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First published online June 28, 2004
doi: 10.1242/10.1242/dev.01202

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Generation of an environmental niche for neural stem cell development by the extracellular matrix molecule tenascin C

Emmanuel Garcion^1,*, Aida Halilagic¹, Andreas Faissner² and Charles ffrench-Constant^1,

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

View larger version (45K): [in a new window]   Fig. 1. Expression of tenascin C (TNC) in subventricular zone (SVZ) cells revealed by lacZ expression from the transgene in TNC heterozygous mice. This was observed within the regions surrounding the ventricle (V) at E17.5, shown in A. (B) The higher level of lacZ expression in the SVZ as compared with the ventricular zone (VZ) immediately adjacent to the ventricle. Scale bars: 250 µm in A; 40 µm in B.

View larger version (154K): [in a new window]   Fig. 2. Acquisition of the epidermal growth factor receptor (EGFR – shown in red with nuclei counterstained in blue) by NSCs is delayed in tenascin C (TNC) null brains. At E12.5, note the absence of EGFR immunostaining in the TNC null mice (K/O, B), whereas EGFR is present in their heterozygous littermates (+/–, A). At later stages (E17.5, C,D; P17, E,F), similar patterns of EGFR immunolabelling are observed in homozygous and heterozygous animals. Similar results were observed in three sets of littermates. Scale bars: 100 µm in A,B; 200 µm in C,D; 80 µm in E,F.

View larger version (21K): [in a new window]   Fig. 3. (A) Numbers of neurospheres from E10.5 rostral telencephalic vesicles of wild type (WT) or tenascin C (TNC) null mice (K/O) grown for seven days in the presence of either fibroblast growth factor (FGF2) or epidermal growth factor (EGF) (20 ng/ml). Note the lack of EGF-responsive neural stem cells in TNC null cells as demonstrated by the absence of neurosphere formation in EGF. Results represent mean±s.e.m. of three independent experiments (Student's t-test: *P<0.001). (B) E10.5 neurospheres were grown for 5 days from heterozygous (+/–) or TNC null (–/–) cells in the presence of FGF2 (20 ng/ml) and 0 or 10 µg/ml TNC as indicated. The Figure shows 4 gel lanes from the same Western blot analysis of neurosphere proteins using antibodies against the EGFR, with a long exposure required to visualise the low levels of EGFR protein at this developmental stage. Note that EGFR is detected in heterozygous cells either with or without added TNC (lanes 1-2). By contrast, FGF2 alone was not sufficient to induce EGFR expression in the null cells after 5 days (lane 3), and that EGFR expression was rescued by the addition of exogenous TNC for the duration of the experiment (lane 4).

View larger version (26K): [in a new window]   Fig. 4. (A) BrdU uptake in E12.5 neurosphere cells in response to fibroblast growth factor (FGF2) (20 ng/ml). (B) BrdU uptake in P0 neurosphere cells to increasing concentration of FGF2. Note the reduction in cells stained for BrdU in the tenascin C (TNC) null neurospheres at both ages. Results represent mean±s.e.m. of three independent experiments. *P<0.05, ***P<0.001, using Student's t-test. (C) Expression of epidermal growth factor receptor (EGFR) in neurospheres grown in FGF2 assessed by Western blotting. One of three independent experiments is presented, with all three giving the result shown. Cells lacking TNC show enhanced sensitivity to the inhibitory effects of bone morphogenic protein 4 (BMP4), as discussed in the main text.

View larger version (38K): [in a new window]   Fig. 5. (A) Phase contrast micrographs of neural stem cell-derived neurospheres from P0 wild type (upper panel) and P0 tenascin C (TNC) null mice (lower panel) - note that null spheres are smaller and more numerous. As discussed in the main text, these differences were also observed in secondary passages of the neurospheres. Scale bar: 80 µm. (B-C) Clonal analysis of fibroblast growth factor (FGF2) (B) and epidermal growth factor (EGF)-responsive (C) neural stem cell numbers in the brain of newborn TNC null mice using a serial dilution neurosphere assay. The slope of the line reflects the proportion of cells plated that form neurospheres; i.e. have stem cell properties. Note that the TNC null cells (broken lines) contain more stem cells that respond to FGF2 than wild-type cells (solid line). This is shown by the steeper gradient of the null-cell line in B. In contrast, the number of stem cells that respond to EGF is not increased in the TNC null cells (C). Linear regression values calculated from analysis of three independent experiments are shown within each graph as means±s.e.m. of wild type (upper Figure) and null (lower Figure) cells. The differences between wild type and null cells grown in FGF2 are significant; P<0.001 using Student's t-test.

View larger version (45K): [in a new window]   Fig. 6. (A-D) RC2 immunostaining on coronal sections of mouse telencephalic vesicles at E13 and P0. (A,C) Heterozygous animals; (B,D) Tenascin C (TNC)-deficient animals. A reduction in the intensity of RC2 staining is observed in the brains of TNC null animals at both ages, but note the normal morphology of the processes at P0. (E) The percentage of RC2-positive cells in dissociated cell populations from heterozygous and homozygous animals during different stages of CNS development. Note the decrease in the proportion of RC2+ cells in the homozygous animals at later stages, showing a reduction in the number of radial glia. *P<0.05. c, cortex; s, striatum; lv, lateral ventricle.

View larger version (67K): [in a new window]   Fig. 7. (A) Tenascin C (TNC) gene expression in neurospheres from P0 wild-type (WT) and P0 TNC null mice (K/O) grown for seven days in either epidermal growth factor (EGF) or fibroblast growth factor (FGF2) (20 ng/ml) assessed by RT-PCR. Note the band corresponding to TNC mRNA in WT but not null neurospheres. ß-actin mRNA is detected as a loading control (ß-a), and sizes shown on the right are in bp. (B-E) Six days after growth factor withdrawal, neurosphere cells differentiate into oligodendrocytes, as shown by Gal-C staining in red (B, WT cells; C, TNC null cells), and neurones, as shown by ßIII-tubulin staining in green (D, WT cells; E, TNC null cells). The increased number of neurones in the absence of TNC can be seen by comparing D (WT) and E (TNC null), and is quantified as a percentage of the total cell numbers in F. Note also the longer oligodendrocyte processes in the TNC null cells (C) as compared with the WT cells (B). Cells were counterstained by DAPI (blue). (F) More neurones develop from TNC null striatal neural stem cells. The graph shows the increase in neurogenesis from TNC null cells in either FGF2 or EGF. Results represent mean±s.e.m. of three independent experiments; **P<0.01, using Student's t-test. (G) The addition of exogenous TNC to neurosphere cultures from TNC-deficient animals (–/–) reverses the increased neurogenesis to a level not significantly different from that seen in heterozygous littermate controls (+/–). Results represent mean±s.e.m. of three independent experiments; *P<0.05, using Student's t-test. Scale bars: 30 µm in B,C; 60 µm in D,E.

View larger version (22K): [in a new window]   Fig. 8. A model to explain the effect of tenascin C (TNC) on stem cell development, based on the modulation of growth factors previously identified as regulators of epidermal growth factor (EGF) receptor expression (Lillien and Raphael, 2000), as discussed in the main text.