1 Departments of Medical Genetics and Pathology, University of Cambridge, Tennis
Court Road, Cambridge CB2 1QP, UK
2 Institute of Cell Biology, Department of Biology, Swiss Federal Institute of
Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland
3 Department of Molecular Medicine, Max-Planck-Institut für Biochemie, Am
Klopferspitz 18a, D-82152 Martinsried, Germany
* Author for correspondence (e-mail: cfc{at}mole.bio.cam.ac.uk)
Accepted 31 March 2004
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SUMMARY |
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Key words: Extracellular matrix, Laminin, Fibronectin, Neurosphere, Cre/lox, Stem cell niche, Ventricular zone, Subventricular zone
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Introduction |
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Although a number of the transcription factors that control neural stem
cell differentiation into neurones and glia have been identified
(Kintner, 2002), the crucial
mechanisms that maintain the neural stem population within the SVZ throughout
life remain undefined. An important contribution to the maintenance of stem
cells in other developmental systems is made by extracellular signals present
in specific microenvironments or `niches'
(Watt and Hogan, 2000
;
Spradling et al., 2001
). So,
for example, BMPs and their analogues have been shown to maintain germline
stem cells in mice (Lawson et al.,
1999
; Ying et al.,
2000
) and Drosophila
(Xie and Spradling, 1998
). The
instructive potential of such extracellular signals for neural stem cells is
illustrated by the finding that oligodendrocyte precursor cells can revert
back to a stem cell state when exposed to appropriate growth factors
(Kondo and Raff, 2000
). These
experiments highlight the need to identify the `niche' signals within the SVZ
and those cell-surface receptors present on neural stem cells that are
required for their recognition.
In addition to growth factors, another potentially important class of
signals are those provided by extracellular matrix (ECM) molecules recognised
by integrin receptors. Integrin signalling pathways are instructive for cell
migration, proliferation, differentiation and survival. These pathways
interact with those downstream of growth factor receptors, so providing
coordinated regulation of cell behaviour by growth factors and the ECM
(Yamada and Even-Ram, 2002).
Integrins are heterodimers of two transmembrane chains,
and ß.
The ß1 subunit is widely expressed and can heterodimerize with at least
12 different
subunits, generating integrins with differing ligand
specificities (Hynes, 1992
).
Integrins containing the ß1 subunit regulate epidermal stem cell
maintenance (Jensen et al.,
1999
; Zhu et al.,
1999
; Raghavan et al.,
2000
). The higher expression of the laminin receptor
6ß1 has been implicated in the maintenance of mouse spermatogonial
stem cells (Shinohara et al.,
1999
) and human embryonic stem cells
(Xu et al., 2001
). Together,
these results suggest that integrin signalling may also play a role in neural
stem cell maintenance. We have shown previously that neural precursor cell
populations containing stem cells express a number of ß1 integrins
(Jacques et al., 1998
). Here
we have examined the function of these ß1 integrins in neural stem
behaviour. We have determined the distribution of ß1 integrins and
extracellular matrix ligands within the germinal neuroepithelium during CNS
development, and have used cell culture assays to demonstrate that
MAPK-dependent signalling pathways downstream of these integrins contribute to
neural stem cell maintenance.
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Materials and methods |
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Neurosphere culture preparation
Primary cultures were prepared from newborn rats and mice (postnatal day
0-2; P0-P2) (Jacques et al.,
1998). Briefly, spheres of neural precursors were grown in EGF or
FGF2 (20 ng/ml) from dissociated P0-P2 rat forebrain in DMEM/Hams-F12
supplemented with B27 (Reynolds and Weiss,
1992
; Svendsen et al.,
1995
). The culture media was changed every 3 days.
Western blots
For protein expression analysis neurospheres were lysed in lysis buffer (10
mM Tris-HCl, 5 mM EDTA, 150 mM NaCl buffer and 1% Triton X-100) containing
proteases and phosphatase inhibitors (5 µg/ml leupeptin, 2 µg/ml
aprotinin, 2 mM PMSF, 1 µg/ml pepstatin, 2 mM sodium fluoride, 2 mM sodium
vanadate; all from Sigma). The supernatant was clarified by centrifugation at
16,000 g for 20 minutes at 4°C. Protein concentrations in
the supernatant were determined using a Bio-Rad protein assay, with BSA as a
standard. For western blotting, 20 µg of protein was loaded in each
condition. Proteins were separated by SDS-PAGE and electroblotted onto
nitrocellulose membranes (Hybond-C, Pharmacia). Membranes were blocked in 10%
non-fat dry milk in Tris-buffered saline (TBS) for 1 hour at room temperature.
Blots were then incubated with the primary antibodies overnight at 4°C in
milk-TBS containing 0.1% Tween-20 (TBS-T), followed by a 2 hour incubation
with the appropriate secondary peroxidase-conjugated antibody (Amersham). To
visualize the immunoreactive proteins the ECL kit was used, following the
manufacturer's instructions (Amersham).
Fluorescence activated cell sorting (FACS) of neural stem cells
Neurospheres were dissociated and incubated with the FITC-conjugated Ha2/5
monoclonal antibody (Pharmingen) in PBS at 4°C for 30 minutes; this
antibody was used previously to study ß1 expression levels in fetal liver
cells (Suzuki et al., 2000).
Control cells were incubated in PBS only. The cells were then counter-stained
with propidium iodide (5 µg/ml, Sigma) for live/dead discrimination and
sorted through a MoFlow (Cytomation). Non-FITC fluorescent cells were excluded
from the sorting. Amongst the fluorescent cells (of either EGF- or FGF2-grown
neurospheres) we defined two groups: a group of very strongly fluorescent
cells and a group of cells with intermediate levels of labelling. The same
number of events was then sorted from each population. Five thousand cells
were deposited in each well of a 24-well plate. The cells isolated by FACS
were then grown for a week in the appropriate medium and the number of
secondary neurospheres formed for each condition was counted. These
experiments were done five times for EGF- and four times for FGF2-grown
neurospheres. Statistical significance was determined using Student's
t-test.
Serial dilution assays
Neurospheres were completely dissociated, and 4000 cells were re-suspended
in 400 µl of culture media with EGF or FGF2. Dilution assays were set up on
96-well plates (Iwaki) as follows: 200 µl of the initial 400 µl
containing the 4000 cells was transferred to the adjacent well, which already
contained 200 µl of media. From this second well another 200 µl was
collected and transferred. This operation was repeated for all the wells in
each line creating a range of cell concentrations from 2000 (first well) to
1-2 cells (twelfth well). Each individual experiment was performed in
triplicate, with the exception of the experiments using U0126/4 which were
performed in duplicate. Serial dilutions were set up in the presence of the
EGF receptor inhibitor AG1478 (20 µM), MAPK inhibitors (PD98059, 50 µM;
U0126/4, 100 µM), the p38 MAPK inhibitor SB203580 (50 µM) and PI3K
inhibitors (wortmannin, 50 nM; LY294002, 2.5 µM). After one week, the newly
formed neurospheres were counted in each well and plotted against the initial
number of cells per well. The resulting slope of the line was used to compare
the different experimental conditions. Statistical analysis of the slopes of
the regression lines was performed using Student's t-test.
Conditional knock-out of ß1 in neurospheres
Neurospheres were prepared from P0 mice bred to have either the floxed
ß1/null or floxed ß1/wild-type (wt) phenotype (by crossing mice
carrying the floxed allele with those heterozygous for a null allele of
ß1), and were grown in both EGF and FGF2 (20 ng/ml). The floxed ß1
allele was generated as previously described
(Brakebusch et al., 2000).
After 10 days in culture, the neurospheres were dissociated, cells were
infected with an adenoviral vector expressing cre recombinase
(Kalamarides et al., 2002
) (a
kind gift from Dr Marco Giovannini, INSERM U434, Paris) and then replated in
the same growth factors. The culture medium was changed after 3 days to medium
without adenovirus. Recombination was confirmed by the expression of
ß-galactosidase in the newly-formed neurospheres, as excision of the
ß1 gene activates a lacZ reporter gene in the floxed allele. The
number of entirely blue neurospheres formed by the floxed ß1/null was
then compared with the number of entirely blue neurospheres made by the
control floxed ß1/wt for an equal number of plated cells (3000 cells/well
of a 24-well plate). Neurospheres in which excision had not occurred or was
incomplete (as evidenced by cells not expressing ß-galactosidase) were
not included in the assay. For the experiments analysing MAPK phosphorylation,
neurospheres generated from floxed ß1/null or floxed ß1/wt cells
were passaged at least ten times and then incubated overnight in the EGF
receptor inhibitor AG1478 (20 µM), in the presence of 20 ng/ml EGF and
FGF2. The spheres were then lysed as above and western blot analysis performed
using MAPK and phosphorylated MAPK antibodies, as described above. Equal
loading was confirmed by protein quantification. The gels were scanned and
quantified using the NIH Image 1.62 software, with the ratio of the band
intensities of total MAPK and phosphorylated MAPK calculated for each
neurosphere cell line, with and without the inhibitor. Three cell lines of
each genotype were analysed. All samples were run on a single gel and
processed simultaneously, with the total MAPK analysis performed by stripping
the membrane after the P-MAPK analysis. Student's t-test was used to
compare the ratio of band intensities following exposure to AG1478 in both of
the two genotypes.
Immunohistochemistry and cell counts
For proliferation assays BrdU was added to the culture media (10-20
µmol/l) 30 minutes, 3 hours or 24 hours before fixation. Neurospheres and
neonatal or embryonic brain tissue were then fixed in 2-4% paraformaldehyde in
PBS (phosphate buffered saline). The samples were then transferred to a 25%
solution of sucrose (w/v) and left overnight at 4°C. Cryostat sections (14
µm) were prepared from imbedded brains or neurospheres (TissueTek-Sakura)
and used for immunohistochemistry. Sections were blocked in PBS (0.1% Triton
X-100) containing normal goat serum and incubated overnight with the
appropriate antibodies at 4°C. After incubation with the appropriate
secondary antibodies and counter-staining with DAPI, pictures were acquired
using a Zeiss fluorescence microscope equipped with a Hamamatsu Orca camera.
Images were processed using Open Lab (Improvision).
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Results |
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To test these predictions we cut cryostat sections of intact neurospheres and immunostained them with anti-ß1 integrin antibodies. These experiments showed that, for spheres prepared from either rat or mouse, cells expressing high levels of ß1 were present only on the edge of the sphere (Fig. 3A,B). We then compared this distribution with that of a stem and early precursor cell marker (the intermediate filament protein nestin, Fig. 3B,C) and with that of markers of committed precursor cells for neurones (ß3 tubulin, Fig. 3F) and astrocytes (GFAP, Fig. 3D,E). Nestin+ cells were present around the edge of the sphere within the population expressing high levels of ß1 integrin (Fig. 3B), whereas the differentiation markers were mainly present inside the sphere (Fig. 3D-F), demonstrating the separation of stem cell and committed precursor populations.
|
Just as in the intact developing CNS, we found that laminin 2 was
present in the region containing the cells that express high levels of ß1
(Fig. 4A,D). Laminin 1, by
contrast, was expressed in the central regions of the sphere
(Fig. 4C), and fibronectin was
diffusely localised in a speckled pattern (not shown) similar to that seen in
the intact CNS. Together, these observations reveal a distribution of the
different molecules within the sphere that mirrors that seen in the intact
CNS; the three-dimensional structure of the spheres is summarised in
Fig. 4B.
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For the genetic studies, we used cre/lox technology to remove the ß1
gene from neurosphere cells and determine the effect on MAPK activation. This
technology has been used previously to excise ß1 integrin efficiently
from chondrocytes (Iba et al.,
2000). Mice containing a floxed ß1 allele
(Fassler and Meyer, 1995
;
Brakebusch et al., 2000
)
generated by homologous recombination were bred with heterozygous ß1-null
mice to generate animals in which the second allele was either ß1-null or
wild type. Neurosphere cultures were prepared from these animals and grown in
EGF and FGF2. The spheres were dissociated, replated at a density of 10
cells/µl and exposed to an adenoviral vector expressing cre recombinase.
Excision of the floxed ß1 gene was confirmed by the activation of a
reporter lacZ gene inserted downstream of the ß1 allele, which
is activated only following excision of the ß1 sequence
(Brakebusch et al., 2000
;
Potocnik et al., 2000
).
Spheres derived from stem cells in which excision had occurred could be
identified by the presence of ß-galactocidase staining in all cells. The
reduction in ß1 integrin following excision was confirmed in three ways.
First, neurospheres exposed to the adenoviral vector but not yet dissociated
and replated were sectioned as above and immunolabelled with anti-ß1
antibodies. Second, lysates from treated and control spheres were used in
western blotting experiments. Third, FACS analysis was performed to detect
changes in the level of ß1 expression. As shown in
Fig. 8, following gene excision
we observed a reduction in ß1 integrin immunolabelling around the edge of
the spheres (Fig. 8B-E), a
reduction in ß1-labelling levels in the FACS analysis
(Fig. 8F,G) and a reduction in
the intensity of the ß1 integrin band, as revealed by western blotting
(Fig. 8H). Having confirmed the
excision procedure, we next performed western blotting experiments using
antibodies against phosphorylated MAPK. These showed a reduction in MAPK
activation in primary neurospheres exposed to the cre-expressing adenovirus
(Fig. 8I). However, subsequent
passages of the ß1-excised spheres showed normal levels of activated MAPK
(Fig. 8J,
Fig. 9), even though the
presence of ß-galactosidase in these spheres and a shift to the left in
the FACS analysis of ß1 expression confirmed that these spheres derived
from cells in which gene excision had occurred (data not shown). We also
counted the number of neurospheres that formed when these cells were plated at
low density. No differences were seen in the number of spheres in these
assays, or when secondary and subsequent passaging of the spheres was
performed.
|
Each line was passaged at least ten times following cre-mediated excision, by which time MAPK levels had returned to the levels seen in the ß1-expressing spheres (Fig. 8J). After an overnight exposure to the inhibitor, levels of total MAPK and phosphorylated MAPK (P-MAPK) were analysed by western blotting (Fig. 9A,B). A marked decrease in P-MAPK was observed in the ß1-excised cells, in contrast to the ß1-expressing cells (Fig. 9A,B). Analysis of all six lines showed a significant reduction in the P-MAPK/total MAPK ratio in the ß1-excised cells (1.05±0.08 to 0.84±0.01 in the presence of AG1478, P=0.005), but no significant reduction in the ß1-expressing cells (1.22±0.15 to 1.23±0.22 in the presence of AG1478), consistent with an increased dependence of MAPK activation on EGF signalling in the ß1-deficient cells, and suggesting that upregulation of growth factor signalling contributes to the compensation for the loss of ß1 integrin. The lack of viability and absence of any sphere formation when cells were grown at high dilutions in the presence of the EGF receptor inhibitor (Fig. 7D) prevents the comparison of the ß1-expressing and ß1-deficient cells grown in EGF required to analyse whether this compensatory upregulation is associated with an increased dependence on EGF signalling for maintenance. However maintenance in the presence of FGF2 was not altered by the addition of the inhibitor to ß1-excised cells (data not shown), showing that self-renewal in response to other growth factors was not reduced.
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Discussion |
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How might ß1/ECM interactions affect stem cell behaviour? Increased
adhesion to niche ECM components could ensure that the cell is held in the
niche and so exposed to other extracellular cues that instruct stem cell
maintenance. One example of such extracellular cues is provided by the
Drosophila protein Upd, which is secreted by hub cells in the testis
and contributes to the maintenance of germline stem cells by activation of the
JAK/STAT signalling pathway (Kiger et al.,
2001). Upd binds to the extracellular matrix
(Harrison et al., 1998
) and
previous studies using embryonic mosaics to examine the role of Upd in
segmentation have noted that upd mutant cells could behave normally
only when immediately adjacent to wild-type cells
(Gergen and Wieschaus, 1986
),
suggesting a limited range of diffusion as a result of tethering to the
matrix. Within the testis germ cell niche, the matrix could limit the range of
Upd diffusion from the hub cell ensuring that only adjacent germline stem
cells held in the niche by appropriate adhesion molecules respond to this
signal.
In addition to a role in simply holding the stem cells within a niche, our
results point to a direct signalling role for ß1 integrins in neural stem
cell regulation, as we have shown that self-renewal, or maintenance, in neural
stem cells is partly regulated by ß1 integrins and growth factors through
a MAPK signalling pathway. A similar role for MAPK in stem cell maintenance
has previously been described in human epidermal stem cells
(Zhu et al., 1999). The
interaction between integrin and growth factor signalling, well described in
other cell types (Lee and Streuli,
1999
; Renshaw et al.,
1999
; Byzova et al.,
2000
; Yamada and Even-Ram,
2002
), provides a mechanism by which neural stem cell behaviour
can be regulated by local ECM molecules, as well as by longer range growth
factor signals. Our results indicate a novel integrative role of MAPK that may
be important in allowing populations of stem cells to change behaviour during
development and repair in response to changes in the growth factor
environment, while at the same time allowing individual stem cells to be fine
tuned by their immediate microenvironment. Such signalling mechanisms with the
potential for compensation between integrin and growth factor signalling
pathways may also be operative in other stem cell types; for example, studies
of haematopoietic stem cells in mice chimeric for ß1 null cells show that
ß1 integrin is not required for stem cell maintenance, but is necessary
for the homing of these cells to the liver
(Hirsch et al., 1996
).
The conclusion that ß1 integrins regulate neural stem cell behaviour
within the CNS niche leads us to question the nature of the ligands recognised
by these integrins. One important group of candidates are the laminins, which
are recognised by at least four ß1 integrins, 1ß1,
3ß1,
6ß1 and
7ß1.
6ß1
expression has previously been shown in other stem cell systems
(Shinohara et al., 1999
,
Xu et al., 2001
), and in
RT-PCR experiments we have found all four alpha subunits to be expressed in
neural stem cells (J. Moore and C.ff.-C., unpublished). Laminins are expressed
from the earliest stages of development, and have been shown to maintain human
ES cells in an undifferentiated state (Xu
et al., 2001
). Proximity to a basement membrane is a feature of
epithelial and germ cell niches, and transplantation experiments using
reconstitution of spermatogenesis as an assay reveal that stem cells can be
enriched from testis cell populations by selecting either for laminin binding
or for expression of ß1 or
6 integrin subunits
(Shinohara et al., 1999
). In
this paper, we have described the expression of the laminin
2 chain in
the developing germinal zone and also around the edge of the neurospheres in
the regions containing the stem cell populations. Laminins containing this
chain therefore provide a potential ß1 integrin ligand for neural stem
cells that is likely, by analogy with the ES cell and spermatogonial stem cell
systems, to play a role in the control of neural stem cell behaviour.
Furthermore, changes in the expression levels of laminin
2 provide an
additional mechanism for the regulation of neural stem cell behaviour. We
observe a decline in laminin
2 expression in the postnatal brain that
may play an instructive role in the reduced level of proliferation within the
germinal neuroepithelium at that time, as laminin can increase the
proliferation of neuroepithelial cells
(Drago et al., 1991a
). Equally,
the increase in laminin chain mRNAs seen within neuroepithelial cells exposed
to bFGF may contribute to the mitogenic effects of the growth factor
(Drago et al., 1991a
;
Drago et al., 1991b
).
The importance of extrinsic signals in the regulation of stem cell
behaviour is emphasized by the argument that, "rather than referring to
a discrete cellular entity, a stem cell most accurately refers to a biological
function" (Blau et al.,
2001). Our results point to a complex interplay between integrin
and growth factor signals in the germinal neuroepithelium being important
regulators of this function, with MAPK being a key integrative signalling
molecule. For future studies examining the developmental significance and
therapeutic potential of these interactions, our work highlights the value of
the analysis of neurosphere structure. The morphological observations
described above suggest that neurospheres derived from postnatal brains can be
used as a model for the developing (midgestation) neuroepithelium. These
spheres develop a three-dimensional structure that is remarkably similar in
ECM composition and distribution of cellular phenotypes to the developing CNS.
In particular the edge of the neurosphere is a complex niche, easily
accessible and amenable to acute biochemical manipulation and analysis using
inhibitors and blocking antibodies that will greatly facilitate further
work.
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ACKNOWLEDGMENTS |
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Footnotes |
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