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First published online 21 April 2004
doi: 10.1242/dev.01114

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Transcription factors Sox8 and Sox10 perform non-equivalent roles during oligodendrocyte development despite functional redundancy

Institut für Biochemie, Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany

View larger version (102K): [in a new window]   Fig. 1. Sox8 expression in the embryonic spinal cord. Sox8-specific ß-galactosidase activity was detected colorimetrically using X-gal substrate in transverse spinal cord sections from the forelimb region of Sox8+/lacZ mice at 12.5 dpc (A), 14.5 dpc (B), 16.5 dpc (C), 18.5 dpc (D) and postnatal day 3 (E).

View larger version (91K): [in a new window]   Fig. 2. Cell type-restricted expression of Sox8 in the postnatal spinal cord. Immunohistological analysis of spinal cord from Sox8+/lacZ mice at postnatal day 1 using antibodies against ß-galactosidase (green) in combination with cell-type specific antibodies (red) NeuN (A), GFAP (B), S100ß (C), Olig2 (D), Sox10 (E) and Mbp (F).

View larger version (68K): [in a new window]   Fig. 3. Oligodendroglial development in the embryonic spinal cord of mice carrying Sox8 and Sox10 null alleles. Immunohistochemistry with antibodies specific for the oligodendrocyte marker Olig2 (A-D), and in situ hybridization with probes specific for Mbp (E-H) and Plp (I-L) were performed on transverse sections from the forelimb region of embryos at 18.5 dpc. (A,E,I) Wild-type spinal cords; (B,F,J), Sox8lacZ/lacZ spinal cords; (C,G,K), Sox10+/lacZ spinal cords; (D,H,L), Sox8lacZ/lacZ, Sox10+/lacZ spinal cords.

View larger version (33K): [in a new window]   Fig. 4. Quantification of oligodendrocyte marker gene expression in the spinal cord of mice carrying Sox8 and Sox10 null alleles. The number of Olig2-positive cells (A,B), Plp-positive (white bars) or Mbp-positive (black bars) cells (C,D) was determined at 18.5 dpc (A,C) and postnatal day 3 (B,D) in wild-type (+/+), Sox8lacZ/lacZ, Sox10+/lacZ and Sox8lacZ/lacZ, Sox10+/lacZ spinal cords, as indicated below the bars. At least 15 separate 20 µm sections from the forelimb region of two independent embryos were counted for each genotype. Data are presented as mean±s.e.m. Differences from the wild type were statistically significant for all mutant genotypes in C,D, as determined by Student's t-test (P0.001).

View larger version (119K): [in a new window]   Fig. 5. In situ hybridization studies of terminal oligodendrocyte differentiation in early postnatal spinal cords of mice carrying Sox8- and Sox10-null alleles. In situ hybridization with probes specific for Mbp (A-D,I-L) and Plp (E-H,M-P) were performed on transverse spinal cord sections from the forelimb region at postnatal day 3 (A-H) and postnatal day 7 (I-P). (A,E,I,M) Wild-type spinal cords; (B,F,J,N), Sox8lacZ/lacZ spinal cords; (C,G,K,O), Sox10+/lacZ spinal cords; (D,H,L,P), Sox8lacZ/lacZ, Sox10+/lacZ spinal cords.

View larger version (64K): [in a new window]   Fig. 6. Competition and interaction of Sox8 and Sox10 on bona fide binding sites from target gene promoters. (A) Electrophoretic mobility shift assays with the Sox10-binding sites 1-3 from the Mbp promoter (Stolt et al., 2002) as probes, and extracts from transfected N2A cells-expressing full-length Sox8 and Sox10 proteins as indicated below the lanes. Antibodies directed against Sox8 (-Sox8) or Sox10 (-Sox10) were added to some reactions during the incubation period as indicated. m, bound monomer; d, bound homodimer. Supershifted complexes are marked with an asterisk. (B) Electrophoretic mobility shift assay with site 2 from the Mbp promoter as probe and Sox8-containing N2A cell extract as protein source. Increasing amounts of a truncated Sox10 (MIC variant) (see Kuhlbrodt et al., 1998b; Schlierf et al., 2002) were added to the reactions as indicated. (C) Electrophoretic mobility shift assay with the prototypic dimeric Sox10-binding site C/C' (Peirano and Wegner, 2000) and extracts from transfected N2A cells expressing a short version of Sox8 and a long version of Sox10 either alone or in combination as indicated below the lanes. The heterodimer is indicated by an arrowhead.

View larger version (17K): [in a new window]   Fig. 7. Activation of myelin gene expression by Sox8, and comparison of ß-galactosidase activities in Sox8+/lacZ and Sox10+/lacZ spinal cords. (A) N2A cells were transfected with reporter plasmids in which the luciferase gene was under control of a long (positions -656 to +31) or a short (positions -256 to +31) version of the rat Mbp promoter. Expression plasmids for Sox10 and Sox8 were co-transfected as indicated below the bars. Luciferase activities in extracts from transfected cells were determined in three independent experiments each performed in duplicates. Data are presented as fold inductions ±s.e.m. with the activity of the promoter in the absence of co-transfected Sox protein (-) arbitrarily set to 1. (B) RT-PCR analysis on cDNA obtained from N2A cells inducibly expressing Sox8. - Doxy, no doxycycline (Sox8 absent); + Doxy, doxycycline added (Sox8 present). Transcript levels of Sox8, Plp, Mbp and ß-actin were compared semi-quantitatively using increasing numbers (n, n+3, n+6) of amplification cycles. -, no cDNA added. (C) The amount of ß-galactosidase present per µg extract from Sox8+/lacZ (white bars) and Sox10+/lacZ (black bars) spinal cords was determined at 14.5 dpc, 16.5 dpc, 18.5 dpc and in adult mice as indicated below the bars.