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First published online 27 June 2007
doi: 10.1242/dev.02871
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1 Chair of Cell Morphology and Molecular Neurobiology, Ruhr-University Bochum,
Building NDEF 05/339, Universitaetsstrasse 150, D-44780 Bochum, Germany.
2 GSF-Institute for Stem Cell Research, Ingolstädter Landstraße 1,
D-85764 Neuherberg, Germany.
3 Institute of Physiology, University of Munich, Schillerstaße 46, D-80634
München, Germany.
Author for correspondence (e-mail:
andreas.faissner{at}ruhr-uni-bochum.de)
Accepted 24 May 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Chondroitinase ABC, Astrocyte differentiation, Stem cell niche, Proteoglycans
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Postnatally, the latter lineage of radial glia transforms into astrocytes
(Doetsch, 2003;
Gotz and Huttner, 2005).
Specialized astrocytes in the subependymal zone (SEZ) of the lateral ventricle
or in the subgranular zone of the hippocampus serve as slowly dividing neural
stem cells (NSCs) in the adult CNS
(Alvarez-Buylla et al., 2001).
Factors that regulate the complex pathway of NSC development begin to emerge.
In their site of origin, NSCs are surrounded by a particular cellular
environment and exposed to differentiation signals such as Notch and Delta
ligand-receptor interactions (Campos et
al., 2006). In addition, NSCs of the mouse respond to fibroblast
growth factor (FGF) and epidermal growth factor (EGF) and sequentially express
the corresponding receptors FGFR and EGFR with ongoing maturation
(Reynolds and Weiss, 1996).
Furthermore, it has been shown that NSCs are located in a structured
pericellular environment that comprises distinct extracellular matrix (ECM)
constituents (Gates et al.,
1995; Quinones-Hinojosa et
al., 2006). Substantial evidence has been provided in recent years
that complex ECM milieus are involved in the regulation of CNS development and
plasticity and in the response to lesion. The neural ECM is composed both of
glycoproteins, such as tenascin-C, and proteoglycans
(Garwood et al., 2001). The
latter can be divided in heparan sulfate proteoglycans (HSPGs), with a
preferential membrane association, and the chondroitin sulfate proteoglycans
(CSPGs) that occur primarily as constituents of the pericellular space
(Bandtlow and Zimmermann,
2000). HSPGs play an important role as accessory factors in
signaling processes, e.g. by exposing growth factors such as bFGF (also known
as FGF2 - Mouse Genome Informatics) to the FGFR. By comparison, less is known
about the CSPGs. We have recently shown that the Mab-473HD epitope, a unique
chondroitin sulfate (CS) structure, is expressed in the germinal layers during
mouse forebrain development (von Holst et
al., 2006), and the release of CSPGs from NSCs has been reported
(Ida et al., 2006). The 473HD
epitope, also known as the DSD-1 epitope
(Faissner et al., 1994), is
expressed on the CSPG phosphacan that represents a soluble, released splice
variant encoded by the receptor protein tyrosine phosphatase ß
(Rptpß) gene (the protein is also known as phosphacan, DSD-1-PG
and PTPRZ1 - Mouse Genome Informatics). The large RPTP-ß receptor is
expressed by NSCs and radial glia during development of the CNS and exposes
the 473HD structure (Garwood et al.,
1999). Interestingly, recent studies have proposed that CSPGs play
important roles in the adult CNS as inhibitors of synaptic plasticity and of
axonal regeneration in scar tissues
(Bradbury et al., 2002;
Pizzorusso et al., 2002). In
the light of these findings, treatment strategies based on the application of
chondroitinase ABC (ChABC), an enzyme that degrades the CS-glycosaminoglycan
(CS-GAG) complement of CSPGs, have been developed
(Houle et al., 2006). On the
other hand, evidence has emerged that neural progenitor cells are recruited to
various types of lesion in the adult CNS
(Arvidsson et al., 2002;
Magavi et al., 2000), which
raises the question as to whether ChABC application might impact on NSC
development in the lesioned tissue
(Dobbertin et al., 2003). We
have, therefore, systematically addressed the question of whether ChABC
affects stem cell behavior. We show here that the selective elimination of
CS-GAGs with ChABC, both in vivo and in vitro, reduces NSC proliferation and
the differentiation of radial glia to neurons, whereas it favors the
maturation of the gliogenic subtype of radial glia and the formation of
astrocytes. This implies a role of CS-GAGs in the regulation of growth and
differentiation factors for NSCs. |
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Experimental procedures were performed in accordance with the Society for
Neuroscience and European Union guidelines and were approved by the animal
care and utilization committees at the GSF and Ruhr University.
Immunological reagents
The following primary antibodies were used. Monoclonal antibodies: 473HD
(rat IgM) (Faissner et al.,
1994), RC2 (mouse IgM; Developmental Studies Hybridoma Bank,
University of Iowa, USA), anti-E-cadherin (mouse IgG; Santa Cruz), anti-nestin
(mouse IgG; Chemicon, Temecula, CA), anti-ßIII-tubulin (mouse IgG; Sigma,
St Louis, MO) and anti-BrdU (mouse IgG; Roche, Mannheim, Germany). Polyclonal
antibodies (all rabbit) against: DSD-1-PG/phosphacan [referred to as
pk-anti-phosphacan; Batch KAF 13(4);
(Faissner et al., 1994)], GFAP
(Dako), atypical PKC
(aPKC; BD Science), phospho-histone H3 (PH3),
laminin, brain-lipid-binding protein (BLBP) and GLAST (all Chemicon).
Secondary antibodies: subclass-specific CY2- or CY3-coupled anti-mouse,
anti-rat and anti-rabbit antibodies (all Dianova, Hamburg, Germany).
Neurosphere (Nsph) cultures
Pregnant animals were sacrificed by cervical dislocation. The embryos were
removed and transferred to phosphate-buffered saline (PBS). Subsequently, the
embryonic brains were dissected, the meninges, hippocampi and olfactory bulbs
were removed, and the cerebral cortices (Cor) and the ganglionic eminence (GE)
were prepared in minimal essential medium (MEM, Sigma). Tissues were
enzymatically digested with 0.05% (w/v) trypsin-EDTA in HBSS (Invitrogen,
Karlsruhe, Germany) for 10 minutes at 37°C to obtain E13 neural cell
suspensions. Digestion was stopped by the addition of 1 ml ovomucoid [1 mg/ml
trypsin inhibitor (Sigma), 50 µg/ml BSA (Sigma), 40 µg/ml DNaseI
(Worthington), in L-15 medium (Sigma)]. After centrifugation for 5 minutes at
1000 rpm (212 g), the cell pellets were suspended in Nsph
medium consisting of DMEM/F12 (1:1) with 0.2 mg/ml L-glutamine (all Sigma), 2%
(v/v) B27, 100 U/ml penicillin, 100 µg/ml streptomycin (all Invitrogen).
Embryonic NSCs were cultured at 37°C, 6% CO2 at a cell density
of 105 cells/ml (bulk culture) in Nsph medium in the presence of
EGF and bFGF, both at 20 ng/ml (Preprotech, Tebu, Germany), unless otherwise
indicated. In general, bFGF-containing cultures were supplemented with 0.5
U/ml heparin (Sigma). In parallel sets, 50 mU/ml chondroitinase ABC (ChABC; EC
4.2.2.4; Sigma) or 50 mU/ml keratanase (Calbiochem, La Jolla, CA) were added.
The formation of secondary Nsphs was assessed in clonal density assays by
plating 2x104 cells in 6 ml of FGF- and EGF-containing
medium. The formation of Nsphs was quantified after 7 days in vitro (div) by
size-dependent (<50, 100, 150, 200 and >200 µm in diameter) counting
of Nsphs in the entire dish area (clonal density assays) or in ten randomly
selected visual fields.
Immunocytochemistry
For immunocytochemical stainings, the acutely dissociated cells originating
from forebrain tissues, from cortical and striatal Nsphs, or obtained by
immunopanning using the Mab 473HD (von
Holst et al., 2006), were plated in FCS-containing medium [1%
(v/v), Seromed] at a density of 5000 cells/well in four-well dishes (Greiner)
coated with 10 µg/ml polyornithine (Sigma) and incubated in a humidified
atmosphere with 6% (v/v) CO2 at 37°C for 1 hour. Cell
suspensions were allowed to adhere, washed twice for 5 minutes in
Krebs-Ringer-Hepes buffer [KRH with 0.1% (w/v) BSA, pH 7.4] at room
temperature (RT) and incubated for 20 minutes at RT with Mab 473HD diluted in
KRH/A (1:250). After washing twice for 5 minutes in KRH, the cells were fixed
with 4% (w/v) paraformaldehyde (PFA) in PBS for 10 minutes at RT, washed twice
with PBT1 [PBS containing 1% (w/v) BSA and 0.1% (w/v) Triton X-100] and
incubated for 30 minutes at RT with antibodies against the intracellular
epitopes RC2 (1:500), nestin (1:500), ßIII-tubulin (1:300), BLBP
(1:1000), GLAST (1:1000) and GFAP (1:250), all diluted in PBT1. After three
further washes with PBS/A, the cells were incubated for 30 minutes at RT with
specific fluorochrome-conjugated secondary antibodies to detect the various
primary antibodies. The last incubation step included bisbenzimide (1:10,000;
Sigma) to label cell nuclei. After final washing with PBS, specimens were
mounted in PBS/glycerol (1:1) and viewed under an Axiophot II fluorescence
microscope (Zeiss, Oberkochen, Germany) using UV epifluorescence. To identify
differentiated cell types after the exposure of cultures to 50 mU/ml ChABC or
50 mU/ml keratanase, the same immunostaining protocol was carried out after 24
hours of incubation. For differentiation assays, individual Nsphs of 150-250
µm diameter were transferred to four-well dishes (Greiner) sequentially
coated with polyornithine (10 µg/ml) and laminin-1 (10 µg/ml; Tebu,
Germany) and incubated in Nsph medium containing 1% (v/v) FCS (see above) for
a further 6 days in a humidified atmosphere with 6% (v/v) CO2 at
37°C. The differentiated cell types were identified by immunocytochemistry
using antibody markers as described in the previous section.
Intracerebroventricular injections (ICVIs) in utero
ICVI into telencephalic ventricles of E13 embryos in utero was performed
with timed-pregnant C57/Bl6 mice. Animals were anesthetized by intraperitoneal
injection of 0.1 ml per 10 g body weight of the narcotic mixture 0.5 mg/kg
medetomidine, 5 mg/ml midazolam, 0.05 mg/kg fentanyl hexal. Uterine horns were
exposed by midline laparotomy and the ICVIs were performed through the uterine
wall at the anterior end of the embryonic forebrain using fine-pulled
microcapillaries (borosilicate glass capillaries, 1.5x0.86 mm;
gc150F-10, Harvard apparatus). ChABC (10 mU/µl) was injected into the
lateral ventricles of all embryos in one uterine horn. Keratanase (10
mU/µl) or artificial cerebrospinal fluid (ACSF) controls were injected into
the lateral ventricles of the embryos in the second uterine horn. Thereafter,
the uterus was reinstated in its physiological site. The incisions of the
abdominal muscle and skin were stitched with separate sutures. Finally, the
anesthesia was reversed by intraperitoneal application of antisedate (2.5
mg/kg antisedan, 0.5 mg/kg flumacenil, 1.2 mg/kg naloxone), and animals were
left to recover in a clean cage. Twenty-four hours after injection, pregnant
mice were sacrificed by cervical dislocation.
Immunohistochemistry
The embryos were removed and transferred to PBS (pH 7.4). Subsequently, the
capita were immersion fixed overnight in 4% (w/v) PFA in PBS at 4°C.
Thereafter, tissues were cryoprotected overnight in 30% (w/v) sucrose in
water, embedded in Tissue Freezing Medium (Jung, Nussloch, Germany) and frozen
on dry ice. Frontal sections (12-14 µm) were cut on a cryostat (Leica). For
immunohistochemistry, slides were rehydrated in PBS with 1.7% (w/v) NaCl and
10% (v/v) normal goat serum (Dianova) for 1 hour at RT and then incubated
overnight (4°C) with one of the following primary antibodies: 473HD
(1:500), RC2 (1:500), anti-nestin (1:500), anti-ßIII-tubulin (1:300),
anti-BLBP (1:1000), anti-PH3 (1:100), anti-GLAST (1:1000), anti-E-cadherin
(1:50), anti-aPKC (1:50) or anti-laminin (1:80). Subsequently, the
sections were washed four times for 5 minutes in PBS and incubated with
appropriate secondary antibodies [1:500 in PBS, 0.1% (w/v) BSA)] for 2 hours
at RT. After incubation with the secondary antibodies, cell nuclei were
labeled with bisbenzimide (diluted 1:10,000). After three further washes in
PBS, the sections were mounted in Immumount (Thermo Electron, Dreieich,
Germany). For cryosectioning, Nsphs were allowed to settle in 15 ml Falcon
tubes for 10 minutes before the culture medium was removed and replaced with
4% (w/v) PFA in PBS for 40 minutes at RT. After fixation, the Nsphs were
cryoprotected with 30% (w/v) sucrose for 4 hours at 4°C. Finally, Nsphs
were embedded in Tissue Freezing Medium, sectioned at 14 µm on a cryostat
(Leica) and processed for immunohistochemistry as described for embryonic
brain sections above.
BrdU pulse-labeling
In vitro labeling of cycling Nsph cells was performed by addition of 10
µM BrdU (5-bromo-2-deoxyuridine, Sigma) 15 hours prior to enzymatic
dissociation. In order to count the number of cells that incorporated BrdU,
Nsphs were dissociated, the single-cell suspension was plated and individual
cells were immunocytochemically stained 1 hour later, as described above and
according to the supplier's protocol (BrdU Labeling and Detection Kit I,
Roche). For analysis of cell proliferation in vivo, the label was introduced
by intraperitoneal injection of 10 mg BrdU/100 g body weight 1 hour prior to
removal of the litter. After cryoprotection of embryonic tissues, cryosections
were cut at 14-18 µm, boiled for 5 minutes in 0.01 M citrate buffer (pH
6.0) and washed twice in PBS before incubation with anti-BrdU (1:20) at
4°C overnight. Primary antibodies were detected using appropriate
secondary antibodies, as described above.
Microscopy
The immunolabeling experiments were analyzed using a fluorescence
microscope equipped with UV epifluorescence (Axioplan 2 imaging, Zeiss).
Images were captured with a digital camera and documented using the Axiovision
3.1 and/or 4.2 programs (AxioCamHRc, Zeiss). In some cases, confocal laser
scanning microscopy was applied (LSM 510 meta, Zeiss). Standard phase-contrast
images of living cells were taken using a digital camera (DP10, Olympus) on an
inverted CK40 microscope (Olympus).
Counting and statistical analysis
The proportion of single- and double-immunolabeled cells collected from
dissociated Nsphs or selectively isolated cells was determined by counting 200
to 400 individual cells per independent experiment and antibody. Statistical
significance of differences (see figure legends) observed between distinct
experimental groups was assessed using an unpaired, two-tailed Student's
t-test.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). NSCs
were grown as freely floating Nsphs in defined medium containing the growth
factors EGF and FGF and were continuously exposed to the ChABC enzyme as added
to the culture medium. The formation of Nsphs was monitored either in the
presence or in the absence of ChABC. The ongoing digestion of CS-GAGs
compromised the generation of Nsphs and caused a nearly complete settling of
the Nsphs on the substrate of the culture dish, where a massive emigration of
Nsph-derived cells was seen (Fig.
1). The efficiency of CS-GAG removal was assessed using antibodies
473HD and anti-phosphacan (KAF13), which detect a CS-GAG structure and the
core glycoproteins of the Rptpß gene products, respectively
(Faissner et al., 1994).
Whereas control cryosections of Nsphs displayed the 473HD epitope, its
complete loss was observed when Nsphs were grown in the presence of ChABC. In
this latter case, many cells still retained the core proteins of
DSD-1-PG/RPTP-ß (Fig. 1A).
Analogous results were obtained when Nsph extracts were examined by western
blotting (Fig. 1B). Culture in
the absence of enzyme or in the presence of keratanase, an enzyme that removes
keratan sulfates but does not affect the 473HD epitope
(Faissner et al., 1994),
served as controls (Fig.
1). The selective cleavage of the CS-GAGs decreased the number of Nsphs per visual field from 9.3±1.7 to 5.1±1.1 (n=10; P<0.001, unpaired t-test) in cortex-derived cultures and from 9.1±1.5 to 4.2±1.1 (n=10; P<0.001, unpaired t-test) in striatum-derived cultures (Fig. 1F and see Fig. S1 in the supplementary material). Parallel experiments were performed with keratanase (50 mU/ml) instead of ChABC. The removal of keratan sulfates did not lead to substrate adherence (Fig. 1E) and the rate of Nsph formation was indistinguishable from the control (Fig. 1F). These findings suggested that CS-GAGs are required to maintain Nsphs in a free-floating state and might also influence neural stem/progenitor cell maintenance. To examine the role of CS-GAGs in Nsph formation and maintenance, an equal number of cells from ChABC-treated and control primary Nsphs were examined at clonal density (see methods) for the generation of secondary Nsphs. According to the view that only the self-renewing stem cells of the primary Nsphs would give rise to secondary Nsphs, this assay allows determination of the number of self-renewing stem cells present in the primary Nsph population. Using this approach, twice as many secondary Nsphs originated from cell suspensions derived from untreated primary cortical and striatal Nsphs, than from Nsphs that had been exposed to ChABC (Fig. 1G).
To further monitor whether not only self-renewal, but also proliferation in general, are affected, BrdU was added to the Nsph cultures 15 hours prior to fixation. The number of BrdU-positive cells decreased significantly in cortical or striatal Nsphs upon ChABC treatment (Fig. 1H,I). In conclusion, chondroitin sulfates promote proliferation and self-renewal of early telencephalic stem/progenitor cells grown in Nsph cultures.
Digestion of chondroitin sulfates modifies the composition of the neural precursor cell pool
It has been reported that only a small percentage of the cells within Nsphs
are NSCs (D'Amour and Gage,
2003), whereas the majority are precursor cells of other types.
Stem cells and/or precursors differentially express the intermediate filament
protein nestin (Lendahl et al.,
1990), and the radial glia markers RC2 (also known as IFAPRC2 -
Mouse Genome Informatics) (Chanas-Sacre et
al., 2000), BLBP (also known as FABP7)
(Feng et al., 1994) and GLAST
(also known as SLC1A3) (Shibata et al.,
1997), similar to the radial glia heterogeneity observed in vivo
(Hartfuss et al., 2001). To
correlate the reduction in self-renewing stem cells upon ChABC treatment with
these precursor markers, sections of control and ChABC-treated Nsphs were
studied by immunohistochemistry (Fig.
2A,B). The continuous digestion of CS-GAGs by ChABC treatment
reduced the number of nestin- and BLBP-positive precursor cells, whereas the
number of RC2- and GLAST-positive radial glia cells increased. In order to
quantify the cellular composition, Nsphs were dissociated and the cell
suspensions examined 2 hours after plating
(Fig. 2C,D,
Table 1). In accordance with
the immunohistochemical analysis, the addition of ChABC decreased the
percentage of nestin-positive cells 2-fold in cortical and striatal Nsphs.
Similarly, the number of BLBP-positive radial glia diminished more than
2-fold, whereas the number of GLAST-positive cells was augmented almost 2-fold
upon ChABC treatment. Under control conditions, more than half of the
nestin-positive cells in Nsphs also contained BLBP and less than one third
contained GLAST. By contrast, after CS-GAG degradation, the proportion of
nestin/BLBP-positive cells was reduced 4-fold, whereas the number of
nestin/GLAST double-labeled neural precursor cells increased. Interestingly,
after ChABC treatment, a 2-fold increase in the RC2-positive cell population
that coexpressed GLAST was limited to cortical Nsphs and did not occur in
striatal Nsphs. Taken together, these results support the notion of precursor
heterogeneity (D'Amour and Gage,
2003; Hartfuss et al.,
2001) and indicate that deglycanation of CSPGs gears the emergence
of neural progenitor sublineages.
|
; von Holst et al.,
2006). A striking feature with regard to topography was that
postmitotic neurons were located on the outer surface of control and treated
striatal Nsphs rather than positioned in the core region, as had been reported
previously for cortical Nsphs (Campos et
al., 2004). Quantitative assessment revealed a 4-fold reduction in
the number of neurons amongst cortical or striatal Nsph cells
(Fig. 3C), whereas the
astroglial population was expanded 3- to 4-fold
(Fig. 3D,
Table 1). Taken together, these
data suggest that chondroitin sulfates promote the proliferation or fate of
neurogenic progenitors in this culture system.
|
). In
the immunoselected cell populations obtained from cortical and striatal Nsphs,
the 473HD epitope and the radial glia marker BLBP were coexpressed by up to
96% of the sorted cells, as previously shown. When this population was
cultivated for 2 days in vitro in serum-containing medium in the presence of
ChABC, neurogenesis was strongly compromised. Thus, the number of neurons
originating from the 473HD-positive cell population dropped substantially in
both cortex or striatum-derived Nsphs (Fig.
3E). In addition, a simultaneous preponderance of astroglial cell
differentiation was obvious and more pronounced for cortical Nsph-derived
473HD-positive progenitors (Fig.
3F). These observations revealed that CS-GAGs are essential for
the timely differentiation of the neurogenic 473HD/BLBP-positive radial glia
into neurons.
|
Chondroitin sulfates are essential components for generation of the neurogenic precursor and their differentiation in the embryonic forebrain in vivo
In order to ascertain the functional significance in vivo, ChABC,
keratanase or artificial cerebrospinal fluid (ACSF) were injected into the
cerebral ventricles of E13-14 embryos in utero
(Grove et al., 1993). After 24
hours, the surviving embryos were fixed and analyzed. The
intracerebroventricular injection (ICVI) of ACSF or keratanase did not notably
perturb the histology of the developing forebrain. By contrast, the
elimination of CS-GAGs caused an apparent disorganization of the germinal
regions, which appeared more loosely packed, rendering the distinction between
ventricular zone (VZ) and subventricular zone (SVZ) almost impossible
(Fig. 4A). In addition, a
compaction of cells in the cortical plate could be distinguished after ChABC
application, whereas the histological organization of the cortical marginal
zone remained unchanged (Fig.
4A). Despite the obvious alterations in histology and cell
morphology, only a few apoptotic cells were detectable in TUNEL stainings (see
Fig. S3 in the supplementary material).
|
)
(Fig. 4B). Upon ICVI of ChABC,
many neuroepithelial cells analyzed 1 day after injection retained the core
proteins of DSD-1-PG/RPTP-ß, as demonstrated by staining with the
anti-DSD-1-PG/phosphacan antibody. Most importantly, the 473HD
immunoreactivity almost completely vanished, with the exception of the
marginal zone, demonstrating that the enzyme diffuses from the ventricle far
into the brain parenchyma (Fig.
4B).
|
In order to gain insight into VZ and SVZ precursor subtypes, the
distribution of the PH3- and BrdU-labeled cells was assessed with reference to
their distance from the ventricular surface
(Fig. 5C,D). Following ChABC
application, a substantial decrease in PH3- and BrdU-labeled cells was
apparent in the area close to the ventricular surface; this was not seen
following injection of ACSF. This result suggested that ChABC exerted its
effect mostly on radial glia precursors with access to the ventricle, whereas
basal precursors dividing at abventricular positions were less affected. Thus,
ChABC treatment seems to affect cells that undergo interkinetic nuclear
migration and possess an apical process with contact to the ventricle. In the
light of the importance of cell polarity for subsequent neurogenic cell
division patterns (Cappello et al.,
2006), we also examined the distribution of E-cadherin (also known
as cadherin 1 - Mouse Genome Informatics) and aPKC (also known as PRKCI)
as apical markers and of laminin as a basolateral marker. No difference in
E-cadherin and aPKC staining was found between ChABC- and control-injected
telencephali (see Fig. S4 in the supplementary material).
|
In the light of the significance of CS-GAGs for NSC proliferation reported earlier, we also examined the potential of forebrain tissues to generate Nsphs after ChABC or ACSF treatment. Cell suspensions were cultivated in the presence of EGF and bFGF for 7 days in vitro and emerging Nsphs were counted. When ChABC had been injected into the forebrain, the number of Nsph-forming cells was significantly reduced to half the yield upon ACSF injection (Fig. 6D). Taken together, the analysis of CS-GAG removal in vivo confirmed the importance of this specific class of carbohydrates for proliferation, cell fate decisions and neurogenesis of neural stem/progenitor cells.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Indeed, during development, neuroepithelial and
radial glial cells share many cell biological and molecular features with
adult NSCs. Both have astroglial properties, and similar molecular
determinants influence neurogenesis and proliferation
(Alvarez-Buylla et al., 2001).
For example, PAX6 promotes neurogenesis in the developing cerebral cortex
(Heins et al., 2002), in
embryonic and adult Nsph cells and in adult SEZ cells in vivo
(Hack et al., 2005;
Hack et al., 2004).
Conversely, overactivation of Notch signaling promotes proliferation and
maintains radial glia and NSCs during development
(Gaiano et al., 2000;
Hitoshi et al., 2002).
Intriguingly, Notch is expressed in a subset of radial glial cells during
development and in adult NSCs, but not in most of the adult parenchymal
astroglia, and positively regulates BLBP
(Anthony et al., 2005;
Hartfuss et al., 2001) (for a
review, see Mori et al.,
2005). Here, we were able to show that removal of CS-GAGs reduced
the number of BLBP- and nestin-positive radial glial cells, while increasing
the GLAST- and RC2-positive fraction. These subtype alterations upon ChABC
treatment were virtually indistinguishable in Nsph cells in vitro and in
radial glial cells in vivo. Moreover, these subtype modifications correlated
with reduced proliferation and a decrease in neurogenesis in vitro and in
vivo, suggesting that the nestin- and BLBP-positive radial glial cells
represent a proliferative, self-renewing, neurogenic precursor population
(Anthony et al., 2004;
Hartfuss et al., 2001). This
is consistent with Notch signaling regulating BLBP and cell proliferation
(Anthony et al., 2005;
Hatakeyama et al., 2004).
Conversely, reduced proliferation and increased gliogenesis is accompanied by
upregulation of GLAST, a molecule that persists in adult parenchymal
astrocytes (Mori et al., 2006;
Shibata et al., 1997). These
results underline the functional relevance of radial glial subtypes with
regard to proliferation and cell fate. Most excitingly, however, our results
show the profound role of CSPGs in governing precursor subtypes, cell
proliferation and cell fate in vitro and in vivo. Indeed, the key role of
modifications of signaling molecules in the ECM is becoming ever clearer
(Carulli et al., 2005;
Deakin and Lyon, 1999;
McLaughlin et al., 2003),
although little is known about the role of chondroitin sulfates in general,
and especially in the regulation of neural stem/progenitor cell fate.
Potential molecular mechanisms of CSPGs in neurogenesis and proliferation
How could CSPGs exert their crucial role in mediating radial glia and NSC
proliferation and neurogenesis, as discovered here? Notably, the experiments
in vivo revealed a clear effect on radial glial cell proliferation and
neurogenesis, whereas the proliferation of basal precursors dividing at
abventricular positions was less affected, despite the penetration of the
enzyme deep into the cortical parenchyma. This is consistent with the finding
that radial glia cell polarity was not notably affected by ChABC treatment, as
deduced from the unaltered expression of the apical markers aPKC and
E-cadherin. Indeed, when polarity is compromised by the conditional deletion
of the Rho GTPase CDC42, an enhanced rate of basal mitoses and a decrease in
apical divisions result (Cappello et al.,
2006). In our case, basal progenitors seemed unperturbed and the
cellular polarity conserved, with reduced proliferation confined to the apical
compartment. These data further support the notion that the pathway(s)
affected is predominantly active in the self-renewing radial glia/precursor
cells, because basal precursors do not self-renew
(Haubensak et al., 2004;
Miyata et al., 2004;
Noctor et al., 2004). Our
interpretation is in agreement with the decrease in BLBP immunoreactivity that
occurs prominently in radial glial cells, but not in basal precursors.
BLBP is regulated by Notch signaling that interacts with FGF and EGF
signaling (Gaiano et al.,
2000; Hasson et al.,
2005; Yoon et al.,
2004). Firstly, Notch signaling might be affected by ChABC
activity. Moreover, EGF in conjunction with Notch signaling promotes radial
glia formation (Gregg and Weiss,
2003). CSPGs bind various growth factors that might impinge on the
proliferation rates of NSCs, and the implication of CSPGs in stem cell
proliferation has recently been documented in the nematode
(Mizuguchi et al., 2003;
Sugahara et al., 2003). Thus,
CS-dependent GAG motifs may serve as docking sites for growth factors and
thereby modulate responsiveness to bFGF in embryonic neural stem/progenitor
cells (Deepa et al., 2002;
Penc et al., 1998). CSPGs
might intervene in the bFGF- or EGF-dependent signaling pathways, and thereby
foster NSC and radial glia proliferation. This could be effected either
through the manner in which CS-GAGs bind and store factors in the pericellular
environment (Properzi et al.,
2005), or by their serving as cis-acting co-factors for growth
factor receptors, analogous to the role played by heparan sulfate
proteoglycans with respect to the FGFR
(Bandtlow and Zimmermann,
2000).
Indeed, the growth factors pleiotrophin and midkine, which have been
associated with the proliferation of NSCs, are also secreted into
Nsph-conditioned medium and strongly interact with the CS-GAGs of the CSPG
phosphacan (Deepa et al.,
2002; Furuta et al.,
2004; Hienola et al.,
2004). The 473HD epitope is expressed on the CSPG phosphacan, an
isoform of the transmembrane-based receptor protein tyrosine phosphatase
(RPTP)-ß/
(Faissner et al.,
1994; Garwood et al.,
1999). Selected RPTP-ß/ isoforms are highly expressed
in VZ in the embryonic brain and in the stem cell niches of the adult CNS, and
hence also represent potential targets of ChABC treatment
(Engel et al., 1996). The
RPTP-ß/ receptor is a potential ligand of the ECM glycoprotein
tenascin-C, which interacts with the CSPG phosphacan
(Milev et al., 1994).
Tenascin-C is highly enriched in the VZ of the developing brain
(Gotz et al., 1997) and is
downregulated in the mature CNS, with the exception of areas of ongoing
precursor cell proliferation, for example the SEZ
(Gates et al., 1995). Deletion
of Tnc interferes with NSC maturation by impairing the
bFGF-stimulated acquisition of the EGFR on NSCs
(Garcion et al., 2004).
Notably, tenascin-C expression is virtually lost in the cerebral cortex of
PAX6 mutant mice, concomitant with a decrease in neurogenesis from radial glia
cells (Gotz et al., 1998;
Stoykova et al., 1997).
Interestingly, tenascin-C and sulfation of the ECM influence the canonical Wnt
signaling pathway (Kakinuma et al.,
2004), a well-known regulator of stem cell fate
(Reya and Clevers, 2005).
Taken together, the profound effect of ChABC treatment is likely to be due to
effects on a multitude of signaling pathways.
Another important aspect that merits consideration is the implication of
ECM components in cell adhesion processes. CSPGs are anti-adhesive for neural
cell types and are involved in inhibition of axon regeneration
(Carulli et al., 2005). It is
noteworthy that Nsphs release large amounts of CSPGs into the culture medium
(Ida et al., 2006). Thus, the
settling and the outgrowth of ChABC-treated Nsphs apparent in our studies
might reflect a reduction in the inhibitory qualities of the substrate as a
consequence of CS-GAG degradation, as has been observed in the lesioned CNS
(Bradbury et al., 2002).
Concomitantly, an increase in adhesive interactions mediated by integrins
might be expected, and this would enhance cell-substrate at the expense of
cell-cell interactions. The potential activation of integrins would lead to
repercussions in signal transduction pathways and activated integrins may
interact with growth factor-related signal transduction mechanisms
(Colognato et al., 2005;
Leone et al., 2005).
Remarkably, the survival and regeneration fostering potency of stem cells can
be boosted by transfection with the L1 cell adhesion molecule of the Ig
superfamily (Bernreuther et al.,
2006; Chen et al.,
2005). Hence, the impact of ChABC activity on cell adhesion
molecule gene families and their downstream signal transduction in the realm
of NSC biology represents a challenging topic for future studies.
Conclusion
The present study suggests a role for CSPGs in stem cell biology. How CSPGs
are integrated into the complex interplay of pericellular determinants of cell
fate remains to be investigated in detail. However, the identification of the
pivotal role of CS-GAGs in regulating stem cell proliferation and neurogenesis
represents an important step forward in identifying key factors of the local
environment that regulate stem cell fate and neurogenesis. Indeed, previous
transplantation experiments have highlighted the local environment as the key
determinant of adult neurogenesis. Glial cells isolated from non-neurogenic
regions of the adult CNS generate neurons when transplanted into a neurogenic
environment (Shihabuddin et al.,
2000), whereas neurogenic precursors isolated from the adult SEZ
do not succeed in generating neurons outside their niche
(Lim et al., 2000). Thus, a
better understanding of the NSC niche is of the utmost importance in the
context of employing NSCs for repair processes
(Scadden, 2006). Our work
demonstrates for the first time that complex CS-GAG carbohydrates play a
pivotal role in the orchestration of the NSC micromilieu.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/15/2727/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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