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Dual origin of spinal oligodendrocyte progenitors and evidence for the cooperative role of Olig2 and Nkx2.2 in the control of oligodendrocyte differentiation

Hui Fu1,*, Yingchuan Qi1,*, Min Tan1,*, Jun Cai1, Hirohide Takebayashi2, Masato Nakafuku3, William Richardson4 and Mengsheng Qiu1,{dagger}

1 Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, KY 40292, USA
2 Department of Pathology and Tumor Biology Graduate School of Medicine, Kyoto University Konoe-chou, Sakyo-ku, Kyoto 606-8501, Japan
3 Department of Neurobiology, University of Tokyo, Graduate School of Medicine, 7-3-1 Hongo, Bunkyoku, Tokyo 113-0033, Japan
4 Wolfson Institute for Biomedical Research, The Cruciform Building, University College London,Gower Street, London WC1E 6AE, UK
* These authors contributed equally to this work



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  		Fig. 1. Pdgfra+ and Sox10+ OLPs are generated from the Olig2+ pMN domain of ventral neuroepithelium in chicken. (A) E3 chicken spinal cords were double-labeled with anti-Nkx2.2 (brown) and Olig2 (blue). (B,C) E3-4 chicken spinal cords were double-stained with anti-Mnr2 (brown) and Olig2 (blue). Mnr+ motoneurons are produced from the Olig2+ domain dorsal to the Nkx2.2 domain. (D-G) Immediately adjacent chicken spinal cord sections from E7 (D), E8 (E,G) and E9 (F) were subjected to in situ hybridization with Pdgfra, Sox10 or Olig2 riboprobes. The ventral half from one side of the stained spinal cord was aligned with that from the same side of the adjacent cord. (H,I) Spinal cord from E7 chicken embryos were double-labeled with anti-Nkx2.2 (brown) and Sox10 (blue in H) or Pdgfra (blue in I). Sox10 and Pdgfra expression is located in the dorsal region of the Nkx2.2+ neuroepithelium (arrows).

 


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  		Fig. 2. Double labeling of Olig2 and Nkx-2.2 from E4 to E12. Spinal cord sections from E4 (A), E5 (B), E6 (C), E7 (D), E8 (E,I), E9 (F,J,K), E10 (G,L) and E12 (H) were subjected to Nkx-2.2 immunohistochemical staining (in brown) followed by in situ hybridization (in blue) with Olig2 riboprobe. Only the ventral half of spinal cords are shown. In A, anti-Nkx2.2 immunostaining and Olig2 in situ hybridization were performed on immediately adjacent slides, and half of the ventral cord is aligned closely for comparison. In B, the overlapping region of the Olig2 domain and Nkx2.2 domain is indicated. The Olig2+/Nkx2.2+ cells and Olig2–/Nkx2.2+ cells are represented by arrows and arrowheads, respectively. I is a higher power view of F. J,K are higher power views of F. L is a higher power view of G.

 


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  		Fig. 3. Double staining for Olig2 and Nkx2.2 in the hindbrain. Sections from E6 (A,B), E7 (C,D) and E8 (E,F) hindbrain tissues were subjected to anti-Nkx-2.2 immunostaining (in brown) followed by Olig2 in situ hybridization (in blue). Olig2 is only expressed in groups of migratory Nkx2.2+ OLP cells, but not in the ventricular cells. The representative double-positive cells are indicated by arrows, whereas the Nkx2.2+/Olig2- cells are represented by arrowheads.

 


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  		Fig. 4. Pdgfra+ OLPs are generated from the Olig2+ pMN domain in mouse. (A,B) Sections from E10.5 mouse spinal cord were subjected to in situ hybridization with Nkx2.2/Olig2 (B) or Olig2/Irx3 (A) probes. Nkx2.2 expression is flanked by Nkx2.2 ventrally and Irx3 dorsally. (C) Double labeling of E12.5 spinal cord with anti-Nkx2.2 (green) and anti-Olig2 (red) antibodies by immunofluorescence. (D-F) E12.5 spinal cord were double-labeled by in situ hybridization with Olig2/Pdgfra (D,E) or Nkx2.2/Pdgfra (F). D,E are from two separate embryos. Pdgfra expression is indicated by arrows.

 


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  		Fig. 5. (A-E) Merging of the Olig2 domain and Nkx2.2 domain in mouse after the onset of emigration of Olig2+ OLPs. Sections from E12.5 (A,B), E13.5 (C,D) and E14.5 (E,F) were labeled by Nkx2.2 (left half in A,C,E), Olig2 (right half in A,C,E) or both (B,D,F). Nkx2.2 expression in the ventricular zone starts to expand dorsally at E13.5 and almost overlaps with the entire Olig2 domain at E14.5. (G-H) Co-expression of Olig2 and Nkx2.2 in OLPs in P1 mouse spinal cord. Spinal cord sections were double-stained with anti-Olig2 (in brown) and Nkx2.2 (in blue). Although double-labeled cells are present throughout the entire cord, pictures were taken only from the lateral (G) and ventral (H) regions. Olig2+ cells with strong Nkx2.2 staining are represented by arrows, while those with weak Nkx2.2 expression are represented by arrowheads.

 


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  		Fig. 6. Co-expression of Olig2 and Nkx2.2 in dissociated spinal cord culture. The dorsal halves of E13.5 mouse spinal cords were isolated, dissociated and cultured on coverslips for 2 days (A-C) or 5 days (D-F). Cells were then simultaneously stained with anti-Nkx2.2 (green) and anti-Olig2 (red) by double immunofluorescence. The Olig2+ cells with strong Nkx2.2 expression are indicated by arrows, while those with weak Nkx2.2 expression are represented by arrowheads.

 


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  		Fig. 7. Expression of Pdgfra, Olig2 and Nkx2.2 in the Pdgfa mutants at E13.5 (A-F) and E16.5 (G-N). (A-F) Immediately adjacent sections from E13.5 wild-type (A-C) and mutant (D-F) spinal cords were probed for Pdgfra (A,D), Olig2 (B,E) and Nkx2.2 (C,F) by in situ hybridization. (G-N) Adjacent sections from E16.5 wild-type (G-J) and mutant (K-N) spinal cords were stained with Pdgfra (G,K), Olig2 (H,L), Nkx2.2 (I,M) and MBP (J,N). At these two stages, expression of Pdgfra and Olig2 is delayed and reduced in the mutants, whereas expression of Nkx2.2 and MBP is not affected. Note the similar patterns of expression of Nkx2.2 and MBP (I-J,M-N).

 


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  		Fig. 8. Expression of Pdgfra, Olig2 and Nkx2.2 in the PDGFA mutants at P0 and P7. (A-H) Immediately adjacent sections from P0 wild-type (A-D) and mutant (E-H) spinal cords were probed with Pdgfra (A,E), Olig2 (B,F), Nkx2.2 (C,G) and MBP (D,H) by in situ hybridization. (I-P) Adjacent sections from P7 wild-type (I-L) and mutant (M-P) spinal cords were stained with Pdgfra (I,M), Olig2 (J,N), Nkx2.2 (K,O) and MBP (L,P). At these two stages, expression of Pdgfra and Olig2 is delayed and reduced in the mutants, whereas expression of Nkx2.2 and MBP is mostly affected in the white matter. Note the similar patterns of expression of Nkx2.2 and MBP, especially in the gray matter, at P0 (C-D,G-H).

 


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  		Fig. 9. Inhibition of GalC+ cells and PLP+ cells in dissociated spinal cord culture by Nkx2.2 and Olig2 antisense oligonucleotide treatment. Dissociated culture from E5 spinal cords were treated with sense control (from Nkx2.2) or antisense oligonucleotides treatment (anti-Nkx2.2, anti-Olig2, or both) for 5 days. The relative numbers of GalC+ cells from different treatments were plotted, with the sense control as 100. For PLP+ cells, the average number of positive cells from each ten-field group (three groups from each treatment) was plotted.

 


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  		Fig. 10. Hypothetical model on the embryonic origin and dynamic gene expression profile of oligodendrocyte progenitors in embryonic spinal cord. (A) During neurogenesis stages, the ventral neuroepithelium can be divided into five domains, and each domain produces a distinct class of ventral interneuron (V0-V3) or motoneurons (MN) (adapted from Briscoe et al. (Briscoe et al., 2000Go). At this stage, Olig2 and Nkx2.2 are expressed in adjacent pMN domain and p3 domain, respectively. (B) Oligodendrogenesis in chicken. OLPs can be generated from both the pMN domain and p3 domain. Right before OLPs are produced from the pMN domain, expression of Nkx2.2 is dorsally expanded into the pMN domain. The OLPs that arise from the p3 domain appear to gain Olig2 expression before their terminal differentiation (see text). (C,D) Oligodendrogenesis in mouse. At the early stage of oligodendrogenesis, Nkx2.2 expression is not dorsally expanded and the Olig2+ OLPs acquire Nkx2.2 expression after migration. At later stages of oligodendrogenesis, Nkx2.2 expression is also upregulated in the pMN domain, similar to the situation in the chicken spinal cord. The p3-derived Nkx2.2+ OLPs might gain Olig2 expression after migration.

 

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