Knockout mice reveal a contribution of the extracellular matrix molecule tenascin-C to neural precursor proliferation and migration
Emmanuel Garcion1,
Andreas Faissner2,* and
Charles ffrench-Constant1,
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
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Fig. 2. Increased OP migration in the optic nerve of homozygous
TN-C-deficient mice when compared with heterozygous littermates. OP
migration analysis was performed as shown for Fig. 1. Data were obtained from experiments
at both P0 and P2, with three heterozygous and four homozygous null
animals with a C57Bl6J/CBA background and from an experiment at P2
with three heterozygous and three homozygous null animals with a 129
background. At both P0 or P2, more PDGF R mRNA-positive cells were
found in each segments of the optic nerves of homozygous
TN-C-deficient mice when compared with heterozygous littermates
(Student’s t test: *P<0.05,
**P<0.01, ***P<0.001).
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Fig. 3. Increased OP cell migration on TN-C-null astroglial ECM
substrate. Measurements of rat OP migration from the edge of an
agarose drop show that after 1 and 2 days, the distance moved by the
cells is greater on the TN-C-null astroglial substrate than on the
wild-type astroglial substrate (Student’s t test:
**P<0.01,
***P<0.001). Note that the addition to the
substrate of purified TN-C at 40 µg/ml before the assay does not
reduce OP cell migration on the TN-C-null substrate. Results shown
represent mean±s.e.m. from three to six independent experiments
(WT, wild type; -/-, null).
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Fig. 4. Reduction in cell proliferation of TN-C-null mice. Proliferating
cells were identified by BrdU uptake in vivo at ages from P0 to P17 in
the SVZ of wild-type (WT) and TN-C-null (-/-) animals with
the original genetic background described by Saga et al. (Saga et al., 1992), and
from P2 to P10 in the SVZ, the cortex, the striatum and the corpus
callosum of heterozygous (+/-) and homozygous TN-C-null
(-/-) littermates with a C57Bl6J/CBA background. (A) The
reduction in BrdU-positive cells in the TN-C null animals, as
described in the text; note that for some values the error bars are
too small to see at this scale (Student’s t test:
*P<0.05, **P<0.01,
***P<0.001). (B) A schematic representation of
a frontal section of the anterior part of the mouse brain with the
boxed area showing the region of the SVZ in which BrdU-positive cells
were counted. (C) The extensive BrdU labelling at P10 in the
dorsolateral part of the SVZ of heterozygous animals. (D)
lacZ expression in the SVZ, derived from the transgene in the
TN-C-null mice, in the region shown in C,E (boxed area). (E) BrdU
labelling of the dorsolateral part of the SVZ in homozygous TN-C-null
littermates of those shown in C; note the reduction in labelling. V,
ventricle. Scale bars: 400 µm in D; 100 µm in C,E.
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Fig. 5. Reduction in cell proliferation of OPs in the CNS of TN-C-null
mice with a 129 genetic background. BrdU (green)/NG2 (red) double
staining experiments at P7, in the SVZ (A) and in the cortex (B) of an
heterozygous animal and in the cortex of a TN-C-null homozygous animal
(C). Very few BrdU-positive cells were NG2 positive in the SVZ, while
in the cortex, the OP (NG2-positive) population represents about a
third of the proliferative (BrdU-positive) cells (B). Note that in
TN-C-null homozygous animals (C), fewer double-stained cells were
found in the cortex in comparison with heterozygous littermates
(B). Scale bar: 50 µm.
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Fig. 6. The mitogenic response of OP cells to PDGF requires TN-C. (A)
Response of wild-type or TN-C-deficient OP cells to increasing
concentrations of the mitogen PDGF (0 to 10 ng/ml) when grown on
wild-type or TN-C-deficient astroglia matrix substrates. Results shown
represent mean±s.e.m. from at least three independent
experiments (WT, wild type; -/-, null; P value
was obtained using Student’s t test). Note the lack of
response of TN-C-null OP cells to PDGF at all concentrations on the
TN-C-null substrate. (B) The lack of proliferative response to PDGF in
the absence of TN-C is rescued by the addition of exogenous purified
TN-C (10 µg/ml) to the TN-C-null astroglia substrate
(Student’s t test: *P<0.05).
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Fig. 7. Requirement of the vß3 integrin for the potentiation by
TN-C of PDGF mitogenic effects. Rat OP cells were plated on PDL
substrata or on PDL substrata with exogenous purified TN-C
(PDL+TN-C) in the presence (F11(+)) or absence
(F11(-)) of a ß3 function-blocking monoclonal
antibody. Cells were then grown in the presence of different
concentrations of PDGF (1, 4, 7, 10: 0 ng/ml; 2, 5, 8, 11: 1 ng/ml; 3,
6, 9, 12: 10 ng/ml) for 18 hours before the addition of BrdU for 6
hours. Results represent mean±s.e.m. of three independent
experiments (Student’s t test:
*P<0.001, comparison between PDL and PDL+ TN-C;
°P<0.001, comparison between F11(+) and
F11(-)).
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Fig. 8. Reduced cell death in the postnatal brain of TN-C-null transgenic
mice. At P5, P10 and P17, TUNEL-positive cells were counted in the
corpus callosum and the cortex of wild-type (WT) and TN-C-null mice
(TN-C-/-) in two separate brain sections from three
different animals of each genotype, and shown as
mean±s.e.m. Note the decrease in cell death at P17 in the
corpus callosum and at P5 in the cortex of TN-C-null mice
(Student’s t test: **P<0.02;
***P<0.01).
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© The Company of Biologists Ltd 2001
