Dermatan sulfotransferase Chst14/D4st1, but notchondroitin sulfotransferase Chst11/C4st1, regulatesproliferation and neurogenesis of neuralprogenitor cells

Shan Bian1,2,*, Nuray Akyuz1,*, Christian Bernreuther1,3,*, Gabriele Loers1, Ewa Laczynska1, Igor Jakovcevski1

and Melitta Schachner1,4,5,`

1Zentrum fur Molekulare Neurobiologie, Universitatskrankenhaus Hamburg-Eppendorf, Universitat Hamburg, Martinistr. 52, D-20246 Hamburg,Germany2Fakultat fur Biologie, Albert-Ludwigs-Universitat Freiburg, Schaenzlestr. 1, D-79104 Freiburg, Germany3Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany4Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA5Center for Neuroscience, Shantou University Medical College, 22 Xin Ling Road, Shantou 515041, P.R. China

*These authors contributed equally to this work.`Author for correspondence (schachner@stu.edu.cn)

Accepted 5 July 2011Journal of Cell Science 124, 4051–4063� 2011. Published by The Company of Biologists Ltddoi: 10.1242/jcs.088120

SummaryChondroitin sulfates (CSs) and dermatan sulfates (DSs) are enriched in the microenvironment of neural stem cells (NSCs) duringdevelopment and in the adult neurogenic niche, and have been implicated in mechanisms governing neural precursor migration,proliferation and differentiation. In contrast to previous studies, in which a chondroitinaseABC-dependent unselective deglycosylation

of both CSs and DSs was performed, we used chondroitin 4-O-sulfotransferase-1 (Chst11/C4st1)- and dermatan 4-O-sulfotransferase-1(Chst14/D4st1)-deficient NSCs specific for CSs and DSs, respectively, to investigate the involvement of specific sulfation profiles of CSand DS chains, and thus the potentially distinct roles of CSs and DSs in NSC biology. In comparison to wild-type controls, deficiency for

Chst14 resulted in decreased neurogenesis and diminished proliferation of NSCs accompanied by increased expression of GLAST anddecreased expression of Mash-1, and an upregulation of the expression of the receptors for fibroblast growth factor-2 (FGF-2) andepidermal growth factor (EGF). By contrast, deficiency in Chst11 did not influence NSC proliferation, migration or differentiation.

These observations indicate for the first time that CSs and DSs play distinct roles in the self-renewal and differentiation of NSCs.

Key words: Chondroitin sulfate, Dermatan sulfate, Sulfotransferases, Neural stem cells, Proliferation, Neurogenesis

IntroductionSelf-renewal and multipotency are two major characteristics of

neural stem cells (NSCs), which are regulated by a combination

of intrinsic pathways modifying the ligand dependent activity

of transcriptional factors and signaling pathways in the

microenvironment of NSCs (Li and Zhao, 2008; Miller and

Gauthier, 2007; Shi et al., 2008). Chondroitin sulfate (CS) and

dermatan sulfate (DS) polysaccharide chains are important

glycosaminoglycans (GAGs) of the extracellular matrix (ECM).

They are composed of the alternating disaccharides N-

acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) or

iduronic acid (IdoA) (Bulow and Hobert, 2006), which can be

sulfated at different positions and have been shown to be present

in the neurogenic regions of the embryonic and adult central

nervous system (Ida et al., 2006; Kabos et al., 2004; Sugahara

and Mikami, 2007).

CSs and DSs have important roles in neural precursor

proliferation, neuronal differentiation, migration and

neuroprotection (Sato et al., 2008; Sirko et al., 2007; Sugahara

and Mikami, 2007; von Holst et al., 2006). Previous studies

indicated that the functions of CSs and DSs on NSCs are

determined by their sulfation patterns. A sulfate-dependent

arrangement of the cell surface-associated CS or DS motifs

recognized by the monoclonal antibody 473HD has been

observed in a subpopulation of radial glial cells and plays an

important role in neurosphere formation (von Holst et al., 2006).

Treatment of NSCs from the embryonic mouse telencephalon

with chondroitinaseABC (ChaseABC), which unselectively

degrades CSs and DSs, resulted in diminished proliferation and

impaired neuronal differentiation of NSCs, with a concomitant

increase in astrogenesis (Sirko et al., 2007).

Because CSs and DSs are composed of various repeats of

sulfated disaccharide units generating CS-A, CS-B (DS), CS-C,

CS-D, CS-E and CS-H patterns, we addressed which specific CS

and DS patterns generated by NSCs themselves – and not by their

microenvironment in the neurogenic niche – influence the

properties of these cells in an autocrine or paracrine fashion. To

investigate the roles of different endogenous CS and DS motifs, we

generated transgenic mice deficient in the key enzymes that

distinctly affect the synthesis of CS versus DS chains.

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The sulfation of CSs and DSs is mediated by several

sulfotransferases. Among the 4-O-sulfotransferases involved in

the sulfation of the carbon 4 position of GalNAc in CSs and DSs,

chondroitin 4-O-sulfotransferase-1 (Chst11/C4st1) (Okuda et al.,

2000b) uses predominantly GalNAc residues flanked by GlcA to

generate CS chains. By contrast, dermatan 4-O-sulfotransferase-1

(Chst14/D4st1) (Evers et al., 2001) far more efficiently transfers

sulfate to GalNAc residues flanked by IdoA to generate DSs

(Mikami et al., 2003). Both Chst11 and Chst14 are expressed

in the neurogenic regions of the embryonic and adult central

nervous system as well as in cultures of neurospheres (Akita et al.,

2008). Using Chst11- and Chst14-deficient mice, we investigated

the roles of these two enzymes in NSC biology and elucidated

the functions of endogenous CS and DS patterns independently

in CNS development, in contrast to the previous ChaseABC-

based investigations that did not discriminate between the

effects of CSs and DSs. Importantly, in addition to previous

investigations, our study addresses the distinct roles of CSs and

DSs not only in vitro, but also in vivo. Our results show that

Chst14 deficiency results in impaired neuronal differentiation

and diminished NSC proliferation both in vitro and in vivo,

associated with an upregulation of the expression of growth

factor receptors and changes in distinct subpopulations of radial

glial cells. By contrast, Chst11 deficiency does not show any

influence on NSC properties. These observations indicate for the

first time that DSs and CSs play distinct roles in the biology of

NSCs.

ResultsDifferential gene expression of the HNK-1 sulfotransferase

family members in neurospheres derived from Chst112/2

or Chst142/2 NSCs

To elucidate whether the ablation of Chst11 or Chst14 causes a

dysregulation of the expression of other members of the HNK-1

sulfotransferase family in NSCs, differential gene expression of

the HNK-1 sulfotransferase family members Chst8/GalNAc-4-st1

(Hiraoka et al., 2001; Okuda et al., 2000a; Okuda et al., 2003; Xia

et al., 2000), Chst9/GalNAc-4-st2 (Hiraoka et al., 2001; Kang

et al., 2001; Okuda et al., 2003), Chst10/HNK1-st (Bakker et al.,

1997; Ong et al., 1998), Chst11/C4st1 (Hiraoka et al., 2000;

Okuda et al., 2000b; Yamauchi et al., 2000), Chst12/C4st2

(Hiraoka et al., 2000), Chst13/C4st3 (Kang et al., 2002) and

Chst14/D4st1 (Evers et al., 2001) was analyzed in NSCs derived

from the ganglionic eminence of E13.5 embryonic brains of

Chst112/2 and Chst142/2 mice versus Chst11+/+ and Chst14+/+

littermates by qRT-PCR.

Expression of Chst8 and Chst13 was below the detection

limit in all groups. As expected, in Chst112/2 NSCs, mRNA

expression of Chst11 was reduced below the detection limit

(Fig. 1A). No differences in expression were detected for Chst9

and Chst10 in NSCs derived from Chst112/2 mice and Chst11+/+

mice, whereas mRNA levels of Chst12 and Chst14 were

significantly reduced in Chst112/2 NSCs versus Chst11+/+

NSCs (Chst12, 0.55±0.04 versus 1±0.23; n54; P,0.01;

Chst14, 0.75±0.08 versus 1±0.14; n54; P,0.05). Furthermore,

Fig. 1. Differential gene expression of the HNK-1

sulfotransferase family members and 473HD epitope in

Chst112/2 and Chst142/2 NSCs. Expression of the HNK-1

sulfotransferase family members in NSCs isolated from Chst11+/+

(+/+) or Chst14+/+ (+/+) and Chst112/2 (2/2) (A) or Chst142/2

(2/2) (B) embryos at day E13.5 was analyzed by qRT-PCR. The

average of the expression levels of these genes in +/+ animals,

respectively, was set to 1.0 and relative expression in homozygous

2/2 NSCs was calculated (mean ± s.d.; n54). (C) Western blot

analysis of expression of the 473HD epitope in neurospheres

isolated from Chst112/2 (mean ± s.d.; n53) or Chst142/2 (mean ±

s.d.; n53) and Chst11+/+ and Chst14+/+ littermate mice at

developmental stage E13.5 is shown. An actin antibody was used as

a control for protein loading. (D) Quantification of western blot

measuring expression of the 473HD epitope in neurospheres. Mean

arbitrary units (AU) are shown. The average of expression levels of

473HD epitope in +/+ animals was set to 1.0 and relative expression

was calculated accordingly. Student’s t-test was performed for

statistical analysis. *P,0.05; **P,0.01; ***P,0.001. n.d.,

not detectable.

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in Chst142/2 NSCs, the expression of Chst14 mRNA wasreduced below the detection limit (Fig. 1B) whereas the mRNAlevels of other HNK-1-ST family members were not altered when

compared with NSCs derived from their Chst14+/+ littermates.These results indicate that other HNK-1 sulfotransferase familymembers are not upregulated in a compensatory manner in

Chst112/2 or Chst142/2 versus Chst11+/+ or Chst14+/+ NSCs.

Expression of the 473HD epitope in Chst112/2 orChst142/2 NSCs

A unique CS–DS structure containing CS-A, CS-D and DSpatterns is recognized by the monoclonal antibody 473HD

(Clement et al., 1998; Ito et al., 2005) as a marker for radialglial cells (Kabos et al., 2004). To investigate the influenceof Chst11 or Chst14 depletion on the expression of CS–DS

structures in neurospheres, western blot analysis was performedusing the 473HD antibody on protein extracts of neurospheresoriginating from the embryonic forebrain of Chst112/2 and

Chst142/2 mice in comparison with their wild-type littermates.After ablation of Chst11, expression of the 473HD epitope wasreduced to 11±6% compared with Chst11+/+ neurospheres

(Fig. 1C,D). In neurospheres generated from Chst142/2 mice,expression of the 473HD epitope was decreased to 58±7% ofthe expression level of Chst14+/+ neurospheres (Fig. 1C,D).This result indicates that deficiency in both Chst11 and Chst14

alters CS–DS structures.

Deficiency in Chst14, but not Chst11, impairs proliferationof NSCs

Because the 473HD epitope is important for neurosphere

formation by NSCs (von Holst et al., 2006) and deficiencyeither in Chst11 or Chst14 reduced expression of the 473HDepitope, NSCs were cultured at a low density in the presence of

growth factors and the number of neurospheres was countedwith regard to size to examine whether the ablation of Chst11 orChst14, respectively, influences neurosphere formation. NSCsderived from Chst142/2 mice generated fewer neurospheres

with a diameter larger than 200 mm (30±8 versus 53±16;Fig. 2B) and with a diameter between 100 and 200 mm (85±21versus 166±18; Fig. 2B) in comparison with Chst14+/+ NSCs.

The total number of neurospheres was not different betweengenotypes. Deficiency in Chst11 did not affect the size ofneurospheres (Fig. 2A).

To investigate whether this difference in neurosphere

formation is caused by differences in cell proliferation orapoptosis, we immunohistochemically analyzed the numbersof Ki67+ and caspase-3+ cells in sections of neurospheres.Chst112/2 neurospheres contained similar numbers of Ki67+

cells as found in Chst11+/+ neurospheres (53±2% versus 53±3%;Fig. 2C), whereas Chst142/2 neurospheres contained fewerKi67+ cells than Chst14+/+ neurospheres did (47±4% versus

56±1%; Fig. 2D). No difference was detected in the numbers ofcaspase-3+ cells between Chst112/2 and Chst142/2 neurospheres(Fig. 2E,F) compared with the corresponding wild-type

neurospheres.

To confirm the effect of Chst14 on proliferation in vitro, NSCswere cultured in adherent cell culture under three differentculture conditions: in medium containing only FGF-2, only EGF,

or both FGF-2 and EGF. After culturing for 24 hours andlabeling with BrdU for 8 hours, Chst112/2 NSCs showed thesame proliferative activity as wild-type controls (Fig. 3A,B),

whereas Chst142/2 NSCs showed decreased numbers of BrdU+

cells (Fig. 3A,C) under the three culture conditions (FGF-

2+EGF, 59±2% versus 70±1%; FGF-2, 49±3% versus 54±3%;

EGF, 52±2% versus 60±3% BrdU+ cells, Chst142/2 versus

Chst14+/+ NSCs), confirming the results obtained in

neurospheres. Thus, DS structures modified by Chst14 but not

CS structures modified by Chst11 play important roles in self-

renewal and proliferation of NSCs.

To analyze whether the effects of Chst14 on proliferation

of NSCs are also seen in vivo, BrdU was administrated

intraperitoneally to pregnant mice at E13.5 and to adult mice to

label the neurogenic regions. Twenty four hours after BrdU

labeling, the density of BrdU+ proliferating cells in the

embryonic ganglionic eminence was slightly, but statistically

not significantly, reduced in Chst14 2/2 embryos when compared

with Chst14+/+ controls (supplementary material Fig. S1). In

adult mice, 4 hours after BrdU labeling, the density of BrdU+

proliferating neural progenitors was reduced in Chst142/2 mice

in comparison with control mice, both in the dentate gyrus of the

hippocampus (58±4 cells/mm2 in Chst14 +/+ versus 39±2 cells/

mm2 in Chst142/2 mice; Fig. 4A,B) and in the subventricular

zone (1777±104 cells/mm2 in Chst14+/+ versus 1321±280 cells/

mm2 in Chst142/2 mice, Fig. 4C,D). In the olfactory bulb, 4

weeks after BrdU labeling, Chst142/2 mice had a lower density

of BrdU+ cells in all regions as well as the total area of the

olfactory bulb compared with Chst14+/+ mice, but only in central

regions of the olfactory bulb (granule cell layer, internal

plexiform layer and mitral cell layer) the difference was

significant (supplementary material Fig. S2). Thus, DS patterns

modified by Chst14 influence proliferation of NSCs in vitro and

in vivo.

Finally, to address the question of whether the reduced cell

proliferation had an effect on neuronal numbers in adult

Chst142/2 mice, we analyzed neuron numbers in several brain

structures. No differences were found in the volume and

thickness of the motor cortex and the density of NeuN+

neurons and S100+ astrocytes between Chst142/2 and Chst14+/+

mice (supplementary material Fig. S3). The volumes of the CA1

and dentate gyrus regions of hippocampus were also analyzed,

and no difference between the two groups of mice was revealed

(supplementary material Fig. S4). Furthermore, the analysis

of the density of the principal neurons in the hippocampal

subregions CA1 (pyramidal cells) and dentate gyrus (granule

cells) also showed no significant difference between the

genotypes (supplementary material Fig. S4). Thus,

morphometrical analysis did not reveal any differences between

adult Chst142/2 and Chst14+/+ mice.

Ablation of Chst14 results in upregulation of growth factor

receptors in neurospheres

Because Chst14 deficiency, but not Chst11 deficiency, negatively

influences proliferation of NSCs cultured in the presence of FGF-

2, EGF, or combined FGF-2 and EGF, expression of growth

factor receptors in neurospheres was analyzed. Western blot

analysis revealed that Chst142/2 neurospheres expressed higher

levels of epidermal growth factor receptor (EGFR) (Fig. 3D) and

fibroblast growth factor receptor 1 (FGFR-1) (Fig. 3E) when

compared with Chst14+/+ neurospheres. By contrast, no

differences in expression of EGFR and FGFR-1 were observed

in Chst112/2 versus Chst11+/+ neurospheres (Fig. 3D,E).

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Deficiency in Chst14, but not Chst11, influencesneurogenesis

To investigate the effects of CS and DS patterns modified byChst11 or Chst14, respectively, on neural fate decision in vitro,we analyzed the percentages of tubulin-bIII+ neurons, GFAP+

astrocytes and CNPase+ oligodendrocytes 7 days after induction of

differentiation by growth factor withdrawal (Fig. 5A). A reduction

in the percentage of neurons among total cells was observed in

Chst142/2 versus Chst14+/+ cells [12.2±1.6% versus 16.7±1.2%

(Fig. 5C)], whereas the percentage of GFAP+ astrocytes was

Fig. 2. Chst14 deficiency impairs

neurosphere formation, whereas Chst11

deficiency does not. (A) Photomicrographs and

size-dependent quantification illustrating the

formation of neurospheres cultured from

Chst112/2 NSCs in comparison with Chst11+/+

NSCs (mean ± s.d.; n53). Note that deficiency

in Chst11/C4st1 does not influence neurosphere

formation. (B) Photomicrographs and size-

dependent quantification illustrating the

formation of neurospheres generated from

Chst142/2 NSCs in comparison to Chst14+/+

NSCs (mean ± s.d.; n54). Note that Chst142/2

NSCs generate fewer large-sized neurospheres

(diameter 100–200 mm and .200 mm) than

Chst14+/+ cells.

(C) Photomicrographs and quantification of

Ki67+ proliferating cells (red) in neurospheres

cultured from Chst112/2 NSCs and Chst11+/+

NSCs (mean ± s.d.; n53). Nuclei were

counterstained with DAPI (blue). Note that

Chst112/2 neurospheres contain a similar

percentage of Ki67+ cells as Chst11+/+

neurospheres. (D) Photomicrographs and

quantification of Ki67+ proliferating cells in

neurospheres cultured from Chst142/2 NSCs

and Chst14+/+ NSCs (mean ± s.d.; n53). Note

that Chst142/2 neurospheres contain fewer

Ki67+ cells than seen in Chst14+/+

neurospheres. (E) Photomicrographs and

quantification of immunostaining of

neurospheres cultured from Chst112/2 NSCs

and Chst11+/+ NSCs (mean ± s.d.; n53) using a

caspase-3 antibody (green). Nuclei are

counterstained with DAPI (blue).

(F) Photomicrographs and quantification of

immunostaining of neurospheres generated

from Chst142/2 NSCs and Chst14+/+ NSCs

(mean ± s.d.; n53) using a caspase-3 antibody.

Statistical significance was assessed by

Student’s t-test. *P,0.05; **P,0.01. Scale

bar: 100 mm. TN, total number.

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slightly but not significantly increased in Chst142/2 compared

with Chst14+/+ cells (Fig. 5C). Oligodendroglial differentiation

was not different between the genotypes (Fig. 5C). No differences

in neural differentiation were observed between Chst112/2 and

Chst11–/– cells (Fig. 5B).

We also analyzed the effects of Chst14 deficiency on neuronal

differentiation in vivo. The percentage of cells positive for both

tubulin-bIII and BrdU (tubulin-bIII+BrdU+) among the total

number of BrdU+ cells in the ganglionic eminence of E14.5

embryonic brains 24 hours after BrdU administration was not

different in Chst142/2 mice versus Chst14+/+ littermates

(supplementary material Fig. S1).

Furthermore, neurogenesis was assessed in the adult CNS in

vivo. In the olfactory bulb, the density and the percentage of

NeuN+ neurons and GFAP+ astrocytes among the total number of

BrdU+ cells (Fig. 6A–D) were not different between Chst142/2

and Chst14+/+ mice 28 days after BrdU injection. By contrast,

immunohistochemical analysis revealed a decreased density of

Dcx+ newborn neurons in the dentate gyrus of the hippocampus

(Fig. 6E) in Chst142/2 versus Chst14+/+ mice (620±67 cells/mm2

versus 736±55 cells/mm2; Fig. 6F), whereas 4 weeks after BrdU

injection, no difference in the number of NeuN+BrdU+ cells was

observed between the genotypes (Fig. 6G). Thus, Chst14 might

accelerate adult hippocampal neurogenesis in vivo, but does not

alter the number of newborn NeuN+BrdU+ neurons. The effect of

Chst11 depletion on neurogenesis could not be investigated in

adult animals, because Chst112/2 mice die at birth.

To investigate whether the differences in neuronal

differentiation observed in vitro and in vivo were influenced by

distinct mechanisms of apoptosis in Chst142/2 versus Chst14+/+

mice or Chst112/2 versus Chst11+/+ mice, the percentage of

caspase-3+ cells was measured in vitro 7 days after induction of

differentiation by withdrawal of growth factors (supplementary

material Fig. S5), as well as in the olfactory bulb in vivo

Fig. 3. Chst14 deficiency diminishes NSC proliferation and upregulates expression of growth factor receptors, whereas Chst11 deficiency does not.

(A) Photomicrographs of dissociated NSCs from Chst112/2 or Chst142/2 (2/2) mice, and Chst11+/+ and Chst14+/+ mice (+/+) cultured in the presence of FGF-2,

EGF, or FGF-2 and EFG together. A BrdU antibody was used to label the proliferating cells (red). Cell nuclei were counterstained with DAPI (blue).

(B,C) Quantification of the percentage of BrdU+DAPI+ cells cultured in FGF-2, EGF, or FGF-2 and EFG together. Chst142/2 cells (C) have fewer BrdU+ cells in

comparison to those in Chst14+/+ (mean ± s.d.; n53), whereas Chst112/2 cells (B) are not different from Chst11+/+ cells (mean ± s.d.; n53). (D) An EGFR

antibody was applied to analyze expression of the EGF receptor during neurosphere formation of NSCs. Neurospheres generated from Chst142/2 mice express

more EGFR in comparison to Chst14+/+ neurospheres (mean ± s.d.; n54), whereas the slightly enhanced expression of EGFR detected in Chst112/2 neurospheres

when compared with Chst11+/+ neurospheres was statistically not significant (mean ± s.d.; n53). (E) An FGFR-1 antibody was applied to analyze expression of

the FGF-2 receptor in neurospheres. Neurospheres generated from Chst142/2 mice express more FGFR-1 than Chst14+/+ neurospheres (mean ± s.d.; n53),

whereas Chst112/2 neurospheres express a similar amount as Chst11+/+ cells (mean ± s.d.; n53). For western blot analysis, an actin antibody was used to

demonstrate equal protein loading for all samples. Mean arbitrary units (AU) are shown. The average expression levels of all target proteins in Chst11+/+ and

Chst14+/+ neurospheres was set to 1.0 and relative expression in Chst11- and Chst14-deficient NSCs was calculated. Statistical significance was assessed by

Student’s t-test. *P,0.05. Scale bar: 100 mm.

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(supplementary material Fig. S6). No differences in the

percentage of caspase-3+ cells were observed betweenChst112/2 or Chst142/2 and the respective wild-type controlsin vitro or in vivo, indicating that reduced neurogenesis in

Chst142/2 NSCs was not due to increased apoptosis duringdifferentiation.

Effects of Chst14 and Chst11 on migration of NSCsThe effects of Chst11 or Chst14 deficiency on migration of NSCs

were examined in vitro by measuring the distance of cells thathad migrated from neurospheres 4 and 24 hours after seeding(supplementary material Fig. S7). No differences were observedin cell migration from Chst112/2 or Chst142/2 compared with

Chst11+/+ and Chst14+/+ neurospheres. Furthermore, migration ofNSCs in the dentate gyrus (supplementary material Fig. S8) andolfactory bulb (supplementary material Fig. S2) did not differ

between adult Chst142/2 and Chst14+/+ mice.

Deficiency in Chst14 influences the composition of distinctsubpopulations of radial glial cellsPrevious studies showed that cleavage of CS-GAGs by

ChaseABC reduced proliferation and neuronal differentiation ofNSCs and increased astrocytic differentiation, associated with achange in the composition of subpopulations of radial glial cells:

After removal of the CSs and DSs, neurospheres contained fewerBLBP+ and nestin+ radial glial cells and more GLAST+ and RC2+

radial glial cells (Sirko et al., 2007). In the present study,

expression levels of different radial glial markers weredetermined in Chst112/2 and Chst142/2 neurospheres.Expression of the transcription factor Mash-1 was decreased to

0.66±0.28% in Chst142/2 versus control neurospheres, whereasGLAST expression was increased 1.34±0.29-fold in Chst142/2

versus Chst14+/+ neurospheres (Fig. 7A,C). The expression of

nestin was not altered. Immunohistochemical analysis ofneurospheres revealed increased percentages of GLAST+ cellsin Chst142/2 neurospheres versus Chst14+/+ neurospheres(40.7±4.8% versus 29.8±3.8%; Fig. 7D,E), although no

remarkable increase was detected in the percentage of RC2+

cells. None of the investigated radial glial cell markers wasdifferentially expressed in Chst112/2 neurospheres compared

with Chst11+/+ neurospheres (Fig. 7A,B). Thus, deficiency inChst14, but not Chst11, alters the composition of distinctsubpopulations of radial glial cells.

DiscussionPrevious studies have shown that multiple chondroitin anddermatan sulfotransferases are expressed in the neurogenic

regions of the embryonic and adult CNS and that complex CSand DS chains play important roles in NSC biology (Akita et al.,2008). Two of those sulfotransferases, Chst11/C4st1 and Chst14/D4st1, belonging to the HNK1-ST family, target different GAG

motifs: Chst11 preferentially sulfates GalNAc residues flankedby GlcA and is therefore involved in CS biosynthesis. Chst14mainly targets GalNAc residues flanked by IdoA and is essential

for the formation of iduronic acid blocks in DS sulfation patterns.Chst14 is indispensable in the biosynthesis of functional sulfationdomains in DSs and can thus not be compensated by other 4-O-

sulfotransferases (Pacheco et al., 2009). Therefore, investigationsof NSCs from Chst112/2 and Chst142/2 mice were expected toprovide insights into the roles of different endogenous CS and DS

Fig. 4. Chst14 deficiency influences

proliferation of NSCs in the dentate gyrus and

subventricular zone of adult brains.

(A–D) Photomicrographs and quantification of

immunohistochemical staining of mouse brain

cryosections from 2- to 3-month-old mice

injected with a single dose of BrdU 4 hours

before sacrifice. Proliferating cells are labeled

with antibodies against BrdU (red) and cell nuclei

are counterstained with bisbenzimide (blue). The

dividing neural precursors in the dentate gyrus

(DG; A) and subventricular zone (SVZ; C) were

identified with a BrdU antibody. Quantification

of the density of BrdU+ cells in the dentate gyrus

(B) and the subventricular zone (D) show a

reduction in Chst142/2 mice compared with

Chst14+/+ mice (mean ± s.d.; n54). Statistical

significance was assessed by Student’s t-test.

*P,0.05. Scale bars: 100 mm.

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sulfation patterns in neural development. The consequences

of this depletion for NSC biology were analyzed in view of

possible compensatory mechanisms exerted by other HNK-1

sulfotransferase family members. Neurospheres derived from

the ganglionic eminence of E13.5 Chst112/2 mice exhibited a

reduced mRNA expression of Chst12 and Chst14, whereas no

differences of other HNK-1 sulfotransferase family members

were detected between Chst142/2 and Chst14+/+ neurospheres.

Because the genes encoding Chst11, Chst12 and Chst14 are

located on different chromosomes, a positional effect at the

genomic level can be excluded. Furthermore, potential feedback

mechanisms among members of the HNK-1 sulfotransferase

family have not yet been described. Based on the similar

substrate specificity of Chst11, Chst12 and Chst14, and the

importance of Chst11 in sulfation of CS chains, it is possible

that chondroitin sulphate proteoglycans (CSPGs) modified by

Chst11 negatively influence the expression of Chst12 and

Chst14.

CS and DS chains show variation in length, arrangement

of repeating disaccharide units and sulfation patterns (Sugahara

and Yamada, 2000). Studies on the dissaccharides of CSs andDSs in the brain indicated that blocks of IdoA, which are the

principal structures of DS patterns, are dramatically increased

during development of the cerebellum, accompanied by a

downregulation of CS patterns (Mitsunaga et al., 2006). These

observations suggest that DS chains play important roles in CNS

development. Recently, the 473HD epitope, a unique CS–DSstructure recognized by the antibody 473HD, was shown to be

expressed in the germinative ventricular zone of E13.5 mouse

brains and in neurospheres derived from embryonic forebrains.

Fig. 5. Chst142/2 NSCs, but not Chst112/2 NSCs, show impaired

neuronal differentiation. (A) Distribution of neurons, astrocytes and

oligodendrocytes differentiated from Chst112/2 or Chst142/2 NSCs

and from Chst11+/+ and Chst14+/+ NSCs were analyzed with

antibodies against different markers: Tuj for neurons (shown in red),

GFAP for astrocytes (green) and CNPase for oligodendrocytes (red).

Nuclei were counterstained with DAPI (blue). (B) Quantification of

differentiation of NSCs derived from Chst112/2 mice and Chst11+/+

mice. Note that NSCs derived from Chst112/2 mice are not different

from Chst11+/+ NSCs in neural differentiation (mean ± s.d.; n53).

(C) Quantification of differentiation of NSCs derived from

Chst142/2 mice and Chst14+/+ mice. Neuronal differentiation is

decreased in Chst142/2 NSCs versus Chst14+/+ NSCs (mean ± s.d.;

n53). Statistical significance was assessed by Student’s t-test.

**P,0.01. Scale bars: 100 mm.

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This epitope, consisting of CS-A, CS-D and DS patterns, has

been shown to be a marker for radial glial cells and to be

crucial for neurosphere formation (von Holst et al., 2006).

Immunoisolated 473HD+ NSCs formed significantly more

neurospheres than control NSCs that had not been enriched for

473HD expression. In the present study, expression of the 473HD

epitope was decreased both in Chst112/2 and Chst142/2 NSCs,

which is consistent with the view that Chst11 and Chst14 are

involved in the biosynthesis of the 473HD epitope. Comparison

between NSCs from Chst112/2 or Chst142/2 mice and NSCs

from their wild-type littermates showed that the expression of the

473HD epitope was decreased by 90% in Chst112/2 NSCs and

by 42% in Chst142/2 NSCs. Ito and colleagues (2005) showed

that the 473HD epitope consists of A–D and/or D–A structures.

The A-unit defines the monosulfated GlcUA-GalNAc(4S) and

the D-unit defines the disulfated GlcUA(2S)–GalNAc(6S)

structures. Additionally, these authors found that the 473HD

epitope was eliminated from DSD-1-PG (also known as protein

tyrosine phosphatase, receptor type Z, polypeptide 1) by

digestion with chondroitinaseB, suggesting the close association

of L-iduronic acid with the 473HD epitope. They also discussed

the probability that the iduronic acid was not directly included in

the epitope, but located on the reducing end in the parent chain.

This could explain why we detect a 90% reduction of the 473HD

epitope expression in Chst112/2 NSCs, because Chst11 is the

predominant enzyme in the production of the A-unit which is

present in the 473HD epitope core, and see only a 50% reduction

in Chst142/2 NSCs because Chst14 is the predominant enzyme

in the production of the B-unit, which is probably exposed on the

reducing end in the parent chain and not on the epitope core

itself.

CS and DS GAGs are important for the self-renewal and

proliferation of NSCs. Removal of CSs and DSs by ChaseABC

treatment, which unselectively cleaves CSs and DSs from CS-

DSPGs, impaired neurosphere formation, decreased proliferation

of NSCs in vitro and in the ventricular zone of E14.5 mouse

forebrains (Sirko et al., 2007), and diminished in vitro

proliferation of NSCs derived from the E16 rat forebrain (Gu

et al., 2009). Although the influence of different CS and DS

patterns on NSCs has been reported by studying isolated CSs

Fig. 6. Chst142/2 mice show decreased neurogenesis in the dentate

gyrus, but no differences in neuronal and astroglial differentiation

are observed in the olfactory bulb. (A) Confocal image of the granule

cell layer (GCL) double-stained with a BrdU antibody (shown in red)

and a NeuN antibody (shown in blue) to quantify neuronal

differentiation. (B) Laser-scanning microscopy of the GCL double-

stained with a BrdU antibody (shown in red) and a GFAP antibody

(shown in green) to quantify astrogenesis. (C) Quantification of the

ratios of newborn neurons and astrocytes in the olfactory bulb

differentiated from NSCs derived from the subventricular zone. Note

that Chst142/2 mice show no influence on the differentiation of NSCs

in the olfactory bulb (mean ± s.d.; n54). (D) Quantification of the

density of newborn neurons and astrocytes in the olfactory bulb

differentiated from NSCs derived from the subventricular zone. Note

that in Chst142/2 mice, no influence is detectable on the

differentiation of NSCs in the olfactory bulb (mean ± s.d.; n54).

(E) Photomicrographs of immunohistochemical stainings of the

dentate gyrus of 2- to 3-month-old mouse brains using a Dcx antibody

(shown in red).

(F) Quantification of the density of Dcx+ immature neurons in the

dentate gyrus from Chst142/2 mice and their Chst14+/+ littermates

(mean ± s.d.; n54).

(G) The number of NeuN+BrdU+ cells counted in the dentate gyrus 4

weeks after BrdU incorporation. No significant difference was

observed between Chst142/2 mice and their Chst14+/+ littermates

(mean ± s.d.; n54). Statistical significance was assessed by Student’s

t-test. *P,0.05. Scale bar: 50 mm.

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and DSs, these investigations made use of either chemically

synthesized CS and DS oligosaccharides or CSs and DSs

extracted mostly from non-neural organs, as well as from

different animal species with features possibly different from

those derived from neural tissues (Gama et al., 2006; Ida et al.,

2006; Sato et al., 2008). These compounds were added to cultures

of NSCs and thus did not allow insights into the potentials of CSs

and DSs endogenously synthesized by NSCs.

GAGs exert their biological effects by interacting with growth

factors or cytokines and their receptors, thereby influencing

various signaling pathways (Sasisekharan et al., 2006). CS and

DS chains interact with several growth factors or cytokines that

regulate maintenance and self-renewal of NSCs (Crespo et al.,

2007): deglycosylation of CS-DSPGs by ChaseABC treatment

impaired FGF-2-mediated neurosphere formation and

proliferation. Furthermore, FGF-2-dependent MAP kinase

activation in mouse embryonic cortical NSCs was diminished

after ChaseABC digestion (Sirko et al., 2010). CS-DSPGs

influence FGF-2- and EGF-dependent signaling pathways,

thereby modulating NSC proliferation by binding to these

molecules in the NSC microenvironment (Properzi et al.,

2005), or by serving as co-factors for growth factor or cytokine

receptors (Bandtlow and Zimmermann, 2000). DSs extracted

from pig skin and highly sulfated CS-E motifs from shark

cartilage promoted FGF-2-mediated proliferation of NSCs (Ida

et al., 2006). CS-E and DS motifs thus serve as docking

molecules for growth factors and thereby regulate their activity

towards NSCs (Deepa et al., 2002; Penc et al., 1998).

In the present study, in vitro analysis revealed that deficiency

in Chst14, but not Chst11, impaired neurosphere formation.

The analysis of NSC proliferation in neurospheres revealed a

decreased proliferation of NSCs in Chst14-deficient versus wild-

type neurospheres making it very likely that the differences in

the formation of neurospheres were caused by differences in

Fig. 7. Chst14 deficiency, but not Chst11 deficiency, alters

the composition in distinct subpopulations of the radial

glial cells. (A) Immunoblot analysis of nestin, Mash-1 and

GLAST expression in neurospheres. (B) Quantitative analysis

of expression of nestin, Mash-1, and GLAST in Chst112/2 and

Chst11+/+ neurospheres (mean ± s.d.; n53). No difference is

detected in expression of these proteins between Chst112/2

and Chst11+/+ neurospheres. (C) Quantitative analysis of

expression of nestin Mash-1, and GLAST in Chst142/2 and

Chst14+/+ neurospheres (mean ± s.d.; n53). Note the

decreased expression of Mash-1 and increased expression of

GLAST in Chst142/2 neurospheres versus Chst14+/+

neurospheres. For western blot analysis, an actin antibody was

used to control protein loading. Mean arbitrary units (AU) are

shown. The average of the expression level of all target

proteins, in +/+ neurospheres was set to 1.0 and relative

expression in 2/2 NSCs was calculated.

(D) Photomicrographs of immunostaining of neurospheres

using different radial glial markers: LeX, nestin, GLAST and

RC2. (E) Quantification of the percentage of different radial

glia subpopulations (mean ± s.d.; n53). Statistical significance

was assessed by Student’s t-test. *P,0.05. Scale bars: 50 mm.

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proliferation of cells and not by differences in the adhesion ofcells. Additionally, deficiency in Chst14, but not Chst11, reduced

proliferation of NSCs cultured in adherent cell culture under theinfluence of FGF-2 or EGF alone, or both FGF-2 and EGF. Invivo, deficiency in Chst14 reduced the density of proliferatingneural progenitors in the subventricular zone of the lateral

ventricles and the hippocampal dentate gyrus of adult mice. Inthe E14.5 ganglionic eminence, a tendency towards a reductionof proliferating cells was observed in Chst142/2 mice. The

expression of growth factor receptors EGFR and FGFR-1 wasupregulated in Chst142/2 NSCs compared with Chst14+/+ NSCsbut was not altered in Chst112/2 versus Chst11+/+ neurospheres

in vitro. Combined with the results from our in vitro proliferationanalysis, upregulation of EGFR and FGFR-1 expression inChst142/2 NSCs suggests that DS motifs synthesized by Chst14modify the interaction between the growth factors EGF and FGF-

2 and their respective receptors. CS and DS variants are involvedin different cellular processes, such as binding to growth factorsand neurotrophic factors. In growth factor binding, variations in

CS lead to varying binding capacities (Crespo et al., 2007),whereas the presence of IdoA results in increased binding levels(Hileman et al., 1998). The reason for the higher binding capacity

to the growth factor is believed to be caused by the tendency ofthe pyranose ring of IdoA in dermatan sulfate to form variousconformations. However, for more efficient binding, a domain

consisting of both GlcA and IdoA appears to be essential(Nandini and Sugahara, 2006), which suggests the importance ofChst14 in the modulation of growth factor binding.

Depletion of Chst14 reduced neuronal differentiation of NSCs

in vitro, associated with a tendency towards an increaseddifferentiation into astrocytes which was, however, statisticallynot significant. In vivo, less Dcx-positive newborn neurons were

observed in the dentate gyrus of the adult hippocampus ofChst142/2 mice, whereas the number of newborn NeuN+BrdU+

neurons was not altered, suggesting that Chst14 might accelerate

adult hippocampal neurogenesis in vivo. Because adultneurogenesis in the hippocampus is important for learning andmemory (Jessberger et al., 2009; Shors et al., 2001), the questionwhether this acceleration effect drived by DS patterns formed by

Chst14 plays a role in learning and memory will be addressed in aseparate study. In the olfactory bulb of adult mice, a statisticallyinsignificant reduction in the density of BrdU+ cells in whole

olfactory bulb was observed in Chst142/2 mice compared withChst14+/+ littermates, whereas the percentages of newlygenerated neurons and newborn astrocytes in the total number

of BrdU+ cells were unaltered.

The question remains how DS motifs modified by Chst14influence the fate of NSCs. Previous studies showed thatthe combined removal of both CS and DSs from NSCs using

ChaseABC inhibits neurogenesis and promotes gliogenesis,which is associated with a reduction of the percentage ofBLBP+ and nestin+ radial glial cells and an increased percentage

of GLAST+ and RC2+ radial glial cells (Sirko et al., 2007).Because BLBP is regulated by Notch signaling, which crosstalkswith FGF and EGF signaling (Anthony et al., 2005), and because

GLAST is expressed in adult parenchymal astrocytes (Mori et al.,2005), it has been suggested that CSs and DSs are involved inNotch signaling, and that treatment with ChaseABC affects the

subpopulations of radial glial cells: nestin+ and BLBP+ precursorsrepresent a proliferative and neurogenic precursor fraction,whereas GLAST+ radial glial cells preferentially show

gliogenesis and decreased proliferation (Sirko et al., 2007). In

the present study, the mechanisms by which Chst14 affects

differentiation of NSCs was analyzed by measuring the

expression of nestin, Mash-1 and GLAST in neurospheres

derived from Chst142/2 mice and Chst14+/+ littermate controls.

Mash-1 is an activator-type basic helix-loop-helix (bHLH) gene

that is critical for neurogenesis, and defects in neurogenesis have

consistently been observed in Mash1-deficient mice (Nieto et al.,

2001; Tomita et al., 2000). Mash-1 is also involved in Notch

signaling, which promotes self-renewal and inhibits neurogenesis

of NSCs by regulating transcription factors such as Hes1, which

inhibits neurogenesis and promotes astrogenesis by repressing

expression of Mash-1 (Gaiano and Fishell, 2002). In the present

study, a downregulation of Mash-1 was associated with an

upregulation of GLAST in Chst142/2 mice, while Chst112/2

neurospheres expressed similar amounts of nestin, Mash-1 and

GLAST as Chst11+/+ neurospheres. Immunocytochemical

analysis of Chst142/2 neurospheres showed that they contained

more GLAST+ cells compared with Chst14+/+ neurospheres,

whereas the percentage of RC2+ cells was not significantly

altered. Considering the diminished proliferation and impaired

neurogenesis in Chst142/2 NSCs, we conclude that

endogenously generated DS patterns modified by Chst14 might

exert their effects on the fate determination of NSCs by

crosstalking with the Notch signaling pathway, thereby

influencing the FGF–EGF signaling pathway and thus

potentially altering the occurrence of the radial glial cell

subpopulations. Because the total number of neurons in the

analyzed brain regions did not differ between Chst142/2 and

Chst14+/+ mice, the influence of Chst14 on neurogenesis might

be transient in that the effects of Chst14 deficiency are

compensated by as yet unknown mechanisms.

CSs and DSs have been proposed to provide repulsive cues that

inhibit cell migration (Carulli et al., 2005). ChaseABC treatment

of NSCs removing CSs and DSs enhances the migration of NSCs

in vitro (Gu et al., 2009; Sirko et al., 2010), a feature that is

influenced by EGF signaling (Deepa et al., 2002). Also, RNA

interference (RNAi) knockdown of uronyl 2-O-sulfotransferase

and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, which

are responsible for the synthesis of highly sulfated CS-D and CS-

E motifs, respectively, resulted in disturbed migration of cortical

neurons. However, knockdown of Chst14 did not lead to

migratory defects (Ishii and Maeda, 2008). These observations

are in agreement with the results of this study, showing that

deficiency in Chst14 did not affect NSC migration. Analysis of

Chst112/2 NSCs indicated that CS patterns modified by Chst11

also did not influence migration of NSCs. The observations that

DSs formed by Chst14, as well as CSs formed by Chst11, did not

affect cell migration indicate that sulfation of the carbon 4

position of GalNAc within CS or DS chains is not crucial for

cell migration. Further investigations of other carbohydrate

sulfotransferases targeting CSs or DSs are expected to allow

insights into the sulfation patterns involved in migration of NSCs.

Materials and MethodsAnimals

Chst11/C4st1-deficient (Chst112/2) and Chst14/D4st1-deficient (Chst142/2) micewere generated by homologous recombination using standard techniques andmaintained in C57BL/6 and 129svj mixed background (supplementary materialFigs S9, S10). Briefly, using the recombination cloning (REC) system (Zhang et al.,2002) a 129/SvJ mouse lKO-2 genomic phage library was screened, two clonescontaining either the Chst11 or Chst14 genomic regions were isolated, the third

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exon of the Chst11 gene and the unique exon of the Chst14 gene were deleted and

a neomycin-kanamycin expression cassette was introduced. These targeting

vectors were used for electroporation of R1 ES cells (Nagy et al., 1993) and

lines of Chst112/2 and Chst142/2 mice were established. The Chst11-deficient

mouse line was backcrossed twice to C57BL/6 and the Chst14-deficient mouseline was backcrossed once to C57BL/6 before conducting experiments. The

expected germline manipulations of the Chst11 and Chst14 loci were confirmed

using multiplex PCR, Southern and northern blot analysis (supplementary material

Figs S9 and S10). Southern and northern blot analyses were performed as

described (Sambrook et al., 1989). The probes were amplified from genomic DNA

or cDNA using the Pfu DNA polymerase (Promega, Mannheim, Germany),

confirmed by sequencing and labelled with a-32P using the Megaprime DNA

labelling kit (Amersham Biosciences, Freiburg, Germany) according to themanufacturer’s instructions. The plasmid pCMV-DsRed-Express (Clontech,

Heidelberg, Germany) was digested with the restriction endonucleases NcoI and

NotI and the 0.7 kb DsRed-express fragment was isolated after agarose gel

electrophoresis and labelled as described above. Multiplex PCR was performed

with Taq DNA polymerase (Invitrogen, Darmstadt, Germany) using standard

cycling parameters to determine the genotype of the offspring (supplementary

material Table S1). All animal experiments were performed according to the rules

and regulations of the University and State of Hamburg Animal Care Committees.

Antibodies

The monoclonal antibodies 487 and 473HD (kindly provided by Andreas Faissner,

Ruhr University Bochum, Bochum, Germany), and polyclonal antibody against theglutamate aspartate transporter (GLAST) (kindly provided by Niels Danbolt,

University of Oslo, Oslo, Norway) have been described (Faissner et al., 1994;

Lehre et al., 1995; Streit et al., 1996). All commercial primary antibodies are listed

in supplementary material Table S2. All secondary antibodies were purchased

from Jackson ImmunoResearch (Newmarket, UK) and Santa Cruz Biotechnology

(Santa Cruz, CA).

Neural stem cell culture

NSCs were isolated from the ganglionic eminence of embryonic day 13.5 (E13.5)

mice and cultured as floating neurospheres in neurosphere formation medium

as described previously (Dihne et al., 2003). The formation of secondary

neurospheres was analyzed as described previously (Sirko et al., 2007). Briefly,dissociated NSCs were seeded at a clonal density of 26104 cells in 6 ml culture

medium in the presence of FGF-2 and EGF (20 ng/ml each; PreproTech, Rocky

Hill, NY), and semi-quantitative determination of the diameter of neurospheres

(,100, 100–200, .200 mm in diameter) was performed after 4 days in culture.

Images were acquired with a Kontron microscope (Carl Zeiss, Jena, Germany) and

data was analyzed with the AxioVision Rel. 4.6 software (Carl Zeiss). All in vitro

analyses were confirmed in at least three independent experiments conducted on

independent cell preparations.

Quantitative real-time PCR

The mRNA expression levels of all HNK-1 sulfotransferase family members were

analyzed by quantitative real time PCR (qRT-PCR). Total RNAs were isolated

from neurospheres generated from Chst112/2 and Chst142/2 brains as well asChst11+/+ and Chst14+/+ littermates using the RNeasy Mini Kit (Qiagen, Hilden,

Germany). cDNA synthesis was performed using 0.5–5 mg total RNA, random

hexamers and SuperScript II RNaseH Reverse Transcriptase (Invitrogen), and

qRT-PCR was performed using 0.025 mg reverse transcribed total RNA, specific

primer pairs (0.1 pM each; Metabion, Martinsried) and qPCR Core kit for SYBER

Green I (Eurogentec, Koln, Germany) in an ABI 7900 HT system (Applied

Biosystems, Foster City, CA) according to the manufacturer’s instructions. The

primer sequences are presented in supplementary material Table S3. The qRT-PCRdata were analyzed using the standard curve method (Applied Biosystems). We

used four housekeeping genes encoding hypoxanthine guanine phosphoribosyl

transferase (Hprt), b-actin, glyceraldehyde-3-phosphate dehydrogenase (Gapdh)

and RNA polymerase II (Polr2a) as endogenous references and calculated the

normalization factor with the software geNORM (Vandesompele et al., 2002).

Proliferation, differentiation and apoptosis of NSCs in vitro

For in vitro analysis, neurospheres were mechanically dissociated and plated at a

density of 56104 cells/well onto 15 mm glass coverslips coated with 0.01% (w/v)

poly-L-lysine (PLL) (Sigma, Munich, Germany). For assessment of proliferation

of NSCs derived from Chst112/2 or Chst142/2 mice and wild-type (Chst11+/+ and

Chst14+/+, respectively) littermates under different culture conditions, dissociatedNSCs were adherently cultured in defined medium containing either FGF-2

(20 ng/ml), or EGF (20 ng/ml), or a mixture of FGF-2 and EGF (each 20 ng/ml)

for 24 hours. One dose of 5-bromo-2-deoxyuridine (BrdU) (10 mM; Sigma) was

added to the medium 8 hours before fixation. Proliferation was analyzed by

determining the percentage of BrdU-immunoreactive cells of all 49,6-diamidino-2-

phenylindole (DAPI) (Sigma)-positive cells.

To assess differentiation and apoptosis, dissociated NSCs were cultured oncoverslips in the absence of growth factors for 7 days. Antibodies recognizingdifferent cell markers and apoptosis marker caspase-3 were applied to determinateNSC differentiation and cell apoptosis.

Migration of NSCs in vitro

To analyze the radial migration of neuronal stem cells, neurospheres were seededonto 0.02% (w/v) PLL-coated coverslips and cultured in the absence of growthfactors. The migration distance was defined as the distance between the cell bodiesof the farthest migrated cells and the edge of the neurosphere. After culture for 4and 24 hours, the longest distances between the edge of a neurosphere and the cellbodies were measured in each quadrant of the sphere perimeter, and at least tenneurospheres were analyzed for each group and each time point. Images wereacquired on a Kontron microscope and analyzed with the AxioVision Rel. 4.6software system (Carl Zeiss).

BrdU Incorporation

Proliferating cells in vivo were labeled by intraperitoneal administration of BrdU,which was diluted in a sterile solution of 0.9% (w/v) NaCl and 1.75% (w/v) NaOH(Sigma). For analysis of incorporation of BrdU by embryonic cells, pregnantheterozygous Chst14 (Chst14+/–) mice were injected intraperitoneally with BrdU(100 mg/kg body weight) at embryonic day 13.5 (E13.5) 24 hours beforecollection of embryos. For adult mice, 2- to 3-month-old Chst142/2 mice andChst14+/+ littermates were injected with BrdU (100 mg/kg body weight). Twodifferent previously described protocols (Saghatelyan et al., 2004) were used: (1)to assess proliferation in the subventricular zone and dentate gyrus of thehippocampus and initial migration of adult NSCs in the dentate gyrus of thehippocampus, a single dose of BrdU was applied to the mice 4 hours beforeperfusion; (2) to determine the combined parameters of proliferation, migration,differentiation and cell survival of newborn cells in the olfactory bulb, whichoriginated from the subventricular zone, four consecutive injections of BrdU weregiven at intervals of 2 hours, and mice were sacrificed 28 days later.

Immunocytochemistry and immunohistochemistry

Immunocytochemistry and immunohistochemistry were performed as previouslydescribed (Hargus et al., 2008). For immunocytochemistry with cell type-specificmarkers, coverslips with adherent cells were washed with phosphate bufferedsaline (PBS) (pH 7.4; PAA Laboratories, Colbe, Germany), fixed with 4% (w/v)formaldehyde (Sigma) in 0.1 M cacodylate buffer (pH 7.3; Sigma) for 30 minutes,blocked with 5% (v/v) normal goat serum (NGS) (Jackson ImmunoResearch) inPBS containing 0.2% (v/v) Triton X-100 and 0.02% (w/v) sodium azide (both fromSigma) for 30 minutes, and incubated with primary antibodies in 0.5% (w/v)lambda carrageenan (Sigma) in PBS containing 0.02% (w/v) sodium azide at 4 Covernight followed with Cy2- or Cy3-coupled secondary antibodies diluted 1:1000in carrageenan buffer for 1 hour. Nuclei were counterstained with 50 mg/ml DAPIat room temperature for 2 minutes and coverslips were mounted withFluoromount-G (Southern Biotech, Birmingham, AL). As primary antibodies,tubulin-bIII, GFAP, CNPase, caspase-3, BrdU, Ki67, nestin, RC2, GLAST andantibody 487 were applied. For the BrdU staining, cells were incubated with 2 MHCl at 37 C for 30 minutes to denature DNA before blocking with 5% (v/v) NGS[in PBS with 0.2% Triton X-100 (v/v) and 0.02% sodium azide] for 1 hour at roomtemperature. On each coverslip at least 1000 DAPI+ cells were counted using theNeurolucida software-controlled computer system (MicroBrightField Europe,Magdeburg, Germany). The immunolabeled slide preparation was scanned usingan Olympus FluoViewTM FV1000 confocal microscope equipped with UVepifluorescence (Olympus, Hamburg, Germany).

For histological analysis of embryos, brains of embryos at E14.5 were fixed with4% (w/v) formaldehyde for 2 hours, incubated sequentially in 15% and 30% (w/v)sucrose in 0.1 M cacodylate buffer (pH 7.3) at 4 C overnight, and cut in serialfrontal sections (14 mm each) on a cryostat (Leica, Wetzlar, Germany) and everyfifth section was used for further analysis. For histological analysis of adult mousebrains, mice were deeply anesthetized with an overdose of Narcoren (Merial,Hallbergmoos, Germany) and perfused intracardially with 4% (w/v) formaldehyde(Sigma). The brains were removed and immersed in the same fixative at 4 Covernight, incubated in 15% (w/v) sucrose in 0.1 M cacodylate buffer at 4 Covernight, cut in serial frontal sections (25 mm each) on a Leica cryostat and everytenth section was used for further analysis. For immunohistochemistry, antigenretrieval was performed by incubation in 10 mM sodium citrate (pH 9.0; Sigma) at80 C for 30 minutes, followed by blocking with 5% (v/v) NGS or normal donkeyserum (NDS) (Jackson ImmunoResearch) at room temperature for 1 hour andincubation at 4 C overnight with the primary antibodies against the followingproteins: BrdU for proliferating cells; caspase-3 for cells undergoing apoptosis;Dcx for immature neurons; BrdU and NeuN double staining or BrdU and GFAPdouble staining for analysis of newly generated neurons and astrocytes,respectively. Subsequently, slides were washed with PBS and incubated withspecific fluorochrome-coupled secondary antibodies for 2 hours at roomtemperature. Slides were labelled with bisbenzimide (1:10,000; Sigma) for10 minutes to identify cell nuclei, and mounted with Fluoromount-G. For BrdU

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immunostaining, slides were incubated in 0.1 M HCl instead of antigen retrievalbuffer at 60 C for 20 minutes to denature DNA before blocking with NGS, and cellnuclei were labeled with bisbenzimide for 20 minutes. The density of single- ordouble-labeled cells was analyzed using the Neurolucida system(MicroBrightField Europe) and an Olympus confocal laser microscope(Olympus) was used for image scanning.

Western blot analysisExpression of growth factor receptors EGFR and FGFR-1 and radial glial cellmarkers including nestin, Mash-1 and GLAST were analyzed by western blotanalysis. Protein extracts were harvested by lysing neurospheres generated fromChst112/2 or Chst142/2 mice and Chst11+/+ and Chst14+/+ animals with RIPAlysis buffer (150 mM NaCl, 1 mM Na4P2O7, 1 mM NaF, 1 mM EDTA, 1 mMPMSF, 2 mM Na3VO4, 1% NP-40, 50 mM Tris, pH 7.5) with complete EDTA-free protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany) at 4 Cfor 1 hour. The protein samples were boiled in SDS sample buffer for 10 minutesbefore loading onto 10% SDS-PAGE gel or Progel Tris-Glycine 4–20% gradientgels (Anamed Elektrophorese, Groß-Bieberau, Germany) as 20 mg for each laneand transferred onto nitrocellulose membrane (Protran, Victoria, Australia). Forimmunoblotting, membranes were blocked with 5% (w/v) non-fat milk powder inPBS and incubated at 4 C overnight with primary antibodies diluted in PBST [PBSwith 0.05% (v/v) Tween-20] against the following proteins: 473HD, actin, EGFR,FGFR-1, GLAST, Mash-1, nestin. After washing with PBST, membranes wereincubated with specific HRP-conjugated secondary antibodies for 1 hour at roomtemperature followed with extended washes with PBST. Immunoblot reactionswere visualized using chemiluminescent substrate (Perbio Science, Bonn,Germany) on Kodak BioMax light films (Kodak, Rochester, USA). Theintensities of the bands were densitometrically quantified with the imagesoftware TINA 2.09.

Statistical analysis

All experiments were repeated independently at least three times and performed ina blinded manner, and analyzed by comparison of the mean values ± s.d. ofdeficient and wild-type mice using Student’s t-test for independent samples.

AcknowledgementsWe are grateful to Jinchong Xu and Yifang Cui for insightfulsuggestions for experimental procedures, Achim Dahlmann forgenotyping, and Andreas Faissner and Niels Danbolt for antibodies.

FundingThis research was supported by grants to M.S. from the DeutscheForschungsgemeinschaft [Scha 185/54-1], the New Jersey Commissionfor Spinal Cord Research and the Li Kashing Foundation.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.088120/-/DC1

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