceminence. Except for C4ST-3, mRNAs of all C/D-STs cloned sofar (C4ST-1 and -2, D4ST-1, C6ST-1 and -2, UA2OST, andGalNAc4S-6ST) could be traced in these tissues and confirmedafter subcloning and DNA sequencing (Fig. 1B). It has beenreported previously that C4ST-3 mRNA is highly upregulated inadult human liver [31]. As a positive control, the expression ofC4ST-3 mRNA was confirmed in adult mouse liver by RT-PCRusing our PCR primers and subsequent DNA sequencing (datanot shown).

More recently, we have found that a unique CS/DS structurerecognized by the mAb 473HD and named the 473HD epitopeis expressed in the ventricular zone of E13 mouse telencephalon[20]. We have shown previously that the 473HD epitope de-pends on selective sulfation of chondroitin sulfate chains thatcomprise CS-A and CS-D motifs, and a chondroitin sulfate Belement [17–19]. The coordinated activity of a restricted set ofC/D-STs is required to synthesize this structure. To elaborate amore detailed picture of the distribution of the cells expressingthe 473HD epitope in this region, the spatial expression patternsof the C/D-ST genes were examined by in situ hybridization.Coronal sections of E13 mouse brain were hybridized with theDIG-labeled riboprobes for C4ST-1, D4ST-1, C6ST-1 and -2,and UA2OST, which are presumably involved in the formationof the 473HD epitope. As exemplified for C4ST-1 (Fig. 2), aprominent expression of all C/D-STs examined was detected inthe ventricular zones of the dorsal and the ventral telencephalon(supplemental online Fig. 1). Hybridization signals were ob-served colocalizing with cell bodies that are positioned adjacentto the ventricular surface but are also located farther from thelining of the ventricular zone. Interestingly, we did not findstriking differences in the distribution pattern of the cells ex-pressing individual sulfotransferase genes, which supports theconcept that the orchestrated activity of multiple enzymes in agiven cell is required for the generation of diverse sulfationpatterns in CS/DS chains. These results clearly suggest that thecell population in the embryonic neurogenic regions is endowedwith the capacity to synthesize a large variety of sulfated CS/DSchains.

Neural stem cell proliferation continues in the adult CNS inrestricted areas called niches [21], where the expression ofdistinct chondroitin sulfates, including the 473HD epitope, haspreviously been recorded [20, 41]. We examined the expressionof C/D-STs in neurogenic regions of the adult brain by in situhybridization using the same riboprobes as above (Fig. 2).Prominent signals for all C/D-STs examined in this study weredetected on the cells residing in the subventricular zone (SVZ)around the anterior lateral ventricle wall. Closer inspection athigher magnification revealed that, in addition, ependymal cellsthat line the ventricle wall express the mRNAs corresponding tothese enzymes. Furthermore, signals were detected in cells of

800 C/D-ST Expression in the Neural Stem Cell Niche

the rostral migratory stream where neuroblasts derived from theSVZ migrate toward the olfactory bulb (data not shown).

Expression of the 473HD Epitope on AlternativelySpliced Isoforms of the RPTP-� Gene Locus inNeurospheres and Their Conditioned Culture MediaNeural stem/progenitor cells can be cultivated in suspension asso-called neurospheres when supplied with a defined mediumthat contains adequate growth factors such as EGF or FGF-2[42]. Neurosphere-forming cells express several CS/DS-PGsincluding phosphacan, one of the alternatively spliced isoformsof the RPTP-� gene [22, 23]. Four variants of RPTP-� havebeen identified in mouse so far [43, 44] and all are expressed onmRNA level in neurospheres (data not shown). Only the largetransmembrane receptor protein tyrosine phosphatase formRPTP-� long and the soluble chondroitin sulfate proteoglycanphosphacan possess the glycosylation sites needed for the co-valent attachment of glycosaminoglycans [44]. We have previ-ously reported that cultured immature glial cells from late em-

bryonic and early postnatal mouse cerebellum express the473HD epitope [17] and that this epitope is carried by RPTP-�long and phosphacan [43, 44].

More recently, we have found that most of mAb 473HDimmunoreactive cells dissociated from the early embryonicforebrain and/or neurospheres show the same characteristics asneural stem cells [20]. However, it remained unclear which ofthe RPTP-� gene products expressed carries the 473HD epitopein E13 cortical neural stem cells. To examine this issue, deter-gent extracts and culture supernatants of neurospheres grown inthe presence of FGF-2 and EGF were immunoprecipitated withpolyclonal anti-phosphacan antibodies (KAF13); the resultingprecipitates were immunoblotted and finally developed withmAb 473HD. As shown in Figure 3A, 473HD-reactive materialwas detected both in the cell lysate and the culture medium ofneurospheres; this material was absent when the KAF13 poly-clonal antibodies had been omitted. This is consistent with theinterpretation that the 473HD epitope is carried on RPTP-� longand phosphacan. In the neurosphere detergent extract, two ad-

Figure 2. In situ hybridization of chondroitin/dermatan sulfotransferases (C/D-STs) in the adult neural stem cell niche. Bright-field photomicro-graphs of coronal adult forebrain cryosections after developing digoxigenin-labeled RNA probes for C4ST-1 (A, A�, a, F, F�), D4ST-1 (B, B�, b),C6ST-1 (C, C�, c), C6ST-2 (D, D�, d), and UA2OST (E, E�, e) are shown. The middle panels (A�–E�) indicate the corresponding sense probes, whichdid not give rise to specific signals. The right panels (a–e) refer to the boxed areas that are enlarged to provide higher-resolution images. Coronalsections were hybridized with antisense (A–E) or sense (A�–E�) probes. Note that mRNAs of all studied C/D-STs were detected in the adult SVZ.Dorsal is shown at the top and lateral at the left in all images. Note that the different sulfotransferase genes remained expressed in the adult neuralstem cell niche. (F, F�): One example of CS-ST expression (C4ST-1) (F) in the germinal layers during forebrain development (E13). Scale bar �50 �m (a–e, F, F�) and 200 �m (A–E, A�–E�). Abbreviations: C4ST, chondroitin 4-O-sulfotransferase; C6ST, chondroitin 6-O-sulfotransferase;CTX, cerebral cortex; D4ST, dermatan 4-O-sulfotransferase; E13, embryonic day 13; GE, ganglionic eminence; LV, lateral ventricle; UA2OST,uronosyl 2-O-sulfotransferase; SVZ, subventricular zone.


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ditional bands with molecular weights of approximately 290 and250 kDa were detected. These 473HD epitope bearing bandsmay correspond to immature, partially glycosylated processingintermediates or to proteolytically degraded fragments of theRPTP-� core protein(s), as discussed previously [45, 46].

Although neurospheres contain not only neural stem/pro-genitor cells but also committed precursor populations [47], ithas been reported that these various cell classes display someterritorial preference within the three-dimensional (3D) struc-ture of neurospheres. The actively cycling neural stem cellpopulation is being localized to the more superficial areas of theneurosphere, and the more differentiated, lineage-committedcell populations are located more toward the core of the neuro-sphere [48, 49]. To compare the localization of the 473HDepitope and RPTP-� isoforms in the 3D structure of neuro-spheres, cryosections were prepared and stained with mAb473HD and KAF13 antibodies. Strong immunoreactivities formAb 473HD and KAF13 were detected on the peripheral area ofneurosphere sections, where most nestin-positive progenitorcells reside, whereas immature �III-tubulin-positive neuronswere found in the core regions (Fig. 3B–3E). In situ hybridiza-tion signals using a riboprobe that recognizes the transmem-brane isoforms of RPTP-� were specifically detected on thecells positioned preferentially in the outer layers of neuro-spheres (Fig. 3F, Ptprz1), as opposed to the more ubiquitouslyexpressed receptor protein tyrosine phosphatase � (Fig. 3G,Ptpru). In addition, immunoreactivities for both mAb 473HDand KAF13 could be visualized to some extent in the centralareas of neurospheres. These results suggest that soluble phos-phacan is deposited in the core region of neurospheres, where itmay diffuse from the outer layers. In contrast, the transmem-brane forms of RPTP-� isoforms were expressed mainly on theperiphery of neurospheres. Thus, RPTP-� isoforms that carryCS/DS chains that make up the 473HD epitope are expressed byneural stem cells and by committed precursor populations in theneurospheres.

Gene Expression of C/D-STs on theNeurosphere-Forming Cells and StructuralCharacteristics of Their CS/DS ChainsWe next chose to investigate the expression of C/D-STs in theneurosphere model [40], where FGF-2-responsive neural stemcells precede the appearance of EGF-responsive precursors dur-ing CNS development [50, 51]. To compare the expression ofC/D-STs in both subpopulations of growth factor-responsiveneural stem cells, cortical and striatal E13 neurospheres weregrown in the presence of EGF alone; FGF-2 plus heparin; orEGF, FGF-2, and heparin. Heparin supports, as an adjuvant,FGF-2-FGFR interactions [52]. The expression levels ofC/D-ST mRNAs were estimated by semiquantitative RT-PCRanalysis. With the exception of C4ST-3, the expression of allpresently known C/D-STs was detected in neurospheres, irre-spective of the culture condition (Fig. 4A, 4B). Semiquantitativeanalysis indicated that expression of C6ST-1 mRNA appearedlower in either EGF- or FGF-2-expanded neurospheres than inneurospheres kept in the presence of both EGF and FGF-2.Expression of GalNAc4S-6ST mRNA appeared to be higher inEGF-expanded than in FGF-2- or EGF and FGF-2-expandedneurospheres. Comparable results were obtained with neuro-spheres prepared from E13 mouse cerebral cortex and gangli-onic eminence. The gene expression levels of the other C/D-STenzymes did not significantly differ between these culture con-ditions. To examine the sulfation patterns resulting from theexpression of these enzymes in more detail, we next analyzedthe disaccharide composition of CS/DS chains produced byneurosphere-forming cells cultured in the presence of different

Figure 3. Expression of the 473HD epitope on receptor protein tyrosinephosphatase (RPTP)-� isoforms in Nsphs. (A): An example of 473HD Westernblot analysis is shown. Neural stem/progenitor cells from embryonic day 13(E13) mouse cerebral cortex were cultured for 6 days as Nsphs in the presenceof fibroblast growth factor-2 and epidermal growth factor. Cond. media anddetergent extracts of Nsphs were immunoprecipitated with (�) or without (�)KAF13 antibodies. Note that the 473HD epitope is carried not only by themembrane-bound but also by soluble RPTP-� isoforms. The arrow indicatesthe top of the separating gel. (B–E): Photomicrographs of immunostainedcryosections of Nsphs from E13 mouse cerebral cortex are depicted. RPTP-�isoforms were revealed with the 473HD (B, E) and the KAF13 antibodies (D)as indicated. For comparison, nestin immunoreactivity (C), that is preferentiallyfound on the outer layers, and �III-tubulin-positive cells in the neurosphere core(E) are also shown. (F–G): Serial sections were also hybridized with digoxi-genin-labeled anti-sense probes, which included the nucleotide sequence of thetransmembrane domain of RPTP-� (F). Note that, similar to the localization ofnestin-positive cells, mRNA signals for the membrane-bound RPTP-� isoforms(labeled Ptprz1 according the gene nomenclature) are prominently detected inthe outer area of neurosphere sections. For comparison, we used a probe forRPTP � (labeled Ptpru according to the gene nomenclature), which appears tobe expressed rather ubiquitously in Nsphs (G). Scale bars � 50 �m. Abbrevi-ations: cond., conditioned; IP, immunoprecipitation; Nsphs, neurospheres.


combinations of growth factors (Table 1). Since it has beenreported that most CS-PGs are detected in phosphate-bufferedsaline-soluble extracts of the developing CNS [53], the CS/DS

chains were prepared starting from conditioned neurosphereculture media. The proportion of nonsulfated disaccharides incultures treated with a combination of EGF and FGF-2 wasapproximately twofold lower than in cases where only EGF orFGF-2 was applied. On the other hand, the proportion of 6-O-sulfated disaccharides in the EGF and FGF-2-treated culturewas up to 1.5-fold higher than in cases where each factor wasused individually. The proportion of 4-O-sulfated disaccharideswas invariant with respect to culture conditions. Disulfateddisaccharides such as �Di-diSD and �Di-diSE were also de-tected, as well as CS/DS chains equivalent to those preparedfrom E13 mouse brain. Notably, the proportion of �Di-diSD

from the EGF plus FGF-2-treated cultures was up to 1.5-foldlower than that from either the EGF- or the FGF-2-treatedculture. Furthermore, it should also be noted that the totalamount of CS/DS chains per milligram of protein in the EGFplus FGF-2-treated cultures was up to 2.4-fold higher than thatin the cultures grown in either EGF or FGF-2 alone.

Localization of the Cells Expressing C/D-STs Genesin the 3D Structure of NeurospheresAs pointed out above, neurospheres represent a complex mix-ture of cells that display territorial preference, with activelycycling neural stem/progenitor cell populations being localizedto the more superficial areas and the more differentiated, lin-eage-committed cell populations toward the core of the neuro-sphere [48, 49]. Therefore, we examined the regional distribu-tion of the cells expressing particular C/D-STs by in situhybridization on neurosphere sections using riboprobes forC4ST-1, D4ST-1, C6ST-1 and -2, and UA2OST. Sections werecollected from neurospheres grown from E13 mouse cerebralcortex in the presence of either EGF alone or FGF-2 in con-junction with heparin. Strong signals for all C/D-ST riboprobesexamined were visible in the circumference of FGF-2-expand-ed-neurospheres, whereas the core region displayed lower ornondetectable levels (Fig. 5). Although neurospheres grown inEGF were smaller, they showed similar patterns of C/D-STexpressions, with prominent signals in the outer layers sugges-tive of neural stem/progenitor cells, as opposed to more differ-entiated cell types. Comparable signals were recorded in striatalneurospheres (supplemental online Fig. 2).

Effect of Sodium Chlorate on NeurosphereFormationTo examine the cell biological roles of sulfated molecules suchas proteoglycans on neurosphere formation, cells from second-ary neurospheres of E13 mouse cerebral cortex were cultured inthe presence of FGF-2, EGF, and sodium chlorate, an inhibitorof sulfation [54]. As expected, the expression of the 473HDepitope on neurospheres was prominently inhibited when grownin the presence of 30 mM sodium chlorate (Fig. 6A). On theother hand, this treatment did not affect the expression of thecore proteins recognized by KAF13, which migrate at compa-rable levels in SDS-polyacrylamide gel electrophoresis [18]. Onthe first day of cultivation in the presence of 30 mM sodiumchlorate, the viability of plated cells was examined using trypanblue staining. No significant difference was observed betweensodium chlorate-treated and control cultures (data not shown).Thus, the addition of sodium chlorate to neurosphere formingassays seemed a promising strategy to examine the specificfunctions of sulfated structures on glycosaminoglycans for theneural stem/progenitor cell population. Therefore, the self-re-newal ability of neural stem/progenitor cells was examined bythe clonal density assay [40] in the presence of sodium chlorate.As shown in Figure 6B, the addition of sodium chlorate toculture medium containing FGF-2 and EGF decreased the num-

Figure 4. Semiquantitative reverse transcription (RT)-polymerase chainreaction (PCR) analysis of chondroitin/dermatan sulfotransferase (C/D-ST)expression in neurospheres. (A): Cortical (C-cortex) and striatal (G-emi-nence) neurosphere cultures were established from embryonic day 13 cellsuspensions that were grown for 6 days in the following growth factorconditions: EFH, FH, or E, as indicated below each panel. RT-PCR wasperformed in the linear range with 29–38 cycles (as shown in supplementalonline data 3) on cDNAs obtained from neurospheres of all three cultureconditions. The amplified products were visualized by electrophoresis on a1.5% agarose gel containing ethidium bromide. Note that with the excep-tion of C4ST-3, amplicons of the known CS-STs were detected in neuro-spheres under all three culture conditions. No bands were obtained incontrol amplifications W/O. (B): The density of PCR band products wassemiquantitatively analyzed using the NIH ImageJ program. The ratio ofeach C/D-ST amplicon versus the corresponding �-actin-band was calcu-lated. To compare the relative expression levels of the three culture condi-tions, the values obtained for the EFH/�-actin ratio were set as the referencevalue 1.0. The relative changes are shown as bar histograms (mean � SEM;n � 3). Note that GalNAc4S–6ST and C6ST-1 mRNA levels were differ-entially regulated in epidermal growth factor alone, whereas all otherCS-STs were not significantly altered independent of the growth factorconditions. Abbreviations: C4ST, chondroitin 4-O-sulfotransferase; C6ST,chondroitin 6-O-sulfotransferase; C-cortex, cerebral cortex; D4ST, derma-tan 4-O-sulfotransferase; E, epidermal growth factor alone; EFH, epidermalgrowth factor and fibroblast growth factor-2 with heparin; FH, fibroblastgrowth factor-2 with heparin; GalNAc4S–6ST, N-acetylgalactosamine4-sulfate 6-O-sulfotransferase; G-eminence, ganglionic eminence; UA2OST,uronosyl 2-O-sulfotransferase; W/O, without template.


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ber of neurospheres in a dose-dependent fashion. The averagesize of neurospheres also seemed to be decreased by this treat-ment, indicating a slowed cell cycle (data not shown). Further-more, sodium chlorate also decreased the number of neuro-spheres in the presence of EGF alone. Notably, the addition ofexogenous heparin to culture medium did not rescue the de-crease in neurosphere formation in the EGF-treated cultures(Fig. 6C). To further investigate the functional implications ofCS-GAGs on neurosphere formation, we added defined, com-mercially available chondroitin sulfates to chlorate-treated neu-rosphere-derived cells to rescue neural stem cell maintenanceand self-renewal. At concentrations that had revealed a functionfor CS-B, CS-D, and CS-E in neurosphere growth and/or pro-liferation [23], none of the CS chains nor heparin resulted inneurosphere numbers that were comparable to those of untreatedcontrol cultures, although an increase was recorded that was notsignificant (Fig. 6E). Thus, neural stem cell maintenance re-quired sulfation, but the simple addition of defined but randomlysulfated GAGs is not sufficient. However, in heparin-, CS-B-,CS-D-, and CS-E-supplemented cultures, we observed a roughlytwofold increase in the size of the neurospheres (Fig. 6D), whichindicates functional activity of defined GAGs with respect tosurvival, growth, or proliferation of neural stem/progenitor cells,as reported previously [23].

DISCUSSION

Multipotent precursor cell populations have been discovered notonly in embryonic brain but also in adult brain. It has beensuggested that the microenvironment surrounding these cells,the neural stem cell niche, constitutes a crucial milieu for theregulation of their self-renewal and multipotent differentiationcapacity [55]. In this context, it seemed worthwhile to studyCS-PGs in the neural stem cell niche, because they represent amajor component of the extracellular matrix of the brain. Inparticular, it has been suggested that the sulfation patterns ontheir glycosaminoglycans play important roles during brain de-velopment [56]. In the present study, we demonstrated thatseveral C/D-ST genes are expressed in the neurogenic regions ofthe embryonic and adult mouse CNS. The synergistic actions ofvarious C/D-STs in an individual cell may underlie the emer-gence of distinct sulfation patterns within the CS/DS chains.Consistent with this scenario, we recently reported that the473HD epitope is expressed in the germinal layers of the em-

bryonic mouse telencephalon and on neurosphere-forming cells[20] (Fig. 3). In the present study, we found that neurosphere-forming cells expressed at least two alternatively spliced iso-forms of RPTP-� modified with the 473HD epitope. Thisepitope, a CS/DS structure recognized by mAb 473HD, includesCS-A, CS-D, and CS-B units [17–19]. Two other mAbs, CS-56and MO-225, also recognize CS structures that comprise CS-Aand CS-D units and are in this respect comparable to mAb473HD [19]. It could previously be ascertained, however, thatthe oligosaccharides recognized by each of these individualmAbs undoubtedly possess individual and unique structuralcharacteristics [19]. Maeda et al. [3] have shown that the CS-56epitope is expressed in the lateral zone adjacent to the lateralventricle in postnatal mouse brain and that immunoreactivity ofMO-225 in this region dramatically increases during develop-ment of the CNS. In contrast, expression of the 473HD epitopedecreases but is constantly detected in the neurogenic CNSregions, irrespective of the developmental stages [20]. Theseobservations suggest that the occurrence of particular sulfatedCS/DS structures is strictly regulated by the expression ofvarious C/D-STs in the neural stem cell niche throughout life.

We detected the gene expression of multiple C/D-STs inneurospheres from E13 mouse cerebral cortex and ganglioniceminence, as well as their expressions in these tissues. It hasbeen postulated that the environment of the neural stem cellniche is mimicked in the outer layer of neurospheres as aconsequence of the formation of a 3D spheroid structure [48].Interestingly, in situ hybridization signals for C4ST-1, D4ST-1,C6ST-1 and -2, and UA2OST were mostly detected in the outerzone of the sectioned neurosphere, suggesting that the neuro-sphere culture constitutes an adequate in vitro model to studythe possible functions of CS/DS-PGs. We found that the ratio ofCS-C/CS-A units in the EGF plus FGF-2-treated neurosphereculture increased 1.4–1.7-fold in comparison with those in theculture treated with either EGF or FGF-2 alone, irrespective ofthe cellular source. It has been suggested that the proportion ofCS-C units decreases during the development of chick and ratbrains in consequence of the downregulation of the C6ST-1gene [1, 25]. In agreement with these reports, the expression ofC6ST-1 mRNA appeared more pronounced in the EGF plusFGF-2-expanded neurospheres than in either EGF- or FGF-2-exposed neurospheres. In contrast, the semiquantitative analysisdid not reveal prominent differences for the expression of theC6ST-2 gene. Therefore, it seems likely that this proportionalincrease of the CS-C unit reflects mainly the enzymatic activity

Table 1. Disaccharide analysis of CS/DS chains produced by neurosphere-forming and E13 mouse brain cells

Origin of theCS/DS chains

Unsaturated disaccharide (%, pmol proportion)Sulfation

degreeGlcUA content

(pmol/mg of protein)�Di-0S �Di-6S �Di-4S �Di-diSD �Di-diSB �Di-diSE

C-cortex (EF) 12.8 17.6 67.7 0.8 0.2 1.1 0.9 85.0C-cortex (F) 26.6 9.7 61.0 2.3 ND 0.6 0.77 31.9C-cortex (E) 25.3 10.3 61.6 1.7 ND 1.1 0.78 33.0G-eminence (EF) 9.3 19.2 69.1 1.4 ND 1.1 0.93 99.6G-eminence (F) 22.3 12.2 62.2 2.2 ND 1.7 0.82 40.7G-eminence (E) 26.9 10.3 58.3 3.6 ND 1.0 0.78 29.5E13 mouse brain 32.6 12.1 51.4 3.1 ND 1.6 0.73 21.5

Cells from E13 mouse C-cortex and G-eminence were cultured in the neurosphere culture medium supplemented with EF, F, or E. After 6days, each spent culture medium was collected for the purification of CS/DS chains. CS/DS chains purified from E13 mouse whole brainwere also analyzed. Sulfation degree was calculated as the average number of sulfate groups per disaccharide unit. The values represent themeans from two independent experiments.Abbreviations: CS/DS, chondroitin/dermatan sulfate; C-cortex, cerebral cortex; �Di-0S, �4,5HexA�1–3-N-acetylgalactosamine; �Di-4S,�4,5HexA�1–3-N-acetylgalactosamine(4-O-sulfate); �Di-6S, �4,5HexA�1–3-N-acetylgalactosamine(6-O-sulfate); �Di-diSB, �4,5HexA(2-O-sulfate)�1–3-N-acetylgalactosamine(4-O-sulfate); �Di-diSD, �4,5HexA(2-O-sulfate)�1–3-N-acetylgalactosamine(6-O-sulfate); �Di-diSE,�4,5HexA�1–3-N-acetylgalactosamine(4,6-O-disulfate); G-eminence, ganglionic eminence; ND, not detected; E13, embryonic day 13; E, EGFalone; F, FGF-2 alone; EF, EGF plus FGF-2.


of C6ST-1. A positive correlation between active mitosis andthe proportional increase of CS-C/CS-A units has been noticedpreviously [1]. The stimulation with EGF in conjunction withFGF-2 produces the largest size and the highest number ofneurospheres from E12–E14 mouse telencephalon [50].

It has been reported that mice lacking the C6ST-1 gene donot display apparent abnormalities, with the exception of a slightdecrease of the immature T-lymphocyte population [57]. Thepresence of CS-C and CS-D units has not been detected in theCS/DS chains prepared from the adult brain of C6ST-1 gene-deficient mice. In this case, however, the situation during em-bryonic development has not been analyzed [57]. In our study,

we detected prominent expression of the C6ST-2 gene in theembryonic germinal and adult subventricular zones. On thebasis of available data, however, it cannot be decided whetherthe lack of C6ST-1 function in the knockout mutant is partiallycompensated by the enzymatic activities of C6ST-2 in selectedareas such as the neural stem cell niche. Alternatively, it may beenvisaged that other glycosaminoglycans including HS chainscompensate for the decrease of CS-C and CS-D units in theCS/DS chains in vivo. The structural basis for this ability ofsubstitution could reside in the possibility that specific sulfatedstructures in CS chains share the binding ligands with HSchains, as discussed below.

Figure 5. In situ hybridization for chondroitin/dermatan sulfotransferases (C/D-STs) in cortical Nsphs. Bright-field photomicrographs of Nsphcryosections after developing digoxigenin-labeled RNA probes for C4ST-1 (A, A�, F, F�), D4ST-1 (B, B�, G, G�), C6ST-1 (C, C�, H, H�), C6ST-2(D, D�, I, I�), and UA2OST (E, E�, J, J�) are depicted. Nsphs were grown for 6 days from single-cell suspensions from E13 mouse cerebral cortexin medium containing either FGF-2 plus heparin (left two columns) or EGF (right two columns), as indicated at the top of the figure. The sectionswere hybridized with antisense (A–J) or sense (A�–J�) probes. Note that mRNAs of all C/D-STs studied were preferentially expressed in the outerlayers of the Nsphs, where the neural stem/progenitor cells reside. Scale bar � 50 �m ([A], applies to all images). Abbreviations: C4ST, chondroitin4-O-sulfotransferase; C6ST, chondroitin 6-O-sulfotransferase; D4ST, dermatan 4-O-sulfotransferase; E13, embryonic day 13; EGF, epidermal growthfactor; FGF, fibroblast growth factor; Nsphs, neurospheres; UA2OST, uronosyl 2-O-sulfotransferase.


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Neurospheres grown in the presence of EGF and FGF-2increased the content of CS/DS chains per milligram of proteinsin the conditioned culture media, suggesting quantitative alter-ations on the glycosaminoglycan and/or core protein levels. Ithas been reported that the expression of the large splice variant

of the RPTP-� gene was prominently detected on the mRNAlevel in the embryonic germinal and postnatal subventricularzones [58, 59]. RPTP-� long and its alternative splice variantphosphacan/DSD-1-PG/6B4-PG can interact with heparin-bind-ing growth-associated molecule (HB-GAM)/pleiotrophin [60].

Figure 6. Sodium chlorate treatment reduces neurosphere formation. (A): To confirm the decrease of sulfation on chondroitin/dermatan sulfatechains, detergent extracts (20 �g of protein) from 30 mM sodium chlorate-treated or untreated neurospheres were subjected to 7% SDS-polyacrylamide gel electrophoresis. Thereafter, the samples were analyzed by immunoblot using the monoclonal antibody 473HD. Note that thetreatment with sodium chlorate diminished the expression of receptor protein tyrosine phosphatase-� isoforms modified with the 473HD epitope,whereas immunoblot analysis using KAF13 antibodies did not show that expression of their core proteins is altered by this treatment. The arrowindicates the top of the separating gel. (B, C): Clonal density assays were performed to examine the effect of sodium chlorate on the self-renewalability of neural stem/progenitor cells. Single-cell suspensions from secondary neurospheres were plated at clonal density (200 cells per cm2) andcultured with FGF-2 plus EGF (square) and EGF alone (circle) in the presence or absence of sodium chlorate for 5 days. The total number ofneurospheres was counted and plotted in the bar histograms (mean � SEM; n � 3). Note that sodium chlorate dose-dependently decreased the numberof neurospheres, which were cultured in the presence of FGF-2 and EGF or EGF alone. Also note that in the EGF-treated culture, the sodiumchlorate-induced decrease of neurosphere number was not recovered even by the addition of exogenous Hep. (D): Photomicrographs of representativeindividual neurospheres grown from chlorate-treated dissociated secondary neurospheres under the indicated conditions. Note that Hep, CS-B, CS-D,and CS-E increased neurosphere size in comparison with CS-A, CS-C, and non-GAG-supplemented control cultures. Scale bar � 100 �m. (E): Effectof chondroitin sulfate units on neurosphere formation. Bar histograms show the total/relative neurosphere numbers quantified in clonal density assays.Neurospheres were grown in the presence of FGF-2 (20 ng/ml), 30 mM chlorate, and the various GAG chains as indicated below the chart. Data areexpressed as mean � SEM from three independent experiments. Note that neurosphere formation appears to be increased after the addition of definedchondroitin sulfate units, but the neurosphere numbers were not significantly different from those of control cultures (no GAGs; i.e., FGF-2 alone orFGF-2 and Hep). Abbreviations: CS-A, GlcUA�1–3GalNAc(4-O-sulfate); CS-B, IdoUA(2-O-sulfate)�1–3GalNAc(4-O-sulfate); CS-C, GlcUA�1–3GalNAc(6-O-sulfate); CS-D, GlcUA(2-O-sulfate)�1–3GalNAc(6-O-sulfate); CS-E, GlcUA�1–3GalNAc(4, 6-O-disulfate); EGF, epidermal growthfactor; FGF, fibroblast growth factor; Hep, heparin.


The affinities of this interaction are positively correlated withthe proportion of CS-D units in the CS chains [3]. We haverecently provided evidence that the binding site of HB-GAM inCS/DS chains overlaps with the 473HD epitope [61]. CS chainsthat contain a high proportion of CS-E units from squid cartilagedisplay high-affinity binding for various heparin-binding factors[12]. Zou et al. [24] have reported a ligand affinity bindingexperiment using a mixture of CS/DS chains prepared from E13mouse brain. In their report, fractions rich in CS-E units wereeluted at 0.7 M NaCl from a midkine-affinity column [24]. Inthe present study, we demonstrated that neurosphere-formingcells possess the capacity to synthesize CS/DS chains thatcontain significant amounts of disulfated disaccharides such asthe CS-D and CS-E units. Unlike the case of the CS-C unit, theproportion of CS-D units detectable in the EGF plus FGF-2-treated neurosphere cultures was up to 1.5-fold lower than upontreatment with either EGF or FGF-2 alone. As outlined above,the proportional increase of CS-D units in the CS/DS hybridchains is likely to affect the binding affinity for HB-GAM.Interestingly, it has been reported that HB-GAM inhibits cellproliferation and enhances differentiation of neural stem/pro-genitor cells [62]. We have recently reported that fewer neuro-spheres were observed in neurosphere cultures grown in thepresence of mAb 473HD [20]. Our current results revealed thatthe 473HD epitope was carried by RPTP-� long and phospha-can. The binding of this antibody to RPTP-� long on cell surfacemay affect its phosphatase activities and consequently intracel-lular signal transduction, as reported for the binding of HB-GAM to its high-affinity cell surface receptor, RPTP-� long[63]. Specific structural motifs in DS chains purified fromporcine intestinal mucosa are required for their interactions withFGF-2 and -7 [64]. Although the CS-B unit was not detected inthe majority of our samples, its absence may reflect limitationsof the detection method. Indeed, the epitope of mAb 473HDcould be documented by immunohistochemistry both in neuro-spheres and in embryonic forebrain [20]; Figure 3).

In this study, we showed that addition of sodium chlorate toneurosphere cultures attenuated the expression of the 473HDepitope on neurosphere-forming cells. Ida et al. [23] have re-ported that addition of exogenous CS-B and CS-E into neuro-sphere culture medium significantly enhances FGF-2-mediatedcell proliferation of neural stem/progenitor cells. Interestingly,the culture of neurospheres in the continuous presence of so-dium chlorate significantly decreased the number of both FGF-2and EGF-expanded neurospheres in a dose-dependent fashion.A similar effect was observed even when EGF alone was usedas exogenous mitogen. Different from the situation with FGFsignaling, HS/heparin is not involved in the binding of EGF tothe EGF receptor and its stabilization [65]. In the course of cellculture, neurosphere-forming cells may, however, endogenouslyproduce some heparin-binding growth factors, as reported in the

case of FGF-2 [66]. In our hands, the maintenance of neuralstem cells in the presence of FGF-2 critically depended onendogenous sulfation. Because the addition of purified GAGchains from various non-CNS sources could not fully reversethe effect of chlorate treatment on neurosphere number, weassume that the sulfation pattern of CNS GAG chains is differ-ent. Thus, neural stem cell maintenance might require a sulfa-tion code, as has been proposed for neurite branching in thenematode [67]. We propose that such a hypothetical code woulddiffer for neural stem cell self-renewal as opposed to theirgrowth and proliferation behavior since the latter could berescued by defined CS-GAGs and heparin. In this way, thepatterned level of sulfation in the glycosaminoglycans of theneural stem cell niche may allow or even instruct neural stemcell behavior by modulating the activities of endogenous growthfactors.

Altogether, we show here for the first time that the expres-sion of multiple C/D-STs is maintained in the neurogenic re-gions of the CNS throughout life. It seems plausible that theirsynergic action renders it possible to synthesize some CS/DS-PGs modified with diversely sulfated CS/DS chains in theneural stem cell niche.

ACKNOWLEDGMENTS

We thank Dr. A. Horvat-Brocker, Department of Cell Morphol-ogy and Molecular Neurobiology, Ruhr-University Bochum,Bochum, Germany, for the support of in situ hybridization andall other members of the department for insightful suggestionsand discussions. This work was supported by the German Re-search Council (Deutsche Forschungsgemeinschaft priority pro-gram SPP 1109 to A.v.H. and A.F.) and by the German Ministryof Research and Technology (Grant BMBF 01GN0503 to A.F.).K.A. was supported by a grant from the German AcademicExchange Program (Deutscher Akademischer Austauschdienst).The work performed in Japan was supported in part by HAIT-EKU (2004–2008) from the Japan Private School PromotionFoundation, Grant in-Aid for Exploratory Research17659020 and the Core Research for Evolutional Science andTechnology of the Japan Science and Technology Agency.K.A. and A.v.H. contributed equally to this work. K.A. iscurrently affiliated with the Department of Biotechnology,Kyoto Sangyo University, Kita-ku, Kyoto, Japan.

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

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