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First published online 15 August 2007
doi: 10.1242/dev.003350

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Hypomorphic Sox10 alleles reveal novel protein functions and unravel developmental differences in glial lineages

¹ Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany.
² Max-Planck-Institut für Neurobiologie, Martinsried, Germany.
³ Institut für Klinische Neurobiologie, Universität Würzburg, Germany.

View larger version (39K): [in this window] [in a new window]   Fig. 1. The Sox10 mutations. (A) Schematic of the aa1 and K2 mutations. (B) Subcellular localization of wild-type Sox10 and the K2 mutant in transiently transfected Neuro2a cells was determined by immunocytochemistry with a Sox10-specific antibody. Nuclei were counterstained by DAPI. (C) Stability of wild-type Sox10 (blue circle) and the K2 mutant (red square) were compared in transiently transfected Neuro2a cells cultured for various times in the presence of cycloheximide as indicated. Extracts were prepared and Sox10 proteins detected by western blot. Relative amounts were quantified from band intensities with the amount in untreated cells set to 100%. (D) The transactivation capacity of the K2 mutant was analyzed in luciferase reporter gene assays. Transient transfections were performed in Neuro2a cells with luciferase reporters under the control of the Mpz promoter (positions -915 to +48), the Dct promoter (positions -3240 to +443) and the Mbp promoter (positions -656 to +31). Luciferase reporters were transfected either alone or in combination with wild-type Sox10, the K2 or the Q377X mutant. Data from three independent experiments each performed in duplicate are presented as fold inductions±s.d., with the activity for each luciferase reporter in the absence of co-transfected Sox10 set to 1.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Targeted replacement of wild-type Sox10 by mutant Sox10 sequences in mice. (A) Schematic showing, from top to bottom, the targeting construct for the Sox10 K2 mutation, the Sox10 wild-type locus and the mutant locus before and after Cre recombination. The Sox10 exons (I-V) and the mutant Sox10 K2 open reading frame (ORF) are shown as boxes, the 4.5 kb and 1.5 kb flanking regions as bars. Regions of homology between wild-type locus and targeting vector are depicted as thick black lines, introns III and IV as thin open boxes and surrounding genomic regions not contained in the targeting construct as dashed lines. Plasmid backbone sequences of the targeting construct are indicated by a thin line. Restriction sites for NcoI (N), BamHI (H) and ScaI (S) are shown, as are the locations of the 5' and 3' probes and the start codon of the Sox10 gene (ATG). The arrowheads indicate the locations of primers 1,2,3,4 used for quantitative RT-PCR. neo, neomycin resistance cassette; loxP, recognition sites for Cre recombinase; Tk, herpes simplex virus thymidine kinase gene cassette. (B) Schematic of the mutant locus for the Sox10 aa1 mutation. Recombination into the Sox10 genomic locus was as depicted for the Sox10 K2 construct. (C) Southern blot analysis of DNA from wild-type (wt) and heterozygous (+/aa1 and +/K2) ES cells digested with NcoI for use with the 5' probe, and with BamHI/ScaI for the 3' probe. The size of bands corresponding to the wild-type (6.6 kb for the 5' probe and 4.6 kb for the 3' probe) and the targeted alleles (10.9 and 5.9 kb, respectively, for the 5' probe; 10.2 and 10.4 kb, respectively, for the 3' probe) are indicated. (D) PCR genotyping of wild-type (wt), heterozygous (+/K2 and +/aa1) and homozygous (K2/K2 and aa1/aa1) mouse embryos at 18.5 dpc. DNA fragments in the size marker (M) are 1.0 kb and 0.5 kb.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Sox10 expression levels and early development of neural crest derivatives in Sox10K2/K2 and Sox10aa1/aa1 embryos. (A) Comparison of Rpl8-normalized Sox10 expression levels in wild-type (set to 1, indicated by dashed line) and age-matched Sox10aa1/aa1 and Sox10K2/K2 mouse embryos at 10.5 and 11.5 dpc following quantitative RT-PCR with primer pairs 1,2 (white bars) and 3,4 (black bars). Experiments were repeated three times with material from two embryos for each genotype and age. (B) Immunoprecipitation of Sox10 from extracts of wild-type (wt) and age-matched Sox10aa1/aa1 (aa1/aa1) embryos at 11.5 dpc. Immunoprecipitated material was detected by western blotting with anti-Sox10 rabbit antiserum. Sox10, Sox10-containing extract from transiently transfected Neuro2a cells. (C) Whole-mount in situ hybridization of wild-type (wt), Sox10K2/K2 and Sox10aa1/aa1 embryos at 10.5 dpc and 11.5 dpc using an antisense riboprobe directed against Sox10. V, trigeminal; VII/VIII, facial/acoustic; IX, glossopharyngeal; X, vagus; ot, otic vesicle.

View larger version (149K): [in this window] [in a new window]   Fig. 4. Analysis of the ENS in Sox10K2/K2 and Sox10aa1/aa1 embryos. Stomach (A), duodenum (B,C) and caecum (D) of wild-type (wt), Sox10K2/K2 and Sox10aa1/aa1 mouse embryos were analyzed at 18.5 dpc by NADPH diaphorase staining. For the duodenum, a region immediately adjacent to the stomach (B) is shown as well as a more distal region (C).

View larger version (95K): [in this window] [in a new window]   Fig. 5. Melanocyte development in Sox10K2/K2 and Sox10aa1/aa1 embryos. (A) Whole-mount in situ hybridizations were performed on wild-type, Sox10K2/K2 and Sox10aa1/aa1 mouse embryos at 12.5 dpc using antisense riboprobes against Dct and c-Kit. (B,C) Dct-positive and c-Kit-positive cells were quantified in the hindlimb region of wild-type embryos (black bars) and their Sox10K2/K2 (B) or Sox10aa1/aa1 (C) littermates (white bars). Three embryos were counted for each genotype. The number of cells counted in the wild type was set to 100%. Cell numbers for the Sox10K2/K2 and Sox10aa1/aa1 embryos are presented relative to the wild type as mean±s.d. Differences were statistically significant between wild type and each mutant as well as between the two mutants as determined by Student's t-test (P0.001).

View larger version (72K): [in this window] [in a new window]   Fig. 6. Analysis of DRG in Sox10K2/K2 and Sox10aa1/aa1 embryos. Immunohistochemistry was carried out on transverse sections through DRG of wild-type, Sox10K2/K2 and Sox10aa1/aa1 mouse embryos at 11.5, 12.5, 14.5 and 18.5 dpc using antibodies against the neuronal marker NeuN, the glial marker B-FABP and against Sox10. The spinal cord is to the right of the dorsal root ganglion.

View larger version (52K): [in this window] [in a new window]   Fig. 7. Peripheral nerve development in Sox10K2/K2 and Sox10aa1/aa1 embryos. Immunohistological analysis of spinal nerves from wild-type, Sox10K2/K2 and Sox10aa1/aa1 mouse embryos at 18.5 dpc was carried out using antibodies against NF165, Sox10, Oct6 and SoxB1 (Sox1/2/3). Expression of Mbp and Mpz was additionally visualized in all three genotypes by in situ hybridization with specific antisense probes.

View larger version (91K): [in this window] [in a new window]   Fig. 8. Development of sympathetic ganglia in Sox10K2/K2 and Sox10aa1/aa1 embryos. Immunohistochemical staining of sympathetic ganglia was performed on wild-type, Sox10K2/K2 and Sox10aa1/aa1 mouse embryos at 12.5 dpc using antibodies against Phox2b, Sox10 and Th.

View larger version (57K): [in this window] [in a new window]   Fig. 9. Development of oligodendrocyte precursors in Sox10K2/K2 and Sox10aa1/aa1 embryos. Specification and subsequent distribution of oligodendrocyte precursors throughout the spinal cord of wild-type, Sox10K2/K2 and Sox10aa1/aa1 mouse embryos was analyzed on transverse sections from the forelimb region at 12.5, 14.5 and 18.5 dpc by immunohistochemistry with antibodies directed against Sox10 (A) and Olig2 (B).

View larger version (73K): [in this window] [in a new window]   Fig. 10. Terminal differentiation of oligodendrocytes in Sox10K2/K2 and Sox10aa1/aa1 embryos. (A) In situ hybridizations with probes specific for Plp and Mbp were performed on transverse spinal cord sections from the forelimb region of wild-type, Sox10K2/K2 and Sox10aa1/aa1 mouse embryos at 18.5 dpc. (B) Plp-positive (black bars) and Mbp-positive (gray bars) cells were counted on at least 20 sections from three independent embryos for wild-type and Sox10K2/K2 spinal cords. The number of cells counted in the wild type was set to 100%. Cell numbers for the Sox10K2/K2 are presented relative to the wild type as mean±s.d. Statistically significant differences were observed using Student's t-test for Plp (P0.001) and Mbp (P0.01). (C) Similar quantifications for Sox10aa1/aa1. No statistical difference in the number of Plp- and Mbp-positive cells was detected compared with the wild type.