1 Department of Medical Genetics and Cambridge
Center for Brain Repair, University of Cambridge, The E.D. Adrian
Building, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK
2 Centre de Neurochimie du CNRS, Laboratoire de
Neurobiologie du Développement et de la
Régénération, UPR 1352, 5 rue Blaise Pascal,
67084 Strasbourg Cedex, France
* Present
address: Department of Molecular Neurobiology, Ruhr University,
Building NDEF 05/593, Universitaetsstr. 150, D44801, Bochum,
Germany
Author for correspondence
(e-mail: cfc{at}mole.bio.cam.ac.uk)
Accepted April 12, 2001
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SUMMARY |
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Key words: Tenascin-C, vß3, Integrin, Proliferation, Central nervous system, Migration, Apoptosis, Mouse
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INTRODUCTION |
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Given the pattern of expression and the multiplicity of effects in cell culture, the observation that TN-C-null transgenic mice show no obvious abnormalities (Forsberg et al., 1996; Saga et al., 1992) was unexpected. More recent studies have revealed behavioural abnormalities, alterations in neurotransmitter levels in the adult CNS (Fukamauchi et al., 1996; Kiernan et al., 1999) and alterations in the pattern of glomerulonephritis that occurs after administration of renal toxins (Nakao et al., 1998); all these are dependent on the genetic background of the mice. Changes in the architecture of the neuromuscular junction have also been described (Cifuentes-Diaz et al., 1998), although these were not confirmed in a separate study (Moscoso et al., 1998), which may also reflect genetic background effects (see Kiernan et al., 1999 for discussion). However, the phenotype as currently described includes none of the CNS abnormalities that might be expected from previous work. There are two possible explanations for this result. Either TN-C is a largely redundant protein, which seems unlikely given the degree of evolutionary conservation (Erickson, 1993a; Erickson, 1993b; Fassler et al., 1996). Alternatively, TN-C does have specific and important roles during early stages of development but later developmental processes can correct for the loss of TN-C function. An example of such developmental correction is provided by transgenic mice that express high levels of platelet-derived growth factor (PDGF). These mice show increased OP proliferation that increases precursor cell number well above normal levels, although final oligodendrocyte numbers are normal as a result of increased cell death (Calver et al., 1998). It follows that defining a role for potentially important ECM glycoproteins within the CNS may require developmental studies of knockout mice, in which one is able to analyse different aspects of precursor cell behaviour within the intact CNS, rather than an examination of the adult phenotype. We now report such an analysis for TN-C knockout mice. To examine precursor cell behaviour we focused on the OP cells that give rise to myelin-forming oligodendrocytes. These cells arise within the germinal zones and then show phases of migration, proliferation and target-dependent programmed cell death that are characteristic of precursor cells in the developing CNS (Calver et al., 1998; Levison et al., 1993; Levison and Goldman, 1993; Levison and Goldman, 1997; Levison et al., 1999). This developmental analysis has revealed contributions of TN-C that are not apparent from studies of the adult mice. We have confirmed the role in OP cell migration suggested by the cell culture studies, have demonstrated a novel role in the regulation of neural precursor cell proliferation and provide evidence that reduced levels of programmed cell death provide a corrective mechanism that explains the normality of the final phenotype.
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MATERIALS AND METHODS |
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PDGFR in situ hybridisation
Optic nerves were removed from heads immersed in 4%
paraformaldehyde (PFA) in phosphate buffer saline pH 7.4 (PBS),
embedded in Tissue-Tek OCT compound (Agar Scientific), frozen on dry
ice and stored at -80°C until use. Coronal serial cryostat
sections (15 µm) were obtained by cutting the optic nerves from
the retinal to the chiasmal end. In situ hybridisation using a 1637 bp
EcoRI cDNA fragment encoding most of the extracellular domain
of mouse PDGF receptor (PDGF
R) cloned into Bluescript KS (a
kind gift from W. Richardson, University College, London) was then
performed as described (Kiernan et al., 1999). Average
cell numbers/section were calculated at different distances from the
retina. This method will slightly overestimate the actual cell
numbers, as discussed by Guillery and Herrup (Guillery and Herrup,
1997), but does allow a direct comparison between
wild-type and knockout animals that can be used to determine the
position of the leading edge of cell migration along the nerve.
Cell migration assays
Cell migration studies using rat oligodendrocyte precursors
migrating out of agarose drops on astroglial matrix derived from
either wild-type or TN-C-deficient mice were performed as described
previously (Milner et
al., 1996).
BrdU incorporation in vivo and immunohistochemistry
Animals received two injections of BrdU (100 µg/g body weight)
at 1 hour intervals. One hour after the last injection, animals were
sacrificed, the brains snap frozen in isopentane cooled by liquid
nitrogen (-30°C to -40°C) and cryosections (10
µm) collected. Frozen sections were thawed and fixed in ethanol
95%/acetic acid 5% for 20 minutes at -20°C,
then washed in PBS and incubated for 1 hour in blocking solution
containing 10% normal goat serum (Sigma) and 0.2%
gelatin (Sigma) in PBS. For BrdU immunostaining, a BrdU detection kit
(Roche) was used, with BrdU-labelled cells revealed using a mouse
monoclonal anti-BrdU antibody followed by an anti-mouse
FITC-conjugated antibody. For BrdU/NG2 double staining experiments,
fixed and blocked sections were first stained for NG2; tissues were
incubated with a rabbit polyclonal anti-NG2 antibody (Chemicon, 1/200
dilution in 0.2% gelatin overnight at 4°C), followed by a
biotin-conjugated anti-rabbit antibody (Vector, 1/100 dilution in
0.2% gelatin for 1 hour at room temperature) and finally
incubated with TRITC-conjugated streptavidin (Amersham, 1/100 in PBS
for 1 hour at room temperature), with PBS washes between each
step. All slides were finally mounted under coverslip in ImmunoFloure
mounting medium (ICN). For quantitation of BrdU and NG2 labelling,
BrdU-positive and NG2-positive cells were counted in seven adjacent
fields of 500 µm x 500 µm each in the subventricular
zone (SVZ). For the corpus callosum, the whole structure in one or
both hemispheres was counted. For the cortex, a region of at least
seven adjacent fields of 500 µm x 500 µm at the external
edge of both cerebral hemispheres was counted. For the striatum, an
area of seven fields of 500 µm x 500 µm each was
counted.
Cell culture
Purified oligodendrocyte precursors from mouse and rat were
obtained by the mechanical dissociation method from cultures of
cerebral cortex as originally described (McCarthy and Vellis,
1980) with minor modifications (Milner and ffrench-Constant,
1994).
Cell proliferation assay in vitro
Freshly purified OP from either wild-type or TN-C-null cultures
were plated onto either wild-type or TN-C-null astroglial matrix
(prepared from the basal monolayer in the described brain cultures by
washing with distilled water for 40 minutes) in SATO medium
(Milner
and ffrench-Constant, 1994) in the presence of varying
concentrations of PDGF. Proliferation of OP was assessed by measuring
BrdU incorporation for 6 hours at 37°C/7.5% CO2
using an immunofluorescence assay kit (Roche) as previously described
for in vivo experiments. Before mounting in ImmunoFloure mounting
medium (ICN), slides were incubated for 10 minutes in propidium iodide
(20 µg/ml) in PBS, in order to allow evaluation of the ratio of
BrdU-positive cells to the total cell population. For the TN-C rescue
experiments, TN-C purified from neonatal mouse brains by
immuno-affinity column chromatography (Faissner and Kruse, 1990)
was placed on TN-C-null astroglia matrix at the concentration of 10
µg/ml in PBS and incubated overnight at 37°C before washing
and cell plating. For the vß3 function blocking experiments,
rat OP cells were used instead of mouse cells, as discussed in the
text. OP cells were plated on LabTek chamber slides (Nunc) either
precoated with PDL alone (control) or PDL followed by an overnight
incubation at 37°C with purified TN-C at 10 µg/ml in PBS. OP
cells were grown for 18 hours in SATO medium in different
concentrations of PDGF, with or without the F11 mouse monoclonal
anti-ß3 antibody (a kind gift from M. Horton, London) at 20
µg/ml, before incubation in BrdU and processing for the
proliferation assay as previously described.
TUNEL labelling in vivo
Cryostat sections (10 µm) of cortex and corpus callosum were
prepared as above and labelled using the terminal deoxyribonucleotide
transferase-mediated dUTP nick end labelling method, using a
commercially available kit (ApopTag, Intergen, NY) according to the
instructions. Labelled cells were counted in two separate frontal
anterior brain sections. For the corpus callosum, the whole region was
counted and for the cortex, an area of seven fields of 500 µm
x 500 µm at the external edge of both cerebral hemispheres
was counted as for the studies on cell proliferation.
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RESULTS |
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To examine OP migration we chose to study the optic nerve. OP
cells start to enter the optic nerve from the chiasm at birth and then
migrate toward the retina (Bartsch et al., 1994). Cell
migration is responsible for all the OP cells within the nerve, and
both the single source of cells and the uniform direction of migration
along a narrow tract makes the optic nerve ideal for studies on
migration in vivo. To compare the distribution of OP cells in
wild-type and TN-C-deficient nerves, we serially sectioned the nerve
for 1.8 mm from the retinal end towards the chiasm and counted the
number of PDGFR mRNA-positive cells in sections at different
distances from the retina as described in the Materials and
Methods. In wild type newborn (P0) animals very few PDGF
R
mRNA-positive cells were detectable in the optic nerves and were all
restricted to regions greater than 1.2 to 1.8 mm from the retina
(Fig. 1, P0). In
contrast, in the knockout animals, PDGF
R mRNA-positive cells were
seen between 0.9 and 1.2 mm from the retina, with significantly more
cells found in the nerve than in wild type (Fig. 1B,C, P0). In postnatal day 2 (P2)
animals, the number of PDGF
R mRNA-positive cells and the distance
travelled from the chiasm increased in both wild-type and knockout
mice, and cells positive for PDGF
R mRNA were first found 300
µm from the retina (Fig. 1, P2). However, the number of
PDGF
R mRNA-positive cells was significantly greater in TN-C-null
mice in each region of the optic nerve (Fig. 1, P2). By P5, the density of PDGF
R
mRNA cells had increased still further in each region of the optic
nerve (Fig. 1, P5) and,
in agreement with previous studies, the OPs had now reached the
retinal end of the optic nerve (Bartsch et al., 1994). We did
not find any differences in cell numbers between TN-C-null mice and
wild-type mice at this (Fig. 1D,E, P5) and later stages (Fig. 1, P12 and adult). However,
from P12 to adult, the number of cells containing PDGF
R mRNA
decreased and, in contrast to earlier developmental stages, the
PDGF
R mRNA-positive cells in the adult optic nerve were now
uniformly distributed along the different segments. At all stages in
both wild-type and knockout mice, no PDGF
R mRNA-positive cells
entered the part of the optic nerve immediately adjacent to the
retina, although a few cells were occasionally seen between 250-300
µm from the retina (Fig. 1).
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During early postnatal development, forebrain OP cells are generated from pre-progenitor cells in the SVZ surrounding the lateral ventricles (Levison et al., 1993; Levison and Goldman, 1993; Levison and Goldman, 1997; Luskin and McDermott, 1994). Consequently, our first experiment was an analysis of BrdU incorporation in this area between P0 to P17. We found a significant reduction at all ages in the number of BrdU-positive cells in the SVZ of TN-C-null mice when compared with wild-type controls (Fig. 4A). This reduction remained significant even after the overall number of BrdU-positive cells decreased later in development in the P17 SVZ (Fig. 4A). It was most apparent in the dorsolateral part of the SVZ (see Fig. 4B,C,E), an area in which TN-C is normally highly expressed, as evidenced by the expression of lacZ from the transgene in sections taken from TN-C-null mice (Fig. 4D).
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We also compared the levels of cell proliferation between heterozygous and TN-C deficient homozygous littermates in the 129 genetic background. Once again we found reduced levels of BrdU labelling in SVZ and cortex at both P7 and P10 and in corpus callosum at P7 (Table 1). This result confirms that the reduced cell proliferation associated with TN-C deficiency is present on at least two genetic backgrounds.
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Involvement of vß3 integrin in TN-C mitogenic
functions in OP cells
The experiments above show that TN-C is required for the mitogenic
response of mouse OP cells to PDGF in cell culture. In order to
investigate the mechanism by which TN-C interacts with PDGF signalling
pathways, we focussed on the vß3 integrin. This integrin is
an established TN-C receptor that has been shown to mediate TN-C
growth control effects in other cell types and to mediate interactions
between integrin and growth factor receptor signalling pathways (for a
review, see Jones
and Jones, 2000). Additionally, we have shown previously
that
vß3 integrin is expressed during OP differentiation and
regulates OP proliferation (Blaschuk et al., 2000; Milner
and ffrench-Constant, 1994; Milner et al., 1997). We
therefore performed proliferation assays in vitro in which BrdU
incorporation into OP cells was determined in the presence or absence
of a function-blocking
vß3 antibody, either on control
substrates (PDL) or on substrates containing purified TN-C
(PDL+TN-C at 10 µg/ml, see Materials and Methods). In the
absence of any well-characterised blocking antibodies against mouse
vß3, we used rat OP cells and the monoclonal anti-rat
vß3 antibody F11 (Helfrich et al., 1992). Initial
experiments established that, as in the mouse cells, the mitogenic
response of rat OP cells to PDGF was enhanced by exogenous TN-C
(Fig. 7). The F11
antibody blocked this TN-C-mediated effect, but had no effect on
control PDL substrates on which PDGF was still able to stimulate OP
cell proliferation (Fig. 7). These data therefore demonstrate
that TN-C acts via one of its receptors,
vß3 integrin, to
enhance the mitogenic effects of PDGF in OP cells.
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DISCUSSION |
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The reduction in cell proliferation in the TN-C-deficient animals was revealed by BrdU studies in vivo, with double labelling experiments showing directly that there was a reduction in OP proliferation. Cell culture studies using cells obtained from either wild-type or knockout animals show that the effect on cell proliferation reflects a change in the sensitivity of the OP cells to the mitogen PDGF. Cells that lack TN-C and are grown on an astrocyte-derived extracellular matrix also derived from TN-C-deficient animals show almost no response to PDGF. Importantly, this response is restored by the addition of exogenous TN-C, confirming that this molecule is necessary for the mitogenic effect of PDGF on OP cells. As experiments using transgenic mice expressing different levels of PDGF have shown that this mitogen is limiting for OP proliferation in vivo (Calver et al., 1998; Fruttiger et al., 1999), any alteration in the sensitivity of the precursor cells to PDGF should alter cell proliferation. We propose, therefore, that the observed changes in the transgenic mice reflect a partial loss of response to PDGF in three areas of the CNS where TN-C is expressed during development: the SVZ, cortex and white matter tracts (Bartsch et al., 1992; Bartsch et al., 1994; Crossin, 1996; Crossin et al., 1989; Gates et al., 1995; Gotz et al., 1997; Jankovski and Sotelo, 1996; Miragall et al., 1990; Mitrovic et al., 1994).
TN-C has been shown to stimulate cell proliferation in a number of other non-neural cell types in vitro (Jones et al., 1997; Ohta et al., 1998; Seiffert et al., 1998), and one previous study has also described reduced cell proliferation in vivo in association with a model of renal glomerulonephritis (Nakao et al., 1998). Interactions such as we document here between TN-C and growth factor signalling have been described in these studies for PDGF, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF; for a review, see Jones and Jones, 2000) and we have recently found that the response of neural stem cells to FGF is also TN-C dependent (E. G., unpublished). Taken together with our results presented here, this suggests that regulation of growth factor responsiveness represents a general mechanism by which TN-C regulates cell proliferation. As a consequence of these interactions, cell proliferation will be regulated both by long-range signals from diffusible growth factors and short-range signals from the ECM. This dual regulation may be an important mechanism for the maintenance of a proliferating stem/precursor cell population in the SVZ and rostral migratory stream, as TN-C expression persists in these areas of the adult brain (Gates et al., 1995; Jankovski and Sotelo, 1996; Miragall et al., 1990). Equally, the upregulation of TN-C by astrocytes in response to injury may be an important component of the repair response by enhancing the sensitivity of precursor cells to the growth factors present in the lesion environment (Crossin, 1996; Deller et al., 1997; Gates et al., 1996; Laywell et al., 1992).
Short-range ECM signals could either derive from adjacent cells or the OP cells themselves. Our own data point to a direct autocrine role for TN-C in OP proliferation. First, we have found previously that purified OPs are immunoreactive for TN-C in vitro (Kiernan et al., 1996). Second, we observed in the present study that the sensitivity to PDGF was more dependent on the genotype of the cells than on the underlying matrix. We interpret this to mean that cells respond more efficiently to TN-C that they themselves have synthesised and secreted. An autocrine role for tenascin-R has also been suggested previously (Pesheva et al., 1994; Probstmeier et al., 1999). Our present work therefore suggests the hypothesis that autocrine effects may be a general property of the tenascin family, and further studies to examine the synthesis of TN-C in vivo are required to determine the relative contributions of astrocytes and OP cells to the TN-C present in the developing CNS.
Three receptors for TN-C that might regulate cell proliferation in
OP cells have been described. First, RPTP-ß/ (Milev et al., 1997; Ranjan and
Hudson, 1996), a protein-tyrosine phosphatase that could
alter signalling downstream of the PDGF receptor as previously
described for another protein-tyrosine phosphatase, SHP-2 (Zhao and Zhao,
1999). Second, annexin II (Chung and Erickson, 1994), which has been shown to mediate a mitogenic effect of TN-C on
endothelial cells (Chung
et al., 1996). Third, the integrin
vß3
(Yokoyama et al.,
2000), previously shown to modulate the EGF-driven growth
responses of smooth muscle cells (Jones et al., 1997) and also to
stimulate cell proliferation in colon cancer cells grown on TN-C
substrates (Yokosaki
et al., 1996). Our experiments demonstrate a role for
vß3, as a well-characterised antibody against this integrin
will block the stimulatory effect of exogenous TN-C on OP cell
proliferation. An association between
vß3 and PDGF signalling
has been described previously, as activated PDGFß receptor will
co-immunoprecipitate with
vß3 and ligand binding to
vß3 will enhance PDGF signalling responses (Schneller et al.,
1997). However, the association between
vß3 and
the PDGF
R suggested by our work has not been shown
previously.
vß3 contributes to the regulation of OP cell
proliferation in cell culture, and we have previously suggested a
model in which the sequential signalling of
vß1,
vß3
and
vß5 regulates OP migration, proliferation and
differentiation, respectively (Blaschuk et al., 2000;
Milner et al.,
1996; Milner and ffrench-Constant,
1994; Milner et al., 1997). Our
demonstration here that TN-C acts through the
vß3 integrin to
potentiate PDGF mitogenic activity in OPs adds significant support to
this hypothesis. It emphasises the role of
vß3 in OP
proliferation, demonstrating the association with PDGF signalling
pathways known to regulate OP proliferation in vivo (Calver et al., 1998;
Fruttiger et al.,
1999) and identifying an essential role for an ECM ligand
of
vß3, which is known to be expressed in the pathways of OP
migration during development (Bartsch et al., 1992; Bartsch et al.,
1994).
We have also examined OP migration during postnatal development in
the TN-C-deficient mice, as TN-C has been shown to inhibit OP
migration and adhesion in cell culture (Frost et al., 1996; Kiernan et al.,
1996). Our studies show that OPs, as identified by the
expression of the PDGFR, have migrated further along the optic
nerve at P0 and P2 in the absence of TN-C, so confirming for the first
time a role for TN-C in the regulation of inhibition of migration in
vivo. Based on cell culture studies, the inhibition of OP migration by
TN-C has been proposed as a mechanism to limit the movement of OPs in
vivo and so prevent myelination in two areas of the CNS, the retina
and the molecular layer of the cerebellum. High concentrations of TN-C
produced by astrocytes at the optic nerve head and at the border of
the molecular layer have been proposed to act as barriers during
development (Bartsch
et al., 1992; Bartsch et al., 1994). Although
we showed previously that adult mice lacking TN-C showed no change in
the pattern of myelination at the optic nerve head and cerebellum
(Kiernan et al.,
1999), we could not exclude the possibility that OPs enter
these regions during development but are then lost by programmed cell
death (Calver et al.,
1998). The present developmental study argues that this is
not the case; even in the optic nerves that lack TN-C, cells arriving
prematurely at the optic nerve head were never seen to enter the
retina. TN-C is not therefore required for the barrier function of
this region, and our finding that OPs also never entered the molecular
layer of the cerebellum (E. G., unpublished) shows that TN-C is also
not required as a barrier to OP migration in this structure.
In contrast to the experiments examining proliferation in which the addition of purified TN-C to the in vitro assays restored OP proliferation to wild-type levels, the addition of exogenous TN-C to our migration assays, either to the substrate or in solution, did not reduce the level of migration. This was surprising given our previous finding that purified TN-C substrates inhibit OP migration (Kiernan et al., 1996), and points to additional indirect effects of TN-C loss mediated by changes either in the levels or in the organisation of other molecules in the ECM. As we have also described previously, the inhibition of OP migration by TN-C is substrate dependent (Frost et al., 1996) and we suggest that the changes in the TN-C-deficient ECM render the cells insensitive to the direct effects of purified TN-C. TN-C interacts with a number of other ECM molecules (Jones and Jones, 2000) and the reorganisation of the matrix associated with TN-C deficiency could therefore alter the 3D architecture and the interactions of cells with these other molecules. This is an important conclusion, as it emphasises that the effects of deficiency deduced from the phenotype of null mice reflect both direct and indirect effects of the loss of any one molecule, with in vitro experiments, such as those performed here, helpful in distinguishing these possibilities.
Given the roles for TN-C in proliferation and migration demonstrated by this present study why, as we have described previously (Kiernan et al., 1999), is the final pattern of myelination normal in TN-C-deficient animals? Our results showing a decrease in TUNEL labelling suggest that this reflects the presence of corrective mechanisms during normal development. Overproduction of OPs normally occurs, with as many as 50% of newly formed oligodendrocytes subsequently undergoing programmed cell death (Barres et al., 1992) regulated, at least in part, by the availability of axonal targets for myelination (for a review, see Barres and Raff, 1999). Variations in the numbers of OP cells in different regions of the CNS that reflect changes in proliferation and migration could therefore be corrected at a later stage of development. In the case of reduced proliferation, the reduction in the level of cell death we observe at the final stages of myelination (at which time axonal targets would normally be limiting) would correct the phenotype. These corrective cellular mechanisms are quite distinct from compensatory molecular mechanisms that may operate within individual cells as has been shown, for example, in the MyoD knockout where Myf5 can compensate for the loss (Rudnicki et al., 1993). They may represent an important cause of the apparent normality of many transgenic mice. Correction may, however, be inadequate in the presence of other genetic or environmental perturbations of development and this may provide the selection pressure required to explain the high degree of conservation seen for TN-C across a range of vertebrate species.
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ACKNOWLEDGMENTS |
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