Fig. 7. Zfp488 cooperates withOlig2 to promote ectopic and precocious oligodendrocyte differentiation. E2.5 chick embryos were electroporated with expression vectors for Olig2 (A,B), Zfp488/Olig2 (C-F,I-J) or Zfp488/Nkx2.2 (G,H), and harvested 3 days later at E5.5. In situ hybridization of the neural tube was performed with the probes as indicated in panels. (A,B) Misexpression of the Olig2 transgene (Tg) alone did not induce oligodendrocyte markers in the dorsal region (red arrow in B), while a small number of Sox10+ cells were detected in the ventral domain (black arrowhead). (C-F) Co-electroporation of Zfp488/Olig2 induced robust ectopic expression of Sox10 (D, arrows), Pdgfra (E, arrows) and Mbp (F, arrows) in the electroporated side of the neural tube. (G,H) Co-electroporation of Zfp488/Nkx2.2 did not induce ectopic Sox10 expression in the transgenic side of the chick neural tube. (I,J) Dorsally confined misexpression of Zfp488 and Olig2 resulted in ectopic Sox10 expression in the dorsal region of the spinal cord (J, red arrow) but not in the ventral region (J, black arrowhead). (K,L) Double in situ labeling for chick ortholog of Zfp488 (purple) and immunostaining of Olig2 (brown) were performed in the chick spinal cord at E18. Co-expression of chick Zfp488 and Olig2 was detected in the spinal cord. L is a high magnification of an area outlined in K, showing the co-labeling of Zfp488 and Olig2 in the same cells of the chick spinal cord (red arrows). White arrowheads in L indicate the cells that express only Olig2. (M) Physical interaction between Zfp488 and Olig2. Vectors expressing Myc-tagged Zfp488 and Flag-tagged Olig2 were co-transfected into Cos7 cells. Co-immunoprecipitation (IP) of cell lysates (600 µg total) 48-hours post-transfection was performed with anti-Flag antibody. Western blot was carried out to detect the input proteins for Zfp488 (upper panel) and Olig2 (middle panel). The immunoprecipitated Myc-Zfp488 was detected by anti-Flag (lower panel, arrow). (N) Absence of Zfp488 and Nkx2.2 interaction. Vectors expressing Myc-tagged Zfp488 and Nkx2.2 were co-transfected into COS7 cells. Co-immunoprecipitation was performed with anti-Myc antibody. Western blot was performed to detect the input proteins for Myc-Zfp488 (upper panel) and Nkx2.2 (middle panel). The co-immunoprecipitated complex was detected by anti-Nkx2.2 (lower panel). Star indicates the prospective Nkx2.2 position.