1 Cambridge Centre for Brain Repair, and Departments of Medical Genetics and
Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP,
UK
2 Department of Molecular Neurobiology, Ruhr University, Building NDEF 05/593,
Universitaetsstraße 150, D44801 Bochum, Germany
Author for correspondence (e-mail:
cfc{at}mole.bio.cam.ac.uk)
Accepted 24 March 2004
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SUMMARY |
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Key words: Tenascin C, Growth factor, Proliferation, Differentiation, Neurogenesis, Gliogenesis, Stem cell, Central nervous system, Neurosphere
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Introduction |
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At the present time, the best-studied factors regulating neural stem cell
behaviour are the growth factors. Epidermal growth factor (EGF), basic
fibroblast growth factor (FGF2) and transforming growth factor alpha
(TGF) are mitogens for neural stem cells both in vivo and in vitro
(Richards et al., 1992
;
Kilpatrick and Bartlett, 1993
;
Gritti et al., 1996
;
Gritti et al., 1999
). Other
growth factors including bone morphogenic proteins (BMPs), platelet-derived
growth factor, ciliary neurotrophic factor and brain-derived neurotrophic
factor can regulate stem cell differentiation
(Gross et al., 1996
;
Johe et al., 1996
;
Benoit et al., 2001
). The
timing of growth factor responses reflects a changing pattern of growth factor
receptor expression during neural stem cell development, driven, at least in
part, by the growth factors themselves. Early embryonic neural stem cells
respond only to FGF2 whereas late embryonic and adult neural stem cells are
responsive to both FGF2 and EGF (Reynolds
and Weiss, 1992
; Kilpatrick
and Bartlett, 1995
; Burrows et
al., 1997
; Qian et al.,
1997
; Alvarez-Buylla and
Temple, 1998
), with the acquisition of EGF-responsiveness
stimulated by FGF2 and inhibited by BMP4
(Lillien and Raphael, 2000
).
The BMPs have been shown to promote proliferation, apoptosis or terminal
differentiation of neural stem cells into either neurons or glial cells at
progressively later stages of embryonic development
(Graham et al., 1996
;
Gross et al., 1996
;
Furuta et al., 1997
;
Li et al., 1998
,
Panchision et al., 2001
).
During the expansion phase of neural stem cell development, precursor cells
express BMPR1A and respond to BMP by proliferation and by upregulation of a
second BMP receptor, BMPR1B. This receptor triggers mitotic arrest once levels
exceed that of BMPR1A, so initiating the differentiation phase of neural stem
cell development (Panchision et al.,
2001
).
The soluble and diffusible properties of growth factors make them capable
of signalling over a long range. The restricted localization of neural stem
cells in embryonic and postnatal development, and their developmental
regulation independent of adjacent regions of the CNS, therefore suggests the
presence of mechanisms whose role is to restrict or amplify growth factor
signalling. One such mechanism is the secretion of short-range inhibitory
factors such as the BMP inhibitor, noggin. The production of noggin by
ependymal cells prevents adjacent neural stem cells from responding to the
gliogenic properties of BMPs in the adult mouse SVZ, so generating a
restricted neurogenic environment (Lim et
al., 2000). Another potential mechanism is the distribution of
extracellular matrix (ECM) molecules. One of these, tenascin C (TNC), is
highly expressed within the SVZ throughout postnatal and adult life
(Gates et al., 1995
). TNC can
control cell behaviour both indirectly by binding other matrix components and
also directly by interactions with specific cell surface receptors
(Jones and Jones, 2000
). We
have recently shown that TNC null mice show a lower level of oligodendrocyte
progenitor proliferation as a result of reduced sensitivity to the mitogen
PDGF (Garcion et al., 2001
).
This evidence that TNC normally amplifies the mitogenic role of PDGF during
the development of myelin-forming cells suggests that TNC and other ECM
molecules present in germinal zones of the CNS could play a role in
restricting or amplifying growth factor responses in neural stem cells. Here
we examine this hypothesis by analysing the behaviour of wild-type and
tenascin C-deficient neural stem cells both in vivo and when grown in cell
culture as neurospheres.
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Materials and methods |
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Cell culture of primary embryonic and postnatal neural stem cell-derived neurospheres
Telencephalic vesicles from E10.5 embryos, and striatum and/or cortex from
E12.5 embryos or newborn animals (plug day designated E0) were dissociated and
neurosphere forming cells grown in 50% DMEM (Sigma)/50% Ham's-F12 (Sigma)
medium with B27 supplement (Gibco) in the presence of 0.2 to 20 ng/ml FGF2
(Peprotech) or 0.2 to 20 ng/ml EGF (Calbiochem). For all experiments with
FGF2, 5 µg/ml heparin (Sigma) was added. Where appropriate, human
recombinant BMP4 (R&D Systems) at 10 ng/ml or TNC purified from neonatal
mouse brains by immuno-affinity column chromatography
(Faissner and Kruse, 1990) was
also added. For the clonal density experiments, 20,000 cells were plated onto
a 100 cm2 plate.
Amplification of TNC mRNA by reverse transcription-polymerase chain reaction (RT-PCR)
RNA from wild type or TNC null neurospheres was isolated using the RNeasy
Mini kit (Qiagen) and reverse-transcribed using First-Strand cDNA synthesis
kit (Pharmacia Biotech). PCR reactions were then performed with 10 pmol of
oligonucleotides hybridizing to the 5' end of the fifth and the 3'
end of the sixth conserved fibronectin type-III domain of mouse TNC
(5'-CAC GTG TGA AGG CAT CCA CG-3' and 5'-TAT CCT TCG GAG AAC
CCA TGG C-3'), with a 5 minutes denaturation step at 95°C followed
by 35 cycles of 1 minute at 95°C, 2 minutes at 60°C, and 2 minutes at
72°C and a final extension step at 72°C for 10 minutes.
Serial dilution assays to measure neural stem cell numbers
Serial dilution analysis to analyse neural stem cell numbers in a specified
number of cells was performed according to Bellows and Aubin
(Bellows and Aubin, 1989) with
minor modifications from Tropepe et al.
(Tropepe et al., 1999
). Cells
isolated from P0 cortical and striatum using papain and mechanical
dissociation were plated in 96-well microwell plates containing either EGF (20
ng/ml) or FGF2 (20 ng/ml), with 4000 cells per well in 400 µl of media.
Serial dilutions were then made to obtain final cell numbers from 2000
cells/well to 1 cell/well in 200 µl media. After 7 days the number of
spheres (nsph) per well was quantified and plotted against the number of cells
plated (ncp) per well. The slope of the regression line obtained for each cell
population reflects the proportion of neurosphere forming cells (stem cells)
present in the entire original cell suspension. For the assays to analyse stem
cell numbers within all cells dissociated from intact cortex and striatum, the
entire cell population obtained from the dissociation was serially diluted in
a 96-well plate in twofold steps with a final volume of 200 µl. Growth
media contained FGF2 (20 ng/ml) and Heparin (5 µg/ml) or EGF (20 ng/ml).
After 7 days the highest dilution at which one or more neurospheres was
observed in the culture well was determined.
Immunohistochemistry and Western blotting
Frozen sections from snap frozen tissues were thawed and fixed in 4%
paraformaldehyde (PFA) in PBS for 10 minutes, then incubated for 1 hour in
blocking solution containing 10% normal goat serum (Sigma), 4% bovine serum
albumin (BSA, Sigma) and 0.1% Triton X-100 (Sigma) in PBS. For EGF receptor
immunostaining, a rabbit polyclonal anti-EGF receptor (sc-03, Santa Cruz
Biotechnology) at 1/100 dilution in 4% BSA and 0.1% Triton X-100 in PBS was
used overnight at 4°C followed by a biotin-conjugated anti-rabbit antibody
(Vector, 1/100 dilution) and TRITC-conjugated streptavidin (Amersham, 1/100).
For detection of radial glia, the RC2 monoclonal antibody
(Edwards et al., 1990) was
used at 1/100 dilution followed by FITC-conjugated anti-mouse IgM secondary
antibody (Sigma, 1/100). The antibody was obtained from the Developmental
Studies Hybridoma Bank developed under the auspices of the National Institute
of Child Health and Human Development (NICHD) and maintained by the University
of Iowa, Department of Biological Sciences (Iowa City, IA, USA). Slides were
finally counterstained with DAPI (1/1000, Sigma) and mounted in ImmunoFloure
mounting medium (ICN). For Western blotting we used the polyclonal rabbit
anti-EGF receptor at 1/500 and a polyclonal donkey
horseradishperoxidase-linked anti-rabbit at 1/2000 (Amersham). After 3-7 days
culture, neurospheres were washed with cell wash buffer (50 mM Tris-HCl pH
7.5, 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2) and lysed on
ice for 30 minutes in extraction buffer (cell wash buffer plus 0.5% NP40, 2 mM
PMSF, 5 µg/ml leupeptine, 2 µg/ml aprotinin, 1 µg/ml pepstatin A, 2
mM sodium fluoride, 2 mM sodium vanadate and 4 mM sodium pyrophosphate). After
clarification by centrifuging at 16,000 g for 10 minutes at
4°C, the sample lysates were then adjusted to 10 µg of protein per lane
and run on SDS-PAGE gels under reducing conditions. Proteins were transferred
and detected using ECL (Amersham).
Cell proliferation assay
Proliferation within neurospheres grown for 7 days in the presence of FGF2
or EGF was assessed by measuring bromodeoxyuridine (BrdU) incorporation after
addition to the culture medium for 2 hours. To count labelled cells,
neurospheres were dissociated with trypsin-EDTA (Sigma), plated onto
poly-D-lysine-coated slides and fixed in ethanol 95%/acetic acid 5% for 20
minutes at 20°C, following which cells could be immunolabelled for
BrdU using an immunofluorescence assay kit (Roche) as previously described
(Garcion et al., 2001). After
counterstaining with propidium iodide (PI, 1/1000, Sigma), the number of BrdU+
cells as well as the number of PI+ cells were counted in at least five
different fields using a x20 objective (corresponding to more than 500
cells) in at least three independent experiments for each condition.
Radial glia assay
The radial glia assay was performed as described previously by Hartfuss et
al. (Hartfuss et al., 2001).
In brief, telencephalic vesicles were dissected out and dissociated. For E10.5
and E13.5 whole vesicles were pooled together, whereas for E18.5 and P0
striatum and cortex were treated separately. Cells were resuspended in 10%
FCS/DMEM and plated on PDL slides for 2 hours followed by a 10-minute fixation
in 4% PFA/PBS. After this, immunocytochemistry was performed using the RC2
monoclonal antibody to detect radial glia cells
(Edwards et al., 1990
). The
proportion of RC2+ cells was calculated in at least three animals at each age,
and significance calculated using Student's t-test.
Neurosphere-derived cell differentiation assay
After seven days of culture, neurospheres derived from cortex or striatum
were plated onto poly-D-lysine-coated slides without mitogens in 50% DMEM/50%
F12 medium with B27 supplement and 0.5% horse serum, and allowed to adhere for
6 days. During this time cells migrate out of the sphere and differentiate.
For the rescue experiments using exogenous TNC, immunopurified mouse TNC was
added throughout both stages of the culture at a concentration of 1 µg/ml.
The cultures were then fixed in 4% PFA and processed for immunofluorescence as
previously described (Garcion et al.,
2001). The following antibodies were used: mouse monoclonal
anti-ßIII-tubulin (ß3-tub, 1/500, Sigma), rabbit polyclonal
anti-glial fibrillary acidic protein (GFAP, 1/750, DAKO), rat polyclonal
anti-myelin basic protein (MBP, 1/100, Serotec) and mouse monoclonal
anti-galactocerebroside-C (Gal-C, 1/50, Sigma). Slides were counterstained
with DAPI (1 µg/ml, Sigma) and mounted in Immunofloure mounting medium
(ICN). Quantification of each cell type (neurons - ß3-tub+, astrocytes -
GFAP+ and oligodendrocytes Gal-C or MBP+) was assessed in three independent
experiments by analysing 10 different fields within the rim of migrating cells
using a x40 objective and OpenLab software (Improvision).
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Results |
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Taken together, the experiments above show that the expression of the EGFR
in neural stem cells is delayed by the absence of TNC. Previous work has shown
that EGF receptor expression is stimulated by FGF2 and inhibited by BMP4
(Lillien and Raphael, 2000).
If, as we have hypothesised, TNC regulates neural stem cell development by
modulating growth factor signalling then we would predict changes in the
response to the effects of FGF2 and/or BMP4 in the TNC null cells. We
therefore examined neural stem cell proliferation and EGFR expression in
response to FGF2 and BMP4, respectively in wild-type and TNC-deficient cells.
As shown in Fig. 4A-B, E12.5
and P0 cells from wild-type mice proliferate more than TNC null cells in
response to FGF2, showing that TNC enhances FGF2 signalling in neural stem
cells.
|
Regulation of neural stem cell differentiation by TNC
Having established a role for TNC in the acquisition of EGFR expression by
neural stem cells, we next examined the consequences of TNC deficiency on
neural stem cell development and differentiation. To do this, we examined in
embryonic and newborn TNC-deficient mice the numbers of FGF2- and
EGF-responsive neural stem cells, the morphology and numbers of radial glial
cells and the ability of the neural stem cells to form neurones and glia.
To count neural stem cells in the mice we used neurosphere-forming assays.
In initial experiments plating cells at clonal density in the presence of
FGF2, we observed more neurospheres in cultures prepared from the newborn
TNC-deficient mice although the spheres from the TNC mice were smaller then
wild-type spheres (Fig. 5A). The same increase in sphere number and reduction in size was also seen in the
populations of secondary neurospheres that grew from single cells replated at
clonal density following dissociation of the primary neurospheres (not shown).
This excludes the possibility that the initial smaller spheres seen with the
TNC-deficient cells represented clusters of precursor cells, as these would
not generate secondary neurospheres following passaging. While the smaller
size of the spheres could reflect a reduced number of precursor cells, whose
proliferation rate is reduced in the absence of TNC, the consistent increase
in sphere numbers suggests that the TNC-deficient CNS (and TNC-deficient
neurospheres) may contain a higher number of neural stem cells. To quantify
this we used a serial dilution assay based on that described originally by
Bellows and Aubin (Bellows and Aubin,
1989) and modified by Tropepe et al.
(Tropepe et al., 1999
) to
assay the neurosphere-forming potential of newborn cell populations. These
assays were performed in two ways. First, we used as the starting population a
fixed number of cells (4000) obtained from dissociated newborn cortex and
striatum. The use of both regions ensured that all the SVZ was included. Cells
were grown in either FGF2 or EGF, and the number of neurospheres formed at
different dilutions counted as described in Materials and methods. As shown in
Fig. 5B, the percentage of
cells with the ability to form neurospheres (indicated by the slope of the
line when the number of spheres formed is plotted against the number of cells
plated) was significantly greater in the TNC-deficient cells when cells were
grown in FGF2. In contrast, no differences between TNC-deficient and wild-type
cells were observed in EGF, showing that the differences in the numbers of
EGF-responsive neural stem cells seen at E10.5 are no longer present at
birth.
|
As another method to analyse the effect of TNC deficiency on neural stem
cell populations, we also examined radial glial cells. These cells, which
represent a subset of the total stem cell population in the embryonic CNS
(Hartfuss et al., 2001;
Heins et al., 2002
), were
identified using the RC2 antibody (Edwards
et al., 1990
). No changes in morphology were seen at either of the
ages examined (E13 and P0), with processes remaining straight and lacking the
sharp turns as seen, for example, in the Pax6 mutant cortex
(Gotz et al., 1998
). However,
the intensity of RC2 labelling was diminished in the TNC-deficient CNS
(Fig. 6A-D). To determine
whether this reflected a reduction in the number of radial glia we performed
RC2 immunocytochemistry on acutely dissociated cell populations
(Fig. 6E). We found a reduction
in RC2+ cells at E13.5, E18.5 and P0, demonstrating a reduction in radial
glial numbers in the TNC-deficient CNS. At E10.5, at which stage RC2 labels
neuroepithelial cells prior to the development of radial glia
(Malatesta et al., 2003
), no
differences were seen between the genotypes.
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Discussion |
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Neural stem cell development is accompanied by changes in response to
growth factors such as FGF, BMPs and EGF as embryonic development proceeds
(Burrows et al., 1997;
Ciccolini and Svendsen, 1998
;
Kalyani et al., 1999
;
Tropepe et al., 1999
;
Lillien and Raphael, 2000
;
Ciccolini, 2001
). We and
others have shown previously that TNC can alter the response of cells to
mitogenic growth factors, and our results here suggest that this provides a
mechanism by which TNC facilitates normal stem cell development. In this model
(Fig. 8), TNC modulates the
sensitivity to the two growth factors, FGF2 and BMP4, that regulate EGF
receptor expression so as to promote EGF receptor acquisition. TNC enhances
sensitivity to FGF2, and the reduced sensitivity to FGF2 in the TNC null mice
may contribute to the reduced level of proliferation we have previously noted
in the SVZ of postnatal TNC-deficient mice
(Garcion et al., 2001
).
However, the fact that we observe an increase rather than a decrease in
FGF2-responsive stem cells in the absence of TNC emphasizes that the
predominant effect of TNC on cells in the SVZ is the regulation of
developmental progression rather than proliferation. In contrast to FGF2, TNC
decreases the sensitivity to BMP4, as we observed in the TNC-deficient mice an
increase in the inhibitory effects of BMP4 on EGF receptor expression. Several
potential mechanisms exist by which these effects of TNC could be mediated. An
extracellular interaction of the growth factor with TNC could inhibit or
potentiate its effect, as seen with the interaction between FGF and heparan
sulphate proteoglycans (Yayon et al.,
1991
; Nurcombe et al.,
1993
; Brickman et al.,
1995
; Caldwell and Svendsen,
1998
). Alternatively, TNC could interact with a specific cell
surface receptor, and intracellular components of the signalling pathway
downstream of this TNC receptor could then interact with growth factor
receptor signalling pathways (Jones and
Jones, 2000
), as we have shown for oligodendrocyte precursor cells
in the TNC null mouse (Garcion et al.,
2001
). Finally, TNC could regulate intracellular signalling by the
interaction with cell surface phosphatases such as members of the
receptor-like protein tyrosine phosphatase family, which could then in turn
regulate the activity of intracellular kinases
(Milev et al., 1997
). In
keeping with this we observed increased levels of Smad1 phosphorylation in
cells from TNC-deficient animals (E.G., unpublished). Smad1 is a downstream
target of BMP4 (Kretzschmar et al.,
1997
) and increased phosphorylation (reflecting increased
signalling activity in this pathway) in the absence of TNC points to an
inhibitory effect of TNC on BMP4 signalling.
|
In this model a critical function for TNC is therefore the acceleration of
the feed-forward mechanism and this may be one role for the observed
maintenance of expression of TNC in the postnatal SVZ
(Gates et al., 1995), so
providing an environment that facilitates the production and differentiation
of precursor cells when required in response to injury or cell loss. In
addition, the expression of TNC by the radial glial cells themselves
(Gotz et al., 1998
) and the
observation from gene expression profiling studies that TNC is very highly
enriched in 8-day-old neurospheres
(Ramalho-Santos et al., 2002
)
shows that TNC is produced by neural stem cells or their progeny, and will
therefore provide an autocrine/paracrine factor in a positive feedback loop
for neural stem cell development. A consequence may be the generation of a
community effect within the germinal zones of the CNS. This effect was
originally defined as a mechanism dependent on cell-cell interactions that
ensured coordinated fate specification in developing cell populations
(Gurdon, 1988
), and cell-cell
interactions have been shown to sharpen the boundaries of dose-response
thresholds in Xenopus mesoderm fate determination
(Green et al., 1994
;
Wilson and Melton, 1994
).
Community effects have also been shown to influence cell fate decisions in
response to TGFß by neural crest progenitors, with either neural fates or
apoptosis obtained within narrow ranges of increasing levels of TGFß when
cells were present in clusters (Hagedorn
et al., 2000
). Similar effects of cell-cell interactions based on
the production of TNC within the germinal zones of the CNS may ensure the
coordinated development of neural stem cells at appropriate FGF2
concentrations, so providing the extracellular matrix with a role in both the
spatial and temporal regulation of neural stem cell development.
TNC also appears to inhibit neurogenesis, as TNC null neurospheres show a
relative increase in the numbers of neurones when grown in conditions that
allow differentiation. This result is surprising in light of the reduction
observed in radial glia numbers in TNC-deficient animals. Radial glia generate
neurones during embryonic development driven, at least in part, by the
transcription factor Pax6 (Heins et al.,
2002). If the reduction in radial glia was the only abnormality
then TNC deficiency might be expected to result in lower, rather than higher,
levels of neurogenesis. However, our work also shows an increase in the
FGF2-responsive stem cell population in association with TNC deficiency, which
could contribute to the increased neurogenesis as previous work has
established that these stem cells are likely to differentiate into neurones
(Levitt et al., 1983
;
Vescovi et al., 1993
;
Kilpatrick and Bartlett, 1995
;
Johe et al., 1996
;
Qian et al., 1997
;
Tropepe et al., 1999
;
Qian et al., 2000
;
Raballo et al., 2000
).
Moreover, this is unlikely to be the only mechanism as spheres grown in EGF
also generate more neurones, and previous studies have suggested that the
EGF-responsive neural stem cell population is distinct from the
FGF2-responsive population (Tropepe et
al., 1999
). Although we cannot determine to what extent the
increased neurogenesis in the absence of TNC reflects changes either in
differentiation or in survival of neuronal and glial precursor populations, we
can conclude from the rescue effect of exogenous TNC that prior differences in
the balance of different stem cell populations are not solely responsible for
the increased neurogenesis observed in the TNC-deficient spheres. TNC is
therefore likely to regulate other transitions within the stem
cell/precursor/neurone lineage in addition to that between FGF- and
EGF-responsive stem cells. Further studies on the relationship between the
FGF- and EGF-responsive neural stem cells and radial glia, and on the
proliferation and differentiation of radial glial cells in the TNC mice will
be required to elucidate the differentiation phenotype we have reported
here.
Taken together our results show how TNC contributes to the regulation of
the self-renewal and output of a stem cell population. Microenvironments that
are able to regulate stem cell self-renewal and output in this way are
commonly defined as niches, and are present in many different vertebrate and
invertebrate stem cell systems. We conclude, therefore, that TNC is an
important component of the neural stem cell niche in the SVZ. TNC is also
present in the haematopoetic stem cell microenvironment
(Klein et al., 1993), and the
observation that mice lacking TNC show reduced haematopoesis
(Ohta et al., 1998
) confirms a
role in the regulation of stem cell output in this example of a stem cell
niche. Together with our present results, these observations raise the
possibility that TNC may play an important general role in stem cell niches as
a modulator of growth factor signalling. If so, however, why have
TNC-deficient mice been shown to have a normal morphological phenotype in many
different systems (Saga et al.,
1992
; Forsberg et al.,
1996
)? This may reflect the compensatory abilities of many
developmental processes. We have described how reduced levels of apoptosis
observed in newly differentiated oligodendrocytes in TNC-deficient mice could
compensate for the reduced proliferation of oligodendrocyte precursor cells
seen earlier in development (Garcion et
al., 2001
). The ability to correct final cell numbers in this way
could also correct any consequences of alterations in precursor numbers
resulting from any changes in stem cell development. We would predict,
however, that the ability of the CNS to respond to injury or perturbations of
development would be compromised by these abnormalities in the stem cell
compartment.
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ACKNOWLEDGMENTS |
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Footnotes |
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