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First published online December 20, 2005
doi: 10.1242/10.1242/dev.02208

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p53 suppresses the self-renewal of adult neural stem cells

¹ Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden.
² Department of Biotechnology, The Royal Institute of Technology, SE-106 91 Stockholm, Sweden.
³ Department of Oncology-Pathology, Karolinska Institute, SE-171 76, Stockholm, Sweden.

View larger version (108K): [in a new window]   Fig. 1. p53 in a neural stem cell niche. (A-C,E-G) Nuclear p53-immunoreactivity is seen in the majority of cells in lateral wall of the lateral ventricle (LV) and to a lesser degree in the rostral migratory stream (RMS), whereas levels are lower or undetectable in most cells in the surrounding areas of the adult mouse brain. (D,H) Three-dimensional reconstructions of confocal stacks demonstrate p53 in musashi 1 (Msi1) and Gfap-immunoreactive cells. Nuclei are labeled with DAPI and appear blue. The orientation of all sections is indicated in A. D, dorsal; M, medial. Scale bars: 10 µm.

View larger version (36K): [in a new window]   Fig. 2. Increased proliferation in a neural stem cell niche in the absence of p53. More cells incorporate BrdU (A-C) and display immunoreactivity to the M-phase specific epitope phospho-histone H3 (D-F) in the lateral ventricle wall (LVW) of p53-/- mice compared with wild-type littermates. The number of TUNEL labeled apoptotic cells was not significantly different between wild type and p53-/- mice. Numbers represent LVW positive nuclei per brain section. Positive nuclei were counted on serial coronal sections of the anterior horn of the lateral ventricle within a defined space of the subventricular zone. (H) Quantification of the proportion of LVW cells capable of forming primary neurospheres. *P<0.05, **P<0.01. Scale bars: 10 µm.

View larger version (72K): [in a new window]   Fig. 3. p53 negatively regulates neural stem cell proliferation, survival and self-renewal. Neurospheres from the lateral ventricle wall of adult p53-null mice grow faster and become larger compared with wild-type neurospheres. (A,B) Secondary neurospheres 6 days after passage. (C) Average volume of these neurospheres. (D) When neurospheres are dissociated, a larger proportion of p53-/- compared with wild-type cells are capable of forming new neurospheres, demonstrating an increased self-renewal capacity. (E,F) Apoptotic cell death, indicated by Annexin V (E) or caspase activity (F) detected by flow cytometry, is decreased in the absence of p53. (G-I) A similar proportion of neurospheres from p53 mutant and wild-type mice are tripotent and form neurons (ßIII-tubulin+), astrocytes (Gfap+) and oligodendrocytes (O4+) upon differentiation. (J,K) BrdU incorporation is increased in p53-/- neurosphere cells compared with cells from wild-type mice (K) and more cells from p53-/- mice have a DNA content corresponding to S and M phases (J). There is no size difference between wild-type and p53 mutant cells (L). All bars in graphs indicate mean±s.d. *P<0.05, **P<0.01, ***P<0.001. Scale bar: 100 µm.

View larger version (39K): [in a new window]   Fig. 4. Transcriptional regulation by p53 in neural stem cells. (A) Volcano plot showing the relative expression levels (%) of genes in p53-/- compared with wild-type neurospheres in relation to the statistical significance of the difference. The cDNA arrays contain two independent p21 probes, which is why this gene appears as two independent measurements. (B) Categorization of dysregulated genes by gene ontology themes indicates overrepresentation of genes regulating proliferation. Green indicates upregulation and red indicates downregulation. (C) Quantitative RT-PCR analysis of the relative (%) mRNA expression of Cip/Kip family members. (D) Representative examples of western blot analysis of p21 protein levels in dissected LVW from wild-type and p53-/- mice. (E) Scheme for genes controlling stem cell self-renewal converging on p53. Green circles indicate genes implicated as positive regulators of self-renewal; red circles indicate putative negative regulators.

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