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First published online 5 November 2003
doi: 10.1242/dev.00868

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Region-specific and stage-dependent regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 homeodomain transcription factor

Rugao Liu^1,*, Jun Cai^1,*, Xuemei Hu^1,*, Min Tan¹, Yingchuan Qi¹, Michael German², John Rubenstein³, Maike Sander⁴ and Mengsheng Qiu^1,

¹ Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, KY 40292, USA
² Department of Medicine, University of California, San Francisco, CA 94143, USA
³ Department of Psychiatry, University of California, San Francisco, CA 94143, USA
⁴ Department of Developmental and Cell Biology, University of California at Irvine, 4228 McGaugh Hall, Irvine CA 92697-2300, USA

View larger version (66K): [in a new window]   Fig. 1. Reduced and delayed Olig2 expression in Nkx6.1 mutant spinal cord at the thoracic region. Transverse spinal cord sections from E12.0 (A,B), E13.5 (C,D), E15.5 (E,F), E17.5 (G,H) and P0 (I,J) wild-type (A,C,E,G,I) and Nkx6.1 (B,D,F,H,J) mutants embryos were subjected to in situ hybridization with riboprobe for Olig2. In Nkx6.1 mutants, the early expression of Olig2 in the ventral ventricular zone is markedly reduced and asymmetrically displayed, and the generation of Olig2+ OPC cells is dramatically delayed and reduced.

View larger version (115K): [in a new window]   Fig. 3. Altered expression of Nkx2.2 in Nkx6.1 mutant spinal cord at the thoracic level. (A-F) Spinal cord sections from E13.5 (A,B), E15.5 (C,D) and E18.5 (E,F), wild-type (A,C,E) and mutant (B,D,F) animals were hybridized with Nkx2.2 riboprobe. (G-L) Double immunofluorescent staining of E13.5 (G,H) and E18.5 (I-L) spinal cord sections with anti-Nkx2.2 and anti-Olig2 (G-J), or with anti-Nkx2.2 and NeuN (K,L). In the ventral gray matter, most Nkx2.2+ cells co-express NeuN but not Olig2. In the white matter, Nkx2.2+ cells co-express Olig2, but not NeuN. Representative Nkx2.2+ cells are indicated by arrowheads, whereas the double-positive cells are indicated by arrows. (M,N) Higher power view of the NeuN/Nkx2.2 double-positive cells (yellow) in the wild-type (M) and Nkx6.1 mutant (N) spinal cords.

View larger version (101K): [in a new window]   Fig. 2. Delayed and reduced production of Pdgfra + (A-H) and NG2+ (I-L) OPCs in Nkx6.1 mutant spinal cords. Spinal cord sections from E13.5 (A,B), E15.5 (C,D,I,J), E17.5 (E,F) and P0 (G,H,K,L) wild-type (A,C,E,G,I,K) and mutant (B,D,F,H,J,L) embryos were subjected to in situ hybridization with Pdgfra riboprobe or double immunofluorescence with anti-NG2 (red) and anti-PECAM1 (green) antibodies. During embryogenesis, NG2 labels both OPC cells and PECAM1+ immature blood vessels (Ozerdem et al., 2001).

View larger version (110K): [in a new window]   Fig. 4. (A-F) Disrupted expression of Mbp and Plp in Nkx6.1 mutant spinal cord. Spinal cord sections at the thoracic level were prepared from E17.5 (A,B) and P0 (C-F) wild-type (A,C,E) and mutant (B,D,F) animals and hybridized with Mbp (A-D) and Plp (E,F) riboprobes. (G-H) Similar MBP expression in spinal cord explants in normal and Nkx6.1 mutants. Spinal cord explants were isolated from the thoracic regions of the E13.5 wild-type (G) and mutant (H) embryos and cultured in vitro for 7 days before whole-mount in situ hybridization with Mbp riboprobe.

View larger version (141K): [in a new window]   Fig. 5. Reduced expression of Nkx2.2 and Mbp in P0 Nkx6.1 mutant spinal cord at the cervical region. Cross-sections of cervical spinal cord from P0 wild-type (A,C,E) and mutant (B,D,F) pups were hybridized with Nkx2.2 (A,B), Mbp (C,D) and Plp (E,F) riboprobes. Arrows indicate the MBP+/PLP+ oligodendrocytes in the mutants.

View larger version (75K): [in a new window]   Fig. 6. Stage-dependent effects of Nkx6.1 overexpression on Olig2, Mnr2/Hb9 and Sox10 gene expression. Spinal cord tissues from E2 chicken embryos were electroporated with RCASBP-Nkx6.1 retroviral expression vector, and then were processed either two days (at E4, A-D) or four days (at E6, E-H) later for gene expression studies by immunofluorescence with anti-Nkx6.1, anti-Olig2, anti-Mnr2/Hb9 antibodies or by in situ RNA hybridization with Sox10 riboprobe. Nkx6.1 overexpression induced Olig2 expression at E4 (A), but inhibited its expression at E6 (E). The endogenous expression of Nkx6.1 in the ventral neuroepithelium was much weaker than the retrovirally mediated expression and was not shown under this specific photographic condition. Induction of gene expression is indicated by arrows, whereas inhibition by arrowhead. Note that sporadic expression of Nkx6.1 was also observed in the control side of the spinal cord (E), likely due to the secondary infection of the Nkx6.1-expressing retroviruses produced by the electroporated side.

View larger version (84K): [in a new window]   Fig. 7. Normal expression of MBP in Nkx6.1 mutant hindbrain. Cross- or sagittal sections of the rostral hindbrain from E15.5 (A,D), E17.5 (B,E) and P0 (C,F) wild-type (A-C) and mutant (D-F) animals were stained with Mbp probe. M, medulla; P, pons; SC, cervical spinal cord.

View larger version (81K): [in a new window]   Fig. 8. Multiple sites of origin of Olig1 and Olig2 expression in the rostral hindbrain during early stages of oligodendrogenesis. (A-H) Adjacent slide sections from E13.5 wild-type (A,C,E,G) and mutant (B,D,F,H) hindbrain at the pontine area were hybridized with Olig1 (A,B), Olig2 (C,D), Pdgfra (E,F) and Nkx2.2 (G,H) probes. Only the tissue staining surrounding the fourth ventricle is shown. In A-D, arrows indicate Olig1 and Olig2 expression in the Nkx6.1/Nkx2.2 domain of the ventral ventricular zone; arrowheads indicate Olig1/2 expression outside the Nkx6.1 domain. (I,J) Slide sections from E13.5 wild-type hindbrain at the same level were double-labeled with Nkx6.1 (blue) and Olig1 (orange), or Nkx2.2 (blue) and Olig1 (orange). In I, the blue arrow indicates the dorsal boundary of Nkx6.1 expression.

View larger version (127K): [in a new window]   Fig. 9. Normal expression of early OPC marker genes in the Nkx6.1 mutant brain. Cross-sections of the brain tissue from E15.5 wild-type (A-D) and mutant (E-H) animals were hybridized with Olig1 (A,E), Olig2 (B,F), Pdgfra (C,G) and Sox10 (D,H) riboprobes. M, medulla; P, pons; T, thalamus.