Fig. 9. BMP4-mediated inhibition of oligodendrocyte development is accompanied, in vitro as in vivo, by strong repression of Olig2 but does not depend on inhibition of Nkx2.2 expression. (A-D) Ventral neuroepithelial explants were dissected from E5 embryos, before oligodendrocyte induction has occurred. They were grown for 2 days in the absence or presence of 12 nM Shh, with or without 0.6 nM BMP4, and double-stained with Olig2 antiserum (green) and anti-Nkx2.2 monoclonal antibody (red). (A) Control explant displaying small numbers of Olig2 and Nkx2.2-positive nuclei. Only few nuclei are double-labelled. (B) BMP4 treatment alone inhibits Olig2 expression but does not significantly affect Nkx2.2 expression. (C) Shh treatment, which induces oligodendrocytes, also enhances the expression of Olig2 and Nkx2.2. Note that most nuclei are double labelled. (D) Co-treatment with Shh and BMP4, which abolishes Shh-mediated oligodendrocyte induction, also strongly inhibits Olig2 expression but does not affect Nkx2.2 expression. Note, in particular, Nkx2.2-positive nuclei in neuroepithelial cells forming the side of the explant (arrowhead). (E) Quantification of the number of Nkx2.2-positive and Olig2-positive nuclei per explant (20-33 explants per experiment). Bars represent the mean±s.e.m. of at least three independent experiments. Significance of the differences between treated and control conditions, assessed by Student's t-test: ^*, P<0.01; ^**, P<0.001. (F-K) Grafts of BMP2-overexpressing Q2bn cells were implanted at E3-E3.5 between neural tube and somites and embryos were fixed at E7-7.5. Transverse spinal cord sections were double stained with Olig2 antiserum (green) and Nkx2.2 antibody (red). (F-H) Control section. (F) Olig2-positive neuroepithelial cell nuclei are found several cell diameters above the floor plate. (G) Nkx2.2-positive nuclei are located more ventral in the neuroepithelium. (H) The overlay shows co-expression of the transcription factors in some nuclei (yellow), indicating that their respective domains of expression overlap at this stage. (I-K) Section at the level of the graft from an embryo implanted with BMP2-Q2bn cells. (I) Expression of Olig2 is totally abolished on the grafted side. (J) By contrast, Nkx2.2 expression appears not to be affected by the implanted BMP2-producing cells. (H) The overlay shows co-expression of the transcription factors in some nuclei on the contralateral side.