Fig. 4. Temporal sequence of the co-expression of Ascl1, Olig2 and Nkx2-2. (A,B) Stage-dependent increase in the number of Ascl1⁺, Olig2⁺ and Nkx2-2⁺ cells (A), and NG2⁺, GalC⁺ and MBP⁺ (B) cells. Cells isolated from rat spinal cords between the upper and lower limbs were subjected to immunostaining. Data are mean±s.d. obtained from three independent experiments. The number of cells triple positive for Ascl1, Olig2 and Nkx2-2 was estimated based on the results of a series of double staining. (C) Immunostaining of dissociated spinal cord cells for various markers at E20.5. Arrowheads indicate cells double positive for respective markers. (D) Developmental changes in the co-expression pattern of various markers. The percentages of cells positive for a given marker (leftmost column) that were double positive for other markers (top column) are shown. Data are the mean of three independent experiments. Scale bars: in C, parts c,f, 20 µm for C, parts a-f; in C, part i, 50 µm for C, parts g-i.