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First published online 11 February 2004
doi: 10.1242/dev.01035

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Roles for p53 and p73 during oligodendrocyte development

Nathalie Billon^1,4,, Alessandro Terrinoni², Christine Jolicoeur^1,*, Afshan McCarthy³, William D. Richardson⁴, Gerry Melino^2,5 and Martin Raff¹

¹ MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, London WC1E 6BT, UK
² Biochemistry Laboratory, IDI-IRCCS, c/o Tor Vergata University, Via Montpellier, 1, 00133 Roma, Italy
³ Breakthrough Breast Cancer Centre, London Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK
⁴ Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK
⁵ MRC Toxicology Unit, Hodgkin Building, Leicester University, Lancaster Road, Leicester LE1 9HN, UK

View larger version (19K): [in a new window]   Fig. 1. Expression of p53, p63 and p73 mRNAs in OPCS. Purified P7 rat OPCs were cultured in PDGF without TH and RA. After 10 days, RNA was extracted and processed for RT-PCR.

View larger version (41K): [in a new window]   Fig. 2. Expression of p53, p63 and p73 proteins in oligodendrocyte lineage cells. Purified P7 rat OPCs were cultured in PDGF without TH and RA for 10 days. They were then cultured for 3 or 5 days in either PDGF alone or PDGF and TH to induce differentiation. (A-C) After 3 days, the cells were fixed and stained by immunofluorescence for p53 (A), p63 (B) or p73 (C). (D) Cells in PDGF and TH for 3 or 5 days were fixed and double-stained for both GC and p73. In all cases, nuclei were counterstained with bisbenzamide. Scale bar: 20 µm.

View larger version (20K): [in a new window]   Fig. 3. Effect of the expression of a wild-type or two dominant-negative forms of p53 transgenes on OPC differentiation. Purified P7 rat OPCs were cultured for 2 days in PDGF without TH and RA. They were then infected for 3 hours with either the pBird control vector encoding GFP alone or a vector encoding GFP and either wild-type (pBird-p53) or a dominant-negative p53 (pBird-DNp53 or pBird-DDp53). After a further day in PDGF alone, the cells were either left in PDGF alone or switched to PDGF and TH, PDGF and RA, or medium without PDGF for a further 3 days to induce differentiation. The percentage of GFP+ cells that had acquired the characteristic morphology of oligodendrocytes was scored in an inverted fluorescence microscope. Similar results were obtained when oligodendrocytes were identified by staining with anti-GC antibody (not shown). In this and the following three Figures, the results are shown as mean±s.d. of at least three experiments.

View larger version (18K): [in a new window]   Fig. 4. Effect of the expression of TAp63 or Np63 transgenes on OPC differentiation. Purified P7 rat OPCs were cultured and analysed as in Fig. 3, except that they were infected with either pBird, pBird-TAp63 or pBird-Np63 vectors. The differences between the pBird and the pBird-Np63 results in PDGF+TH are not statistically significant when analysed by Student's t-test (P>0.07).

View larger version (17K): [in a new window]   Fig. 5. Effect of the expression of TAp73 or Np73 transgenes on OPC differentiation. Purified P7 rat OPCs were cultured and analysed as in Fig. 3, except that they were infected with either pBird, pBird-TAp73 or pBird-Np73 vectors. The differences between the pBird and the pBird-TAp73 results in PDGF, PDGF+TH and PDGF+RA are statistically significant when analysed by Student's t-test (p=0.03, 0.03 and 0.02, respectively). The differences between the pBird and the pBird-Np73 results in PDGF, PDGF+TH, PDGF+RA and without PDGF are also statistically significant (p=0.04, 0.004, 0.03 and 0.001, respectively).

View larger version (18K): [in a new window]   Fig. 6. Effect of Np73 transgene on the differentiation of wild-type and p53-/- OPCs. Cells were dissociated from P7 mouse optic nerve and cultured in PDL-coated Nunc slide flasks for 3 days in PDGF alone. They were then infected for 3 hours with either the pBird control vector or the pBird-Np73 vector. After a further day in PDGF alone, they were then either kept in PDGF alone (A) or switched to either PDGF and TH (B) or medium without PDGF (C). After various times, they were fixed and stained for GC. The differences between the pBird and the pBird-Np73 results in all cases are statistically significant when analysed by Student's t-test (in A, p=0.02 for p53+/+ and 0.05 for p53-/-; in B, p=0.002 for p53+/+ and 0.01 for p53-/-; in C, p=0.03 for p53+/+ and p=0.02 for p53-/-).

View larger version (14K): [in a new window]   Fig. 7. Comparison of the proportions of oligodendrocytes, OPCs and astrocytes in the P7 optic nerve of p53+/+ and p53-/- mice. P7 optic nerve cells were isolated, counted and cultured on PDL-coated coverslips for 3-4 hours. The cells were then fixed and stained for GC to identify oligodendrocytes (A), for NG2 to identify OPCs (B) or for GFAP to identify astrocytes (C). Between 5 and 15 pups were analysed for each genotype. Each pair of optic nerves was processed separately, and 2 coverslips were counted for each pair. The total number of cells isolated per nerve was not significantly different in the two genotypes (not shown). The results are expressed as the mean±s.e.m. The differences between the p53+/+ and p53-/- results in Fig. 7A and 7B are statistically significant when analysed by Student's t-test (p=0.02 and 0.04, respectively).