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First published online 21 June 2006
doi: 10.1242/dev.02477

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Replacement of the Sox10 transcription factor by Sox8 reveals incomplete functional equivalence

Susanne Kellerer^1,*, Silke Schreiner^1,*, C. Claus Stolt¹, Stefanie Scholz¹, Michael R. Bösl² and Michael Wegner^1,

¹ Institut für Biochemie, Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany
² Max-Planck-Institut für Neurobiologie, Martinsried, Germany.

View larger version (24K): [in a new window]   Fig. 1. Targeted replacement of Sox10 by Sox8 in mice. (A) Schematic representation from top to bottom of the targeting construct, the Sox10 wild-type locus and the mutant locus before and after Cre recombination. The Sox10 exons (I-V) and the Sox8 open reading frame (ORF) are shown as boxes, 4.5 kb and 1.5 kb flanking regions as bars. Regions of homology between wild-type locus and targeting vector are depicted as black bars, introns 3 and 4 as open bars and surrounding genomic regions not contained in the targeting construct as stippled bars. 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 well as the localization of 5' and 3' probes and the start codon of the Sox10 gene (ATG). The arrowheads indicate the localization of primers A-D used for quantitative RT-PCR. IRES-EGFP, EGFP open reading frame with preceding internal ribosomal entry site; neo, neomycin resistance cassette; loxP, recognition sites for Cre recombinase; Tk, Herpes simplex virus thymidine kinase gene cassette. (B) Southern blot analysis of DNA from wild-type (wt) and heterozygous (+/ki) ES cells digested with NcoI for use of the 5' probe and BamHI/ScaI for the 3' probe. The size of bands corresponding to the wild-type (6.6 and 4.6 kb) and the targeted allele (6.0 and 5.4 kb) are indicated. (C) Southern blot analysis of DNA from wild-type (wt), heterozygous (+/ki) and homozygous (ki/ki) mice digested with NcoI for use of the 5' probe. (D) PCR analysis of Cre-mediated deletion of the neomycin resistance cassette in mice carrying a Sox10Sox8ki allele (+/ki and ki/ki). ES cells with a Sox10Sox8ki allele (+/ki) still contained the neomycin resistance cassette and served as control. M, size marker. (E) PCR genotyping of wild-type (wt), heterozygous (+/ki) and homozygous (ki/ki) mice. DNA fragments in the size marker (M) are 1.0 kb and 0.5 kb.

View larger version (58K): [in a new window]   Fig. 2. Genotype distribution and SoxE gene expression in mouse embryos in which Sox10 is replaced by Sox8. (A) Embryos from crosses of Sox10+/Sox8ki males and females were sorted according to their genotype (wt, +/ki, ki/ki) as indicated below the bars. Embryos with an age between 10.5 and 12.5 dpc (white bars, n=150) were separately grouped from those between 14.5 and 18.5 dpc (black bars, n=111). (B) Comparison of expression levels for Sox10 in wild-type embryos at 10.5 and 12.5 dpc (white bars) and for Sox8 specifically expressed from the Sox10 locus in age-matched Sox10Sox8ki/Sox8ki embryos (black bars) with primers recognizing a common sequence in both transcripts using quantitative Lightcycler-RT-PCR. Transcript levels in each sample were normalized to ß-actin. After normalization, transcript levels in the wild type were arbitrarily set to 1. Experiments were repeated twice with material from two independently obtained embryos for each genotype and age. (C) Determination of the relative contribution of the wild-type allele to overall expression from the Sox10 locus in the indicated tissues of Sox10+/Sox8ki embryos at 13.5 dpc and 18.5 dpc. Values were obtained by comparing transcript levels from the wild-type allele to the ones from both alleles by quantitative Lightcycler-RT-PCR. Transcript levels were normalized to rpl8 and to corresponding transcript levels in tissues from age-matched wild-type embryos. sc, spinal cord; sn, sciatic nerve; st, stomach. (D) Western blot of nuclear extracts prepared from brains of 18.5 dpc wild-type (wt) and Sox10Sox8ki/Sox8ki (ki/ki) embryos with antibodies directed against SoxE proteins (Sox8/Sox10), Sox8 and RNA polymerase II (RNA Pol II). RNA polymerase II served as loading control. (E) Immunohistological analysis of transverse sections from wild-type and Sox10Sox8ki/Sox8ki embryos at 12.5 dpc using antibodies against Sox8 and Sox10. Additionally, EGFP autofluorescence was documented in both genotypes. Sox8 expression in the Sox10Sox8ki/Sox8ki embryos is essentially a composite of Sox8 and Sox10 expression in the wild type.

View larger version (105K): [in a new window]   Fig. 3. Development of cranial ganglia in Sox10Sox8ki/Sox8ki embryos. Whole-mount EGFP autofluorescence of the cranial region of Sox10Sox8ki/Sox8ki embryos (A,D) and their Sox10+/Sox8ki (B,E) and wild-type Sox10+/+ (C,F) littermates at 10.5 dpc. (D-F) Higher magnifications of A-C. V, trigeminal; VII/VIII, facial/acoustic; IX, glossopharyngeal; X, vagus; ot, otic vesicle.

View larger version (78K): [in a new window]   Fig. 4. Analysis of the enteric nervous system in Sox10Sox8ki/Sox8ki mice. Oesophagus, stomach and small intestine of wild-type embryos and Sox10Sox8ki/Sox8ki littermates were analyzed at 18.5 dpc by whole-mount immunohistochemistry with PGP9.5 antibodies (A) or by NADPH diaphorase staining (B). A region of the small intestine is shown that is immediately adjacent to the stomach. No immunohistochemical or NADPH diaphorase staining was observed in the gut distal to the stomach.

View larger version (103K): [in a new window]   Fig. 5. Melanocyte development in Sox10Sox8ki/Sox8ki mice. (A) Comparison of 7-day old Sox10Sox8ki/Sox8ki mice (top) and wild-type littermates (bottom). Sox10Sox8ki/Sox8ki mice are white and smaller than wild type. (B) Skin preparations from 7-day-old wild-type mice and Sox10Sox8ki/Sox8ki littermates shown from the side and from below to visualize hair and hair follicle. (C) Whole-mount in situ hybridization was performed on wild-type and Sox10Sox8ki/Sox8ki embryos at 12.5 dpc using antisense riboprobes against Dct, Kit and Mitf. The regions from which pictures were taken are boxed in the embryo in the lower left corner.

View larger version (101K): [in a new window]   Fig. 6. Analysis of dorsal root ganglia in Sox10Sox8ki/Sox8ki mice. Immunohistochemistry was carried out on transverse sections through dorsal root ganglia of wild-type and Sox10Sox8ki/Sox8ki embryos at 12.5 dpc (A) and 18.5 dpc (B) using antibodies against the glial marker B-FABP and the neuronal markers NeuN and Brn3.0. Occurrence of Sox8 and Sox10 was additionally documented in both genotypes with specific antibodies.

View larger version (43K): [in a new window]   Fig. 7. Peripheral nerve development in Sox10Sox8ki/Sox8ki mice. (A-J) Immunohistological analysis of spinal nerves from wild-type (A,C,E,G,I) and Sox10Sox8ki/Sox8ki (B,D,F,H,J) embryos at 18.5 dpc was carried out using antibodies against NF-165 (A,B), Oct6 (C,D) and Krox20 (E,F). EGFP autofluorescence (G,H) and occurrence of Sox10 (I,J) was additionally visualized in both genotypes. (K-N) Immunohistochemistry on transverse sections of peripheral nerves from wild-type (K,M) and Sox10Sox8ki/Sox8ki (L,N) mice at postnatal day 3 using antibodies against MBP (K,L) and MPZ (M,N).

View larger version (60K): [in a new window]   Fig. 8. Development of sympathetic ganglia in Sox10Sox8ki/Sox8ki mice. Immunohistochemical staining of sympathetic ganglia was performed on wild-type (A,C,E,G) and Sox10Sox8ki/Sox8ki (B,D,F,H) embryos at 12.5 dpc using antibodies against Sox8 (A,B), Sox10 (C,D), NF-165 (E,F) and Phox2b (G,H). Sympathetic ganglia in Sox10Sox8ki/Sox8ki embryos are slightly smaller and contain fewer Phox2b-positive cells.

View larger version (72K): [in a new window]   Fig. 9. Development of oligodendrocyte precursors in Sox10Sox8ki/Sox8ki mice. Specification and subsequent distribution of oligodendrocyte precursors throughout the spinal cord of Sox10Sox8ki/Sox8ki embryos (A-C, G-I, M-O, S-U) and wild-type littermates (D-F, J-L, P-R) was analyzed on transverse sections from the forelimb region at 12.5 dpc (A,D,G,J,M,P,S), 14.5 dpc (B,E,H,K,N,Q,T) and 18.5 dpc (C,F,I,L,O,R,U) using EGFP autofluorescence (A-C) and immunohistochemistry with antibodies directed against Sox8 (D-I), Sox10 (J-O) and Olig2 (P-U).

View larger version (102K): [in a new window]   Fig. 10. Terminal differentiation of oligodendrocytes in Sox10Sox8ki/Sox8ki mice. In situ hybridization with probes specific for MBP (A,B,E,F) and PLP (C,D,G,H) were performed on transverse spinal cord sections from the forelimb region at 18.5 dpc (A-D) and postnatal day 7 (E-H). (A,C,E,G) Wild-type spinal cords; (B,D,F,H) Sox10Sox8ki/Sox8ki spinal cords.