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First published online December 13, 2007
Stem Cells Vol. 26 No. 3 March 2008, pp. 798 -809
doi:10.1634/stemcells.2007-0448; www.StemCells.com
© 2008 AlphaMed Press

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THE STEM CELL NICHE

Expression of Multiple Chondroitin/Dermatan Sulfotransferases in the Neurogenic Regions of the Embryonic and Adult Central Nervous System Implies That Complex Chondroitin Sulfates Have a Role in Neural Stem Cell Maintenance

Kaoru Akitaa, Alexander von Holsta, Yoko Furukawab, Tadahisa Mikamib, Kazuyuki Sugaharab,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 of Life 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

Correspondence: Correspondence: Prof. Andreas Faissner, M.D., Department of Cell Morphology and Molecular Neurobiology, Faculty of Biology, Ruhr-University Bochum, NDEF 05/594, Universitätsstrasse 150, D-44780 Bochum, Germany. Telephone: 49-234-3223851; Fax: 49-234-3214313; e-mail: andreas.faissner{at}ruhr-uni-bochum.de

Received on June 12, 2007; accepted for publication on November 27, 2007.

First published online in STEM CELLS EXPRESS  December 13, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Chondroitin/dermatan sulfotransferases (C/D-STs) underlie the synthesis of diverse sulfated structures in chondroitin/dermatan sulfate (CS/DS) chains. Recent reports have suggested that particular sulfated structures on CS/DS polymers are involved in the regulation of neural stem cell proliferation. Here, we examined the gene expression profile of C/D-STs in the neurogenic regions of embryonic and adult mouse central nervous system. Using reverse transcription-polymerase chain reaction analysis, all presently known C/D-STs were detected in the dorsal and ventral telencephalon 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)-1 and -2, and uronosyl 2-O-sulfotransferase revealed a cellular expression of these sulfotransferase genes in the embryonic germinal zones of the forebrain. The expression of multiple C/D-STs is maintained on cells residing in the adult neural stem cell niche. Neural stem cells cultured as neurospheres maintained the expression of these enzymes. Consistent with the gene expression pattern of C/D-STs, disaccharide analysis revealed that neurospheres and E13 mouse brain cells synthesized CS/DS chains containing monosulfated, but also significant amounts of disulfated, disaccharide units. Functionally, the inhibition of sulfation with sodium chlorate resulted in a significant, dose-dependent decrease in neurosphere number that could not be rescued by the addition of individual purified glycosaminoglycan (GAG) chains, including heparin. These findings argue against a simple charge-based mechanism of GAG chains in neural stem cell maintenance. The synergistic activities of C/D-STs might allow for the adaptive modification of CS/DS proteoglycans with diversely sulfated CS/DS chains in the extracellular microenvironment that surrounds neural stem cells.

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


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Various biological roles of proteoglycans (PGs) have been reported in the developing and mature central nervous system (CNS). Their functions are partially mediated by the glycosaminoglycan chains that are covalently bound to the core protein. Recently, evidence has revealed that sulfation of glycosaminoglycans is spatiotemporally regulated in the brain [1–4]. The variation of this modification allows for a considerable structural diversity of glycosaminoglycans, which may constitute the basis for diverse biological roles. It is established that heparan sulfate (HS) chains modulate the activities of various growth and morphogenetic factors [5, 6]. Among them, fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR) signaling represents one of the crucial effectors for the neural stem cell population. For example, FGF-2 not only promotes cell proliferation of embryonic cortical stem cells [7] but also controls their differentiation into neuronal and glial lineages [8]. Developmental stage-specific structural differences of HS chains that serve as temporal regulators to form the active FGF-FGFR signaling complex have been described in various progenitor cell populations, including the neuroepithelial layers of the brain [2]. Therefore, it has been suggested that the expression of selected heparan sulfotransferases is temporally regulated in neural precursor cells [9]. HS chains that are expressed on embryonic day 9 (E9) to E11 mouse neural precursor cells exhibit differential binding affinities for FGF-1 and -2 [10]. Notably, the binding of cytokines and growth factors is not limited to HS-type glycosaminoglycans. Oversulfated chondroitin/dermatan sulfate (CS/DS) chains from different sources also interact with several heparin-binding growth factors with nanomolar affinities [11–14].

Previously, we have established that the monoclonal antibody 473HD is directed against phosphacan/DSD-1-PG/6B4-PG [15–17], a splice variant of receptor protein tyrosine phosphatase (RPTP)-β that has been purified from postnatal rodent CNS [17]. Subsequent analysis of the 473HD epitope (or DSD-1 epitope) has shown that the antibody recognizes a unique CS/DS glycosaminoglycan 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 represent 2-O-, 4-O-, and 6-O-sulfate, respectively [18, 19]. We recently reported that CS/DS-PGs bearing this unique sulfated structure are expressed in the periventricular germinal zones of the developing mouse CNS and in the subventricular zone of the adult mouse CNS [20], where multipotent stem cells reside [21]. The expression of some CS/DS-PG core proteins has been detected in the embryonic neural stem cell niche and in neurospheres [22, 23]. Furthermore, consistent with the expression of the 473HD epitope, various mono- and disulfated disaccharide units have also been identified by the compositional analysis of CS/DS chains purified from the embryonic mammalian CNS [11, 13, 23–25]. Interestingly, cell surface expression of the 473HD epitope is prominently observed on the neural stem cell population, and the addition of the monoclonal antibody (mAb) 473HD into neurosphere culture medium decreases the number of neurospheres [20]. Using the neurosphere culture system, Ida et al. [23] have recently reported that particular sulfated structures on CS/DS chains such as the IdoUA(2-O-sulfate){alpha}1-3GalNAc(4-O-sulfate) (CS-B), GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) (CS-D), and GlcUAβ1-3GalNAc(4, 6-O-disulfate) (CS-E) units possess the potential to promote FGF-2-mediated cell proliferation of rat embryonic neural stem/precursor cells. These findings suggested that the sulfation profile on CS/DS chains is one of the crucial factors regulating cell proliferation of neural stem cells in the CNS.

Sulfate groups are transferred from 3'-phosphoadenosine 5'-phosphosulfate to the specific acceptor sites in CS/DS chains by chondroitin/dermatan sulfotransferases (C/D-STs) that are located in the Golgi apparatus [26–28]. As illustrated in Figure 1A, these enzymes are classified into the following four groups: chondroitin/dermatan 4-O-sulfotransferase (C4ST/D4ST), chondroitin 6-O-sulfotransferase (C6ST), uronosyl 2-O-sulfotransferase (UA2OST), and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST). Three C4ST isoforms [29–32], two C6ST isoforms [33, 34], D4ST-1 [35], UA2OST [36], and GalNAc4S-6ST [37] have been identified in mammals. It has been reported that gene expression levels of some enzymes correlate with the amount of sulfated products that corresponded to each enzymatic activity [1, 25], which holds the promise that studies of gene expression of C/D-STs will yield more detailed insights about the sulfation profiles in mixed CS/DS chains.


Figure 1
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  		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 consisting of glucuronic acid (GlcUA) (white hexagons) and N-acetylgalactosamine (GalNAc) (light gray hexagons) are depicted. These CS-disaccharide units are modified by four different sulfotransferases: C4ST, C6ST, UA2OST, and GalNAc4S–6ST, as indicated in the scheme. The activity of the chondroitin sulfotransferases leads to the addition of sulfate groups at defined positions (black circles), which results in the generation of specified CS units as shown in the figure (underlined). In case GlcUA is converted to iduronic acid (IdoUA) (dark gray hexagons) by its C-5 epimerization, the enzyme D4ST preferentially adds a sulfate group at the C4 position of GalNAc, which is adjacent to IdoUA. The detailed substrate specificities for each enzyme are further explained in the text and the selected references. (B): Analysis of chondroitin/dermatan sulfotransferase expression in the embryonic day 13 (E13) mouse telencephalon by reverse transcription-PCR. cDNA was synthesized using total RNA purified from E13 C-cortex and E13 G-eminence. PCR was performed with a serial number of cycles (20, 24, 28, 32, and 36). The amplified products were visualized by electrophoresis on a 1.5% agarose gel containing ethidium bromide. Note that with the exception of C4ST-3, mRNAs of all C/D-STs cloned so far were detected in these tissues. Abbreviations: C4ST, chondroitin 4-O-sulfotransferase; C6ST, chondroitin 6-O-sulfotransferase; C-cortex, cerebral 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.

 
To begin to elucidate how the sulfation on CS/DS chains is regulated in the neural stem cell niche, we first studied the gene expression patterns of a range of C/D-STs in the neurogenic regions of the embryonic and adult CNS. The mRNAs of these sulfotransferases were also examined in neurospheres obtained from E13 mouse telencephalon. Furthermore, we analyzed the structural characteristics of the CS/DS carbohydrate chains prepared from the conditioned neurosphere culture medium and from E13 mouse brain. Our results suggest that expression of multiple C/D-STs is maintained in the cells residing in the neurogenic regions of embryonic and adult CNS, rather than displaying temporal regulation of the expression of these enzymes. Furthermore, we show that the addition of sodium chlorate inhibited the expression of the 473HD epitope on neurosphere-forming cells concomitant with a decrease in the number of neurospheres, implying that defined sulfated molecules such as PGs are closely involved in cell proliferation and maintenance of neural stem/progenitor cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Animals and Antibodies
NMRI mice with timed pregnancies were obtained from Charles River Laboratories (Wilmington, MA, http://www.criver.com), and the ages of the embryos were verified according to Bard et al. [38]. The antibodies 473HD and KAF13 and their specificities have been described [17, 20]. All commercial antibodies were obtained from Chemicon (Temecula, CA, http://www.chemicon.com), Roche Diagnostics (Basel, Switzerland, http://www.roche-applied-science.com), or Dianova (Hamburg, Germany, http://www.dianova.de) as indicated in supplemental online data 4.

Primary Neurosphere Culture
Primary neurospheres were grown from E13 forebrain cell suspensions as described previously [20, 39]. The cultures were supplemented 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 used clonal density assays [40] (200 cells per cm2) to assess the effect of sodium chlorate on neurosphere formation by adding 5 or 30 mM chlorate in the presence of EGF, FGF-2, or both. For rescue experiments, GlcUAβ1–3GalNAc(4-O-sulfate) (CS-A), CS-B, GlcUAβ1–3GalNAc(6-O-sulfate) (CS-C), CS-D, and CS-E (all from Seikagaku, Tokyo, http://www.seikagaku.co.jp/english) were added at 10 µg/ml in the continued presence of 30 mM sodium chlorate. After 5 days of cultivation, the total number of neurospheres under control and treatment conditions was microscopically determined.

Reverse Transcription-Polymerase Chain Reaction and Semiquantitative Analysis
Briefly, total RNA was isolated and reverse transcribed as previously described [39]. The polymerase chain reactions (PCRs) were performed in the linear range with primer sequences and PCR conditions as detailed in supplemental online data 3 and 4. All amplicons were cloned and verified by sequencing. For semiquantitative analysis, the density of the amplified products was measured (NIH ImageJ version 1.36) and plotted as ratio of the β-actin band.

In Situ Hybridization
The probes for mRNA of the sulfotransferases C4ST-1, D4ST, C6ST-1 and -2, and UA2OST, as well as RPTP-β, were obtained by reverse transcription (RT)-PCR. Digoxigenin (DIG)-labeled antisense and sense riboprobes were generated according to the manufacturer's instructions (DIG RNA Labeling Kit; Roche Diagnostics). Cryosections were hybridized with the riboprobes at 50°C overnight. After washing and blocking, the sections were incubated with an alkaline phosphatase-conjugated anti-DIG antibody (1:2,000) overnight at 4°C. The probes were visualized using Nitro Blue Tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, and color development was stopped at variable time points to obtain reasonable signal-to-noise ratios.

Immunoblot Analysis
Detergent extracts of samples were obtained, separated, blotted, and developed as described previously [39]. For immunoprecipitation, 4 ml of KAF13 was added to neurosphere lysates or conditioned medium. After incubation at 4°C overnight, 10 µl of protein A-Sepharose was added and incubated at 4°C for 2 hours. Immunoprecipitates were spun down and further treated as the protein lysates (details given in supplemental online data 4).

Immunostaining of Neurosphere Sections
Neurosphere sections were prepared as described for the in situ hybridizations, and the immunohistochemical stainings were performed as described previously [20].

Disaccharide Analysis of CS/DS Chains from Embryonic Brain and Conditioned Neurosphere Culture Media
Disaccharide analysis of CS/DS chains from E13 mouse brain was performed as described previously [11]. Using the same analytical method, disaccharide compositions of CS/DS chains from conditioned neurosphere culture media were also analyzed.

Other Methods
The protein concentration was determined using BCA protein assay kit (Bio-Rad) with bovine serum albumin as the standard. Student's t test was used for statistical analysis of the experiments. p < .05 was taken as the minimal level of significance. A detailed description of all methods can be found in supplemental online data 4.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Gene Expression Patterns of C/D-STs in the Neurogenic Regions of the Embryonic and Adult Mouse Brain
To examine which C/D-STs are expressed in the neurogenic regions at embryonic stages, we performed RT-PCR using total RNA purified from E13 mouse cerebral cortex and ganglionic eminence. Except for C4ST-3, mRNAs of all C/D-STs cloned so far (C4ST-1 and -2, D4ST-1, C6ST-1 and -2, UA2OST, and GalNAc4S-6ST) could be traced in these tissues and confirmed after subcloning and DNA sequencing (Fig. 1B). It has been reported previously that C4ST-3 mRNA is highly upregulated in adult human liver [31]. As a positive control, the expression of C4ST-3 mRNA was confirmed in adult mouse liver by RT-PCR using our PCR primers and subsequent DNA sequencing (data not shown).

More recently, we have found that a unique CS/DS structure recognized by the mAb 473HD and named the 473HD epitope is expressed in the ventricular zone of E13 mouse telencephalon [20]. We have shown previously that the 473HD epitope depends on selective sulfation of chondroitin sulfate chains that comprise CS-A and CS-D motifs, and a chondroitin sulfate B element [17–19]. The coordinated activity of a restricted set of C/D-STs is required to synthesize this structure. To elaborate a more detailed picture of the distribution of the cells expressing the 473HD epitope in this region, the spatial expression patterns of the C/D-ST genes were examined by in situ hybridization. Coronal sections of E13 mouse brain were hybridized with the DIG-labeled riboprobes for C4ST-1, D4ST-1, C6ST-1 and -2, and UA2OST, which are presumably involved in the formation of the 473HD epitope. As exemplified for C4ST-1 (Fig. 2), a prominent expression of all C/D-STs examined was detected in the ventricular zones of the dorsal and the ventral telencephalon (supplemental online Fig. 1). Hybridization signals were observed colocalizing with cell bodies that are positioned adjacent to the ventricular surface but are also located farther from the lining of the ventricular zone. Interestingly, we did not find striking differences in the distribution pattern of the cells expressing individual sulfotransferase genes, which supports the concept that the orchestrated activity of multiple enzymes in a given cell is required for the generation of diverse sulfation patterns in CS/DS chains. These results clearly suggest that the cell population in the embryonic neurogenic regions is endowed with the capacity to synthesize a large variety of sulfated CS/DS chains.


Figure 2
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  		Figure 2. In situ hybridization of chondroitin/dermatan sulfotransferases (C/D-STs) in the adult neural stem cell niche. Bright-field photomicrographs 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, which did not give rise to specific signals. The right panels (a–e) refer to the boxed areas that are enlarged to provide higher-resolution images. Coronal sections 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 neural stem cell niche. (F, F'): One example of CS-ST expression (C4ST-1) (F) in the germinal layers during forebrain development (E13). Scale bar = 50 µm (a–e, F, F') and 200 µm (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.

 
Neural stem cell proliferation continues in the adult CNS in restricted areas called niches [21], where the expression of distinct chondroitin sulfates, including the 473HD epitope, has previously been recorded [20, 41]. We examined the expression of C/D-STs in neurogenic regions of the adult brain by in situ hybridization using the same riboprobes as above (Fig. 2). Prominent signals for all C/D-STs examined in this study were detected on the cells residing in the subventricular zone (SVZ) around the anterior lateral ventricle wall. Closer inspection at higher magnification revealed that, in addition, ependymal cells that line the ventricle wall express the mRNAs corresponding to these enzymes. Furthermore, signals were detected in cells of the rostral migratory stream where neuroblasts derived from the SVZ migrate toward the olfactory bulb (data not shown).

Expression of the 473HD Epitope on Alternatively Spliced Isoforms of the RPTP-β Gene Locus in Neurospheres and Their Conditioned Culture Media
Neural stem/progenitor cells can be cultivated in suspension as so-called neurospheres when supplied with a defined medium that contains adequate growth factors such as EGF or FGF-2 [42]. Neurosphere-forming cells express several CS/DS-PGs including phosphacan, one of the alternatively spliced isoforms of the RPTP-β gene [22, 23]. Four variants of RPTP-β have been identified in mouse so far [43, 44] and all are expressed on mRNA level in neurospheres (data not shown). Only the large transmembrane receptor protein tyrosine phosphatase form RPTP-β long and the soluble chondroitin sulfate proteoglycan phosphacan possess the glycosylation sites needed for the covalent attachment of glycosaminoglycans [44]. We have previously reported that cultured immature glial cells from late embryonic and early postnatal mouse cerebellum express the 473HD epitope [17] and that this epitope is carried by RPTP-β long and phosphacan [43, 44].

More recently, we have found that most of mAb 473HD immunoreactive cells dissociated from the early embryonic forebrain and/or neurospheres show the same characteristics as neural stem cells [20]. However, it remained unclear which of the RPTP-β gene products expressed carries the 473HD epitope in E13 cortical neural stem cells. To examine this issue, detergent extracts and culture supernatants of neurospheres grown in the presence of FGF-2 and EGF were immunoprecipitated with polyclonal anti-phosphacan antibodies (KAF13); the resulting precipitates were immunoblotted and finally developed with mAb 473HD. As shown in Figure 3A, 473HD-reactive material was detected both in the cell lysate and the culture medium of neurospheres; this material was absent when the KAF13 polyclonal antibodies had been omitted. This is consistent with the interpretation that the 473HD epitope is carried on RPTP-β long and phosphacan. In the neurosphere detergent extract, two additional bands with molecular weights of approximately 290 and 250 kDa were detected. These 473HD epitope bearing bands may correspond to immature, partially glycosylated processing intermediates or to proteolytically degraded fragments of the RPTP-β core protein(s), as discussed previously [45, 46].


Figure 3
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  		Figure 3. Expression of the 473HD epitope on receptor protein tyrosine phosphatase (RPTP)-β isoforms in Nsphs. (A): An example of 473HD Western blot analysis is shown. Neural stem/progenitor cells from embryonic day 13 (E13) mouse cerebral cortex were cultured for 6 days as Nsphs in the presence of fibroblast growth factor-2 and epidermal growth factor. Cond. media and detergent extracts of Nsphs were immunoprecipitated with (+) or without (–) KAF13 antibodies. Note that the 473HD epitope is carried not only by the membrane-bound but also by soluble RPTP-β isoforms. The arrow indicates the top of the separating gel. (B–E): Photomicrographs of immunostained cryosections of Nsphs from E13 mouse cerebral cortex are depicted. RPTP-β isoforms were revealed with the 473HD (B, E) and the KAF13 antibodies (D) as indicated. For comparison, nestin immunoreactivity (C), that is preferentially found on the outer layers, and βIII-tubulin-positive cells in the neurosphere core (E) are also shown. (F–G): Serial sections were also hybridized with digoxigenin-labeled anti-sense probes, which included the nucleotide sequence of the transmembrane domain of RPTP-β (F). Note that, similar to the localization of nestin-positive cells, mRNA signals for the membrane-bound RPTP-β isoforms (labeled Ptprz1 according the gene nomenclature) are prominently detected in the outer area of neurosphere sections. For comparison, we used a probe for RPTP {lambda} (labeled Ptpru according to the gene nomenclature), which appears to be expressed rather ubiquitously in Nsphs (G). Scale bars = 50 µm. Abbreviations: cond., conditioned; IP, immunoprecipitation; Nsphs, neurospheres.

 
Although neurospheres contain not only neural stem/progenitor cells but also committed precursor populations [47], it has been reported that these various cell classes display some territorial preference within the three-dimensional (3D) structure of neurospheres. The actively cycling neural stem cell population is being localized to the more superficial areas of the neurosphere, and the more differentiated, lineage-committed cell populations are located more toward the core of the neurosphere [48, 49]. To compare the localization of the 473HD epitope and RPTP-β isoforms in the 3D structure of neurospheres, cryosections were prepared and stained with mAb 473HD and KAF13 antibodies. Strong immunoreactivities for mAb 473HD and KAF13 were detected on the peripheral area of neurosphere sections, where most nestin-positive progenitor cells reside, whereas immature βIII-tubulin-positive neurons were found in the core regions (Fig. 3B–3E). In situ hybridization signals using a riboprobe that recognizes the transmembrane isoforms of RPTP-β were specifically detected on the cells positioned preferentially in the outer layers of neurospheres (Fig. 3F, Ptprz1), as opposed to the more ubiquitously expressed receptor protein tyrosine phosphatase {lambda} (Fig. 3G, Ptpru). In addition, immunoreactivities for both mAb 473HD and KAF13 could be visualized to some extent in the central areas of neurospheres. These results suggest that soluble phosphacan is deposited in the core region of neurospheres, where it may diffuse from the outer layers. In contrast, the transmembrane forms of RPTP-β isoforms were expressed mainly on the periphery of neurospheres. Thus, RPTP-β isoforms that carry CS/DS chains that make up the 473HD epitope are expressed by neural stem cells and by committed precursor populations in the neurospheres.

Gene Expression of C/D-STs on the Neurosphere-Forming Cells and Structural Characteristics of Their CS/DS Chains
We next chose to investigate the expression of C/D-STs in the neurosphere model [40], where FGF-2-responsive neural stem cells precede the appearance of EGF-responsive precursors during CNS development [50, 51]. To compare the expression of C/D-STs in both subpopulations of growth factor-responsive neural stem cells, cortical and striatal E13 neurospheres were grown in the presence of EGF alone; FGF-2 plus heparin; or EGF, FGF-2, and heparin. Heparin supports, as an adjuvant, FGF-2-FGFR interactions [52]. The expression levels of C/D-ST mRNAs were estimated by semiquantitative RT-PCR analysis. With the exception of C4ST-3, the expression of all presently known C/D-STs was detected in neurospheres, irrespective of the culture condition (Fig. 4A, 4B). Semiquantitative analysis indicated that expression of C6ST-1 mRNA appeared lower in either EGF- or FGF-2-expanded neurospheres than in neurospheres kept in the presence of both EGF and FGF-2. Expression of GalNAc4S-6ST mRNA appeared to be higher in EGF-expanded than in FGF-2- or EGF and FGF-2-expanded neurospheres. Comparable results were obtained with neurospheres prepared from E13 mouse cerebral cortex and ganglionic eminence. The gene expression levels of the other C/D-ST enzymes did not significantly differ between these culture conditions. To examine the sulfation patterns resulting from the expression of these enzymes in more detail, we next analyzed the disaccharide composition of CS/DS chains produced by neurosphere-forming cells cultured in the presence of different combinations of growth factors (Table 1). Since it has been reported that most CS-PGs are detected in phosphate-buffered saline-soluble extracts of the developing CNS [53], the CS/DS chains were prepared starting from conditioned neurosphere culture media. The proportion of nonsulfated disaccharides in cultures treated with a combination of EGF and FGF-2 was approximately twofold lower than in cases where only EGF or FGF-2 was applied. On the other hand, the proportion of 6-O-sulfated disaccharides in the EGF and FGF-2-treated culture was up to 1.5-fold higher than in cases where each factor was used individually. The proportion of 4-O-sulfated disaccharides was invariant with respect to culture conditions. Disulfated disaccharides such as {Delta}Di-diSD and {Delta}Di-diSE were also detected, as well as CS/DS chains equivalent to those prepared from E13 mouse brain. Notably, the proportion of {Delta}Di-diSD from the EGF plus FGF-2-treated cultures was up to 1.5-fold lower than that from either the EGF- or the FGF-2-treated culture. Furthermore, it should also be noted that the total amount of CS/DS chains per milligram of protein in the EGF plus FGF-2-treated cultures was up to 2.4-fold higher than that in the cultures grown in either EGF or FGF-2 alone.


Figure 4
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  		Figure 4. Semiquantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis of chondroitin/dermatan sulfotransferase (C/D-ST) expression in neurospheres. (A): Cortical (C-cortex) and striatal (G-eminence) neurosphere cultures were established from embryonic day 13 cell suspensions that were grown for 6 days in the following growth factor conditions: EFH, FH, or E, as indicated below each panel. RT-PCR was performed in the linear range with 29–38 cycles (as shown in supplemental online data 3) on cDNAs obtained from neurospheres of all three culture conditions. The amplified products were visualized by electrophoresis on a 1.5% agarose gel containing ethidium bromide. Note that with the exception of C4ST-3, amplicons of the known CS-STs were detected in neurospheres under all three culture conditions. No bands were obtained in control amplifications W/O. (B): The density of PCR band products was semiquantitatively analyzed using the NIH ImageJ program. The ratio of each C/D-ST amplicon versus the corresponding β-actin-band was calculated. To compare the relative expression levels of the three culture conditions, the values obtained for the EFH/β-actin ratio were set as the reference value 1.0. The relative changes are shown as bar histograms (mean ± SEM; n = 3). Note that GalNAc4S–6ST and C6ST-1 mRNA levels were differentially regulated in epidermal growth factor alone, whereas all other CS-STs were not significantly altered independent of the growth factor conditions. Abbreviations: C4ST, chondroitin 4-O-sulfotransferase; C6ST, chondroitin 6-O-sulfotransferase; C-cortex, cerebral cortex; D4ST, dermatan 4-O-sulfotransferase; E, epidermal growth factor alone; EFH, epidermal growth factor and fibroblast growth factor-2 with heparin; FH, fibroblast growth factor-2 with heparin; GalNAc4S–6ST, N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase; G-eminence, ganglionic eminence; UA2OST, uronosyl 2-O-sulfotransferase; W/O, without template.

 


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

 
Localization of the Cells Expressing C/D-STs Genes in the 3D Structure of Neurospheres
As pointed out above, neurospheres represent a complex mixture of cells that display territorial preference, with actively cycling neural stem/progenitor cell populations being localized to the more superficial areas and the more differentiated, lineage-committed cell populations toward the core of the neurosphere [48, 49]. Therefore, we examined the regional distribution of the cells expressing particular C/D-STs by in situ hybridization on neurosphere sections using riboprobes for C4ST-1, D4ST-1, C6ST-1 and -2, and UA2OST. Sections were collected from neurospheres grown from E13 mouse cerebral cortex in the presence of either EGF alone or FGF-2 in conjunction with heparin. Strong signals for all C/D-ST riboprobes examined were visible in the circumference of FGF-2-expanded-neurospheres, whereas the core region displayed lower or nondetectable levels (Fig. 5). Although neurospheres grown in EGF were smaller, they showed similar patterns of C/D-ST expressions, with prominent signals in the outer layers suggestive of neural stem/progenitor cells, as opposed to more differentiated cell types. Comparable signals were recorded in striatal neurospheres (supplemental online Fig. 2).


Figure 5
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  		Figure 5. In situ hybridization for chondroitin/dermatan sulfotransferases (C/D-STs) in cortical Nsphs. Bright-field photomicrographs of Nsph cryosections 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 cortex in medium containing either FGF-2 plus heparin (left two columns) or EGF (right two columns), as indicated at the top of the figure. The sections were hybridized with antisense (A–J) or sense (A'–J') probes. Note that mRNAs of all C/D-STs studied were preferentially expressed in the outer layers of the Nsphs, where the neural stem/progenitor cells reside. Scale bar = 50 µm ([A], applies to all images). Abbreviations: C4ST, chondroitin 4-O-sulfotransferase; C6ST, chondroitin 6-O-sulfotransferase; D4ST, dermatan 4-O-sulfotransferase; E13, embryonic day 13; EGF, epidermal growth factor; FGF, fibroblast growth factor; Nsphs, neurospheres; UA2OST, uronosyl 2-O-sulfotransferase.

 
Effect of Sodium Chlorate on Neurosphere Formation
To examine the cell biological roles of sulfated molecules such as proteoglycans on neurosphere formation, cells from secondary neurospheres of E13 mouse cerebral cortex were cultured in the presence of FGF-2, EGF, and sodium chlorate, an inhibitor of sulfation [54]. As expected, the expression of the 473HD epitope on neurospheres was prominently inhibited when grown in the presence of 30 mM sodium chlorate (Fig. 6A). On the other hand, this treatment did not affect the expression of the core proteins recognized by KAF13, which migrate at comparable levels in SDS-polyacrylamide gel electrophoresis [18]. On the first day of cultivation in the presence of 30 mM sodium chlorate, the viability of plated cells was examined using trypan blue staining. No significant difference was observed between sodium chlorate-treated and control cultures (data not shown). Thus, the addition of sodium chlorate to neurosphere forming assays seemed a promising strategy to examine the specific functions of sulfated structures on glycosaminoglycans for the neural stem/progenitor cell population. Therefore, the self-renewal ability of neural stem/progenitor cells was examined by the clonal density assay [40] in the presence of sodium chlorate. As shown in Figure 6B, the addition of sodium chlorate to culture medium containing FGF-2 and EGF decreased the number of neurospheres in a dose-dependent fashion. The average size of neurospheres also seemed to be decreased by this treatment, indicating a slowed cell cycle (data not shown). Furthermore, sodium chlorate also decreased the number of neurospheres in the presence of EGF alone. Notably, the addition of exogenous heparin to culture medium did not rescue the decrease in neurosphere formation in the EGF-treated cultures (Fig. 6C). To further investigate the functional implications of CS-GAGs on neurosphere formation, we added defined, commercially available chondroitin sulfates to chlorate-treated neurosphere-derived cells to rescue neural stem cell maintenance and self-renewal. At concentrations that had revealed a function for CS-B, CS-D, and CS-E in neurosphere growth and/or proliferation [23], none of the CS chains nor heparin resulted in neurosphere numbers that were comparable to those of untreated control cultures, although an increase was recorded that was not significant (Fig. 6E). Thus, neural stem cell maintenance required sulfation, but the simple addition of defined but randomly sulfated GAGs is not sufficient. However, in heparin-, CS-B-, CS-D-, and CS-E-supplemented cultures, we observed a roughly twofold increase in the size of the neurospheres (Fig. 6D), which indicates functional activity of defined GAGs with respect to survival, growth, or proliferation of neural stem/progenitor cells, as reported previously [23].


Figure 6
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  		Figure 6. Sodium chlorate treatment reduces neurosphere formation. (A): To confirm the decrease of sulfation on chondroitin/dermatan sulfate chains, detergent extracts (20 µg 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 the treatment with sodium chlorate diminished the expression of receptor protein tyrosine phosphatase-β 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 arrow indicates the top of the separating gel. (B, C): Clonal density assays were performed to examine the effect of sodium chlorate on the self-renewal ability of neural stem/progenitor cells. Single-cell suspensions from secondary neurospheres were plated at clonal density (200 cells per cm2) and cultured with FGF-2 plus EGF (square) and EGF alone (circle) in the presence or absence of sodium chlorate for 5 days. The total number of neurospheres was counted and plotted in the bar histograms (mean ± SEM; n = 3). Note that sodium chlorate dose-dependently decreased the number of neurospheres, which were cultured in the presence of FGF-2 and EGF or EGF alone. Also note that in the EGF-treated culture, the sodium chlorate-induced decrease of neurosphere number was not recovered even by the addition of exogenous Hep. (D): Photomicrographs of representative individual 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 µm. (E): Effect of 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 are expressed as mean ± SEM from three independent experiments. Note that neurosphere formation appears to be increased after the addition of defined chondroitin sulfate units, but the neurosphere numbers were not significantly different from those of control cultures (no GAGs; i.e., FGF-2 alone or FGF-2 and Hep). Abbreviations: CS-A, GlcUAβ1–3GalNAc(4-O-sulfate); CS-B, IdoUA(2-O-sulfate){alpha}1–3GalNAc(4-O-sulfate); CS-C, GlcUAβ1–3GalNAc(6-O-sulfate); CS-D, GlcUA(2-O-sulfate)β1–3GalNAc(6-O-sulfate); CS-E, GlcUAβ1–3GalNAc(4, 6-O-disulfate); EGF, epidermal growth factor; FGF, fibroblast growth factor; Hep, heparin.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Multipotent precursor cell populations have been discovered not only in embryonic brain but also in adult brain. It has been suggested that the microenvironment surrounding these cells, the neural stem cell niche, constitutes a crucial milieu for the regulation of their self-renewal and multipotent differentiation capacity [55]. In this context, it seemed worthwhile to study CS-PGs in the neural stem cell niche, because they represent a major component of the extracellular matrix of the brain. In particular, it has been suggested that the sulfation patterns on their glycosaminoglycans play important roles during brain development [56]. In the present study, we demonstrated that several C/D-ST genes are expressed in the neurogenic regions of the embryonic and adult mouse CNS. The synergistic actions of various C/D-STs in an individual cell may underlie the emergence of distinct sulfation patterns within the CS/DS chains. Consistent with this scenario, we recently reported that the 473HD epitope is expressed in the germinal layers of the embryonic 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 isoforms of RPTP-β modified with the 473HD epitope. This epitope, a CS/DS structure recognized by mAb 473HD, includes CS-A, CS-D, and CS-B units [17–19]. Two other mAbs, CS-56 and MO-225, also recognize CS structures that comprise CS-A and CS-D units and are in this respect comparable to mAb 473HD [19]. It could previously be ascertained, however, that the oligosaccharides recognized by each of these individual mAbs undoubtedly possess individual and unique structural characteristics [19]. Maeda et al. [3] have shown that the CS-56 epitope is expressed in the lateral zone adjacent to the lateral ventricle in postnatal mouse brain and that immunoreactivity of MO-225 in this region dramatically increases during development of the CNS. In contrast, expression of the 473HD epitope decreases but is constantly detected in the neurogenic CNS regions, irrespective of the developmental stages [20]. These observations suggest that the occurrence of particular sulfated CS/DS structures is strictly regulated by the expression of various C/D-STs in the neural stem cell niche throughout life.

We detected the gene expression of multiple C/D-STs in neurospheres from E13 mouse cerebral cortex and ganglionic eminence, as well as their expressions in these tissues. It has been postulated that the environment of the neural stem cell niche is mimicked in the outer layer of neurospheres as a consequence 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 outer zone of the sectioned neurosphere, suggesting that the neurosphere culture constitutes an adequate in vitro model to study the possible functions of CS/DS-PGs. We found that the ratio of CS-C/CS-A units in the EGF plus FGF-2-treated neurosphere culture increased 1.4–1.7-fold in comparison with those in the culture treated with either EGF or FGF-2 alone, irrespective of the cellular source. It has been suggested that the proportion of CS-C units decreases during the development of chick and rat brains in consequence of the downregulation of the C6ST-1 gene [1, 25]. In agreement with these reports, the expression of C6ST-1 mRNA appeared more pronounced in the EGF plus FGF-2-expanded neurospheres than in either EGF- or FGF-2-exposed neurospheres. In contrast, the semiquantitative analysis did not reveal prominent differences for the expression of the C6ST-2 gene. Therefore, it seems likely that this proportional increase of the CS-C unit reflects mainly the enzymatic activity of C6ST-1. A positive correlation between active mitosis and the proportional increase of CS-C/CS-A units has been noticed previously [1]. The stimulation with EGF in conjunction with FGF-2 produces the largest size and the highest number of neurospheres from E12–E14 mouse telencephalon [50].

It has been reported that mice lacking the C6ST-1 gene do not display apparent abnormalities, with the exception of a slight decrease of the immature T-lymphocyte population [57]. The presence of CS-C and CS-D units has not been detected in the CS/DS chains prepared from the adult brain of C6ST-1 gene-deficient mice. In this case, however, the situation during embryonic development has not been analyzed [57]. In our study, we detected prominent expression of the C6ST-2 gene in the embryonic germinal and adult subventricular zones. On the basis of available data, however, it cannot be decided whether the lack of C6ST-1 function in the knockout mutant is partially compensated by the enzymatic activities of C6ST-2 in selected areas such as the neural stem cell niche. Alternatively, it may be envisaged that other glycosaminoglycans including HS chains compensate for the decrease of CS-C and CS-D units in the CS/DS chains in vivo. The structural basis for this ability of substitution could reside in the possibility that specific sulfated structures in CS chains share the binding ligands with HS chains, as discussed below.

Neurospheres grown in the presence of EGF and FGF-2 increased the content of CS/DS chains per milligram of proteins in the conditioned culture media, suggesting quantitative alterations on the glycosaminoglycan and/or core protein levels. It has been reported that the expression of the large splice variant of the RPTP-β gene was prominently detected on the mRNA level in the embryonic germinal and postnatal subventricular zones [58, 59]. RPTP-β long and its alternative splice variant phosphacan/DSD-1-PG/6B4-PG can interact with heparin-binding growth-associated molecule (HB-GAM)/pleiotrophin [60]. The affinities of this interaction are positively correlated with the proportion of CS-D units in the CS chains [3]. We have recently provided evidence that the binding site of HB-GAM in CS/DS chains overlaps with the 473HD epitope [61]. CS chains that contain a high proportion of CS-E units from squid cartilage display high-affinity binding for various heparin-binding factors [12]. Zou et al. [24] have reported a ligand affinity binding experiment using a mixture of CS/DS chains prepared from E13 mouse brain. In their report, fractions rich in CS-E units were eluted at 0.7 M NaCl from a midkine-affinity column [24]. In the present study, we demonstrated that neurosphere-forming cells possess the capacity to synthesize CS/DS chains that contain significant amounts of disulfated disaccharides such as the CS-D and CS-E units. Unlike the case of the CS-C unit, the proportion of CS-D units detectable in the EGF plus FGF-2-treated neurosphere cultures was up to 1.5-fold lower than upon treatment with either EGF or FGF-2 alone. As outlined above, the proportional increase of CS-D units in the CS/DS hybrid chains is likely to affect the binding affinity for HB-GAM. Interestingly, it has been reported that HB-GAM inhibits cell proliferation and enhances differentiation of neural stem/progenitor cells [62]. We have recently reported that fewer neurospheres were observed in neurosphere cultures grown in the presence of mAb 473HD [20]. Our current results revealed that the 473HD epitope was carried by RPTP-β long and phosphacan. The binding of this antibody to RPTP-β long on cell surface may affect its phosphatase activities and consequently intracellular signal transduction, as reported for the binding of HB-GAM to its high-affinity cell surface receptor, RPTP-β long [63]. Specific structural motifs in DS chains purified from porcine intestinal mucosa are required for their interactions with FGF-2 and -7 [64]. Although the CS-B unit was not detected in the majority of our samples, its absence may reflect limitations of the detection method. Indeed, the epitope of mAb 473HD could be documented by immunohistochemistry both in neurospheres and in embryonic forebrain [20]; Figure 3).

In this study, we showed that addition of sodium chlorate to neurosphere cultures attenuated the expression of the 473HD epitope on neurosphere-forming cells. Ida et al. [23] have reported that addition of exogenous CS-B and CS-E into neurosphere culture medium significantly enhances FGF-2-mediated cell proliferation of neural stem/progenitor cells. Interestingly, the culture of neurospheres in the continuous presence of sodium chlorate significantly decreased the number of both FGF-2 and EGF-expanded neurospheres in a dose-dependent fashion. A similar effect was observed even when EGF alone was used as exogenous mitogen. Different from the situation with FGF signaling, HS/heparin is not involved in the binding of EGF to the EGF receptor and its stabilization [65]. In the course of cell culture, neurosphere-forming cells may, however, endogenously produce some heparin-binding growth factors, as reported in the case of FGF-2 [66]. In our hands, the maintenance of neural stem cells in the presence of FGF-2 critically depended on endogenous sulfation. Because the addition of purified GAG chains from various non-CNS sources could not fully reverse the effect of chlorate treatment on neurosphere number, we assume that the sulfation pattern of CNS GAG chains is different. Thus, neural stem cell maintenance might require a sulfation code, as has been proposed for neurite branching in the nematode [67]. We propose that such a hypothetical code would differ for neural stem cell self-renewal as opposed to their growth and proliferation behavior since the latter could be rescued by defined CS-GAGs and heparin. In this way, the patterned level of sulfation in the glycosaminoglycans of the neural stem cell niche may allow or even instruct neural stem cell behavior by modulating the activities of endogenous growth factors.

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


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Dr. A. Horvat-Bröcker, Department of Cell Morphology and Molecular Neurobiology, Ruhr-University Bochum, Bochum, Germany, for the support of in situ hybridization and all other members of the department for insightful suggestions and discussions. This work was supported by the German Research Council (Deutsche Forschungsgemeinschaft priority program SPP 1109 to A.v.H. and A.F.) and by the German Ministry of Research and Technology (Grant BMBF 01GN0503 to A.F.). K.A. was supported by a grant from the German Academic Exchange Program (Deutscher Akademischer Austauschdienst). The work performed in Japan was supported in part by HAITEKU (2004–2008) from the Japan Private School Promotion Foundation, Grant in-Aid for Exploratory Research 17659020 and the Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency. K.A. and A.v.H. contributed equally to this work. K.A. is currently affiliated with the Department of Biotechnology, Kyoto Sangyo University, Kita-ku, Kyoto, Japan.


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 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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