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Control of oligodendrocyte differentiation by the Nkx2.2 homeodomain transcription factor

Yingchuan Qi1,*, Jun Cai1,*, Yuanyuan Wu2, Rui Wu1, Jeffrey Lee2, Hui Fu1, Mahendra Rao2, Lori Sussel3, John Rubenstein4 and Mengsheng Qiu1,{ddagger}

1 Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, KY 40292, USA
2 Department of Neurobiology and Anatomy, University of Utah, School of Medicine Salt Lake City, UT 84132, USA
3 Barbara Davis Center for Childhood Diabetes, University of Colorado, Denver, CO 80262, USA
4 Department of Psychiatry, University of California, San Francisco, CA 94143, USA
* The first two authors contributed equally to this work



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  		Fig. 1. Expression of Nkx2.2 in developing mouse spinal cords shown by in situ hybridization. (A) E10.5; (B) E13.5; (C) E15.5; (D) P0; (E) P4; (F-H) P8. (G,H) Higher magnification of F at the lateral (G) and ventral (H) positions. Nkx2.2-positive cells start to migrate away from the ventricle at E13.5 into both gray matter (arrow, B) and the ventral and lateral white matter (arrowheads, B,C).

 


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  		Fig. 2. Selective expression of Nkx2.2 in isolated A2B5 glial progenitor cells, but not in dissociated neurons or astrocytes. (A-C) Immortalized GRP cells were stained simultaneously with DAPI for labeling nuclei (A) and with an antibody against Nkx2.2 (B). A superimposed image (C) revealed that a subset of A2B5 cells express Nkx2.2. (D) Immunopurified A2B5 cells were co-stained with DAPI, anti-Nkx2.2 and A2B5. Again, a subpopulation of cells express Nkx2.2 (arrows). (E,F) Cultured cells from dissociated E13.5 rat spinal cord were labeled simultaneously with DAPI, anti-Nkx2.2 and anti-ßIII tubulin (E) or anti-GFAP (F). Nkx2.2 staining (arrows) is not found in ßIII tubulin-positive neurons or GFAP-positive astrocytes.

 


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  		Fig. 3. Retarded differentiation of MBP-positive and PLP-positive oligodendrocytes in Nkx2.2 mutant spinal cords. Transverse sections from various stages of wild-type (A-I) and mutant (A'-I') littermates were hybridized with MBP (A-E) or PLP-DM20 (F-I) riboprobes by in situ hybridization. Note the reduced and delayed expression of MBP and PLP in the white matter and the lack of MBP-positive and PLP-positive cells in the gray matter of the mutant spinal cords.

 


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  		Fig. 4. Defective oligodendrocyte differentiation in the Nkx2.2 mutant brains. (A,B) Sagittal sections of the E17.5 wild-type (A) and mutant (B) hindbrain/midbrain tissues were subject to in situ hybridization with MBP probe. Many fewer MBP-positive cells are detected in the ventral brain stem of the mutant. (C-F) Coronal sections of P7 wild type (C,E) and mutant (D,F) were hybridized with MBP (C,D) and PLP-DM-20 (E,F) riboprobes. In the wild-type mice, numerous MBP-positive and PLP-DM20-positive oligodendrocytes are found in association with corpus callosum (cc) and anterior commissure (ac), whereas in the mutants, no clear labeling of MBP and PLP-DM20 is observed.

 


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  		Fig. 5. Differentiation of GFAP-positive astrocytes appears normal in the Nkx2.2 mutants. Spinal cord sections from E15.5 (A,B) and E18.5 (C,D) wild-type (A,C) and Nkx2.2 mutants (B,D) were immunolabeled with anti-GFAP polyclonal antibody. Only a small number of GFAP-positive astrocytes can be observed in the most ventral white matter at E15.5 (indicated by arrows) in both normal and mutant embryos. At E18.5, GFAP-positive astrocytes can be seen in most of the white matter regions.

 


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  		Fig. 6. Nkx2.2 mutation does not affect spinal cord expression of neuregulin 1 (Nrg1). Nrg1 is primarily expressed in motor neurons in the ventral spinal cord at E10.5 (A), E13.5 (C) and E14.5 (B). There is no discernible difference in the expression of Nrg1 between wild type and mutants at E13.5 (C,D) and E17.5 (not shown).

 


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  		Fig. 7. Ventral expansion of Olig1/Olig2 expression in neuroepithelium and increased production of oligodendrocyte progenitor cells in the Nkx2.2 mutant spinal cord. Spinal cord sections from E13.5 (A-D,G-H) and E15.5 (E,F) wild-type (A,C,E,G) and mutant (B,D,F,H) embryos were labeled for Olig1 (A,B) or Olig2 (C-F) or double-labeled for Olig2 and Shh (G,H). In the wild-type embryos, Olig1 and Olig2 are expressed in the same ventricular cells, which are separated from the Shh-expressing floor plate by the Nkx2.2 domain (arrow, G). In the mutants, the Olig1/Olig2 domain is ventrally expanded into the Nkx2.2 domain and becomes directly adjacent to the floor plate (H). The number of Olig1/Olig2-positive cells in the surrounding regions is accordingly increased.

 


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  		Fig. 8. Production of Pdgfra-positive progenitors is not compromised in the Nkx2.2 mutant spinal cord. Spinal cord sections from E13.5 (A,B), E15.5 (C,D) and E18.5 (E,F) wild-type and mutant embryos were hybridized with a riboprobe for Pdgfra. The production of Pdgfra-positive oligodendrocyte progenitors is neither reduced nor delayed in the Nkx2.2 mutants.

 


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  		Fig. 9. Normal production of the Pdgfra-positive and Olig2-positive oligodendrocyte progenitors in the Nkx2.2 mutant brain regions. (A-D) Sagittal sections of the E17.5 wild type (A,C) and Nkx2.2 mutants (B,D) were hybridized with riboprobes for Pdgfra (A,B) or Olig2 (C,D). The number of Olig2-positive and Pdgfra-positive oligodendrocyte progenitors appears not to be reduced.

 


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  		Fig. 10. Nkx2.2 can drive the expression of GFP under the control of the PLP promoter. NIH 3T3 cells were transiently transfected with Nkx2.2 alone (A), PLP-GFP alone (B'), GFAP-GFP alone (C'), Nkx2.2 and PLP-GFP (B) and Nkx2.2 and GFAP-GFP (C). Few, if any, cells are GFP positive in cells transfected with Nkx2.2 alone, PLP-GFP alone or GFAP-GFP alone. In the presence of Nkx2.2 (stained in red), the number of GFP-positive cells (stained in green, indicated by arrows) is dramatically increased by co-transfection with PLP-GFP, but with not GFAP-GAP. (D) The sum of GFP-positive cells in various experiments. In the Nkx2.2/PLP-GFP co-transfection experiment, the vast majority of cells that were GFP positive were also Nkx2.2 positive (n=244, blue bar), although a small number of cells were Nkx2.2/GFP positive (n=8, red bar). For each transfection experiment, 50 random fields of cells were counted for GFP-positive cells.

 

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