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First published online 9 January 2008
doi: 10.1242/dev.010454

Development 135, 637-646 (2008)
Published by The Company of Biologists 2008
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Sox9 and Sox10 influence survival and migration of oligodendrocyte precursors in the spinal cord by regulating PDGF receptor {alpha} expression

Markus Finzsch, C. Claus Stolt, Petra Lommes and Michael Wegner*

Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany.


Figure 1
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  		Fig. 1. Sox9 deletion in the oligodendrocyte lineage. (A) Schematic representation of Sox9 and Sox10 alleles and the Sox10::Cre transgene used in this study. Boxes represent exons with translated parts being filled or expression cassettes for neomycin resistance (neo), β-galactosidase (lacZ) and Cre recombinase (Cre). Ellipses indicate positions of loxP sites, arrowheads those of genotyping primers a-i. (B-M) Sox9 immunoreactivity (red) was detected at 12.5 dpc (B-G) and 15.5 dpc (H-M) alone (B,E,H,K) or together with Olig2 (C,F,I,L) or Sox10 (D,G,J,M) immunoreactivity (green) in transverse spinal cord sections from the forelimb region of wild-type (B,C,D,H,I,J) or Sox9loxP/loxP, Sox10::Cre (Sox9{Delta}/{Delta}) (E,F,G,K,L,M) embryos. The region from which I,J,L,M were taken is boxed in H and K.

 

Figure 2
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  		Fig. 2. Progression of Sox9 deletion in Sox9loxP/loxP, Sox10::Cre spinal cords. Sox9 immunoreactivity (red) was detected and compared with Sox10 immunoreactivity (green) at 13.5 dpc (A-H) and 14.5 dpc (I-P) in transverse spinal cord sections from the forelimb region of wild-type (A-D,I-L) or Sox9loxP/loxP, Sox10::Cre (Sox9{Delta}/{Delta}) (E-H, M-P) embryos. Both low-magnification overviews (A,E,I,M) and high-magnifications (B,C,D,F,G,H,J,K,L,N,O,P) are provided. A,B,E,F,I,J,M,N show the Sox9 staining; C,G,K,O show the Sox10 staining; D,H,L,P represent merged pictures.

 

Figure 3
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  		Fig. 3. Oligodendrocyte development in Sox9loxP/loxP, Sox10::Cre spinal cords. Immunohistochemistry with antibodies specific for Sox10 (A-D), Olig2 (E-H), Pdgfra (I-L) and in situ hybridizations with a Plp-specific probe (M,N) were performed on transverse sections from the forelimb region of wild-type (A,C,E,G,I,K,M) or Sox9loxP/loxP, Sox10::Cre (Sox9{Delta}/{Delta}) (B,D,F,H,J,L,N) embryos at 15.5 dpc (A,B,E,F,I,J) and 18.5 dpc (C,D,G,H,K,L,M,N).

 

Figure 4
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  		Fig. 4. Correlation of Sox9 and Pdgfra expression. Co-immunohistochemistry was performed on transverse spinal cord sections from the forelimb region of wild-type (A,E), Sox9loxP/loxP, Sox10::Cre (Sox9{Delta}/{Delta}) (B,F), Sox10lacZ/lacZ (Sox10-/-) (C,G) and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (Sox9{Delta}/{Delta} Sox10-/-) (D,H) embryos at 14.5 dpc (A-D) and 15.5 dpc (E-H) with antibodies directed against Sox9 (red) and Pdgfra (green). Nuclei were counterstained with DAPI.

 

Figure 5
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  		Fig. 5. Oligodendrocyte development in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords. Immunohistochemistry with antibodies specific for Sox10 (A,E,I,M,Q), β-galactosidase (B,F,J,N,R) and Olig2 (C,D,G,H,K,L,O,P,S,T) were performed at 12.5 dpc (A-D), 13.5 dpc (E-H), 14.5 dpc (I-L), 15.5 dpc (M-P) and 18.5 dpc (Q-T) on transverse sections from the forelimb region of wild-type (A,C,E,G,I,K,M,O,Q,S) or Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (Sox9{Delta}/{Delta} Sox10-/-) (B,D,F,H,J,L,N,P,R,T) embryos. Subcellular localization of Sox10 (nuclear) and β-galactosidase (cytoplasmic) causes a slightly altered appearance of cells in A,E,I,M,Q when compared with B,F,J,N,R. (U-W) OLP numbers were determined at 12.5 dpc (U), 13.5 dpc and 14.5 dpc (V) as Sox10- or β-galactosidase-positive cells, and at 15.5 dpc and 18.5 dpc (W) as Olig2-positive cells in wild-type (white bars), Sox9loxP/loxP, Sox10::Cre (light-grey bars), Sox10lacZ/lacZ (dark-grey bars) and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (black bars) spinal cords as indicated. For these quantifications, at least 30 separate 10 µm sections from the forelimb region of two independent embryos were counted for each genotype. Data are presented as mean±s.e.m. Differences to the wild type were statistically significant for oligodendrocyte numbers in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords from 14.5 dpc onwards, as determined by Student's t-test (***P≤0.001).

 

Figure 6
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  		Fig. 6. Oligodendroglial proliferation and survival in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords. (A) Oligodendroglial proliferation was measured at 15.5 dpc and 18.5 dpc as the percentage of Sox10-positive cells (in the wild type, white bars) or β-galactosidase-positive cells (in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre embryos, black bars) that also expressed Ki67. (B) The fraction of Ki67-positive cells also labelled by a 1 hour pulse of BrdU was determined in the wild-type and in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre embryos as a measure of mitotic rates and cell cycle length. (C) The Ki67-negative fraction of cells labelled by a single BrdU pulse 24 hours before embryo preparation was determined in the wild-type and in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre embryos as a measure of cell cycle exit rates. (D-G) TUNEL was performed on transverse sections from the forelimb region of wild-type (D,F) and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (E,G) embryos at 15.5 dpc (D,E) and 18.5 dpc (F,G). (H) Apoptosis was quantified as the number of TUNEL-positive cells in wild-type (white bars) and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (black bars) spinal cords at 15.5 dpc and 18.5 dpc. (I) Apoptosis in OLPs was determined at 15.5 dpc and 18.5 dpc as the number of TUNEL-positive, β-galactosidase-labelled cells in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords (black bars) relative to the number in the Sox10lacZ/+ spinal cords (grey bars) which was arbitrarily set to 1. For all quantifications, at least 30 separate 10 µm sections from the forelimb region of two independent embryos were counted for each genotype. Data are presented as mean±s.e.m. Differences were statistically significant for apoptosis rates in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords, as determined by Student's t-test (***P≤0.001).

 

Figure 7
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  		Fig. 7. Absence of ectopic marker gene expression in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords. In co-immunohistochemistry on transverse spinal cord sections from the forelimb region of Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (Sox9{Delta}/{Delta} Sox10-/-) embryos at 15.5 dpc (A-C) and 18.5 dpc (D-F). OLPs were identified by Olig2 (A,B,D,E) or β-galactosidase (C,F) expression (in green), and analyzed for the simultaneous occurrence of the cell-type specific markers (in red) NeuN (A,D), Glast (B,E) and B-FABP (C,F). Nuclei were counterstained with DAPI.

 

Figure 8
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  		Fig. 8. Oligodendrocyte marker gene expression in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords. (A-D) Quantification of oligodendroglial marker gene expression at 18.5 dpc as the percentage of Olig2-positive cells that also expressed NG2 (A), DCC (B), O4 (C) and Nkx2.2 (D) in wild-type (white bars), Sox10lacZ/lacZ (Sox10-/-; dark grey bars) and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (Sox9{Delta}/{Delta}, Sox10-/-; black bars) spinal cords at 18.5 dpc following immunocytochemistry on acutely dissociated spinal cord cultures (A-C) or immunohistochemistry on transverse spinal cord sections (D). For all quantifications, at least 10 separate cover slips or 30 separate 10 µm sections from the forelimb region were counted from two independent embryos for each genotype. Data are presented as mean±s.e.m. Differences from the wild-type were statistically significant for Nkx2.2 in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords, as determined by Student's t-test (***P≤0.001). (E) Co-immunohistochemistry with antibodies specific for Olig2 (in red) and Nkx2.2 (in green) were performed on transverse sections from the forelimb region of wild-type, Sox10lacZ/lacZ and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords at 18.5 dpc. Comparable regions of the ventral marginal zone are shown. (F) In situ hybridization with a Plp-specific probe was performed on transverse sections from the forelimb region of wild-type, Sox10lacZ/lacZ and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords at 18.5 dpc.

 

Figure 9
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  		Fig. 9. Correlation of Olig2 and Pdgfra expression. Co-immunohistochemistry was performed on transverse spinal cord sections from the forelimb region of wild-type (A,E), Sox9loxP/loxP, Sox10::Cre (Sox9{Delta}/{Delta}) (B,F), Sox10lacZ/lacZ (Sox10-/-) (C,G) and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (Sox9{Delta}/{Delta} Sox10-/-) (D,H) embryos at 15.5 dpc (A-D) and 18.5 dpc (E-H) with antibodies directed against Olig2 (red) and Pdgfra (green).

 

Figure 10
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  		Fig. 10. Pdgfra expression in Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre spinal cords. In situ hybridizations with a Pdgfra-specific probe (A,B,C,D,E,F,I,J,M,N) and immunohistochemistry with antibodies specific for Pdgfra (G,H,K,L,O,P) was performed at 13.5 dpc (A-D), 14.5 dpc (E-H), 15.5 dpc (I-L) and 18.5 dpc (M-P) on transverse sections from the forelimb region of wild-type (A,E,I,M,G,K,O), Sox9loxP/loxP, Sox10::Cre (Sox9{Delta}/{Delta}) (B), Sox10lacZ/lacZ (Sox10-/-) (C) and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (Sox9{Delta}/{Delta} Sox10-/-) (D,F,H,J,L,N,P) embryos. The arrowhead in D indicates the Pdgfra-positive cells. (Q) Chromatin immunoprecipitation was performed on formaldehyde-fixed spinal cords from four day old wild-type mice in the absence (-) and presence of antibodies (IgG, control IgG; {alpha}-Sox9, anti-Sox9 antibodies). PCR was applied on the immunoprecipitate to detect evolutionary conserved sequence elements C1, C2 and C3 in the proximal 5' flanking region of the Pdgfra gene, as well as non-conserved regions N1 and N2 in the distal 5' flanking region. These fragments were also amplified from 1/20 of the material used for immunoprecipitation (Input). H2O: water control. (R) OLP numbers were determined in cultures of dissociated spinal cords from wild-type (white bars) and Sox9loxP/loxP, Sox10lacZ/lacZ, Sox10::Cre (Sox9{Delta}/{Delta} Sox10-/-) (black bars) 18.5-day-old embryos 3 hours after seeding or after 48 hours in the absence or presence of 20 ng/ml PDGF-AA. At least 30 visual fields were counted from two independent embryos for each genotype. Data are presented as mean OLP number per visual field±s.e.m.

 




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