Oligodendrocytes maturation is defective in b-raf ^Δ/Δneu mice. Immature, PDGFRα+ oligodendrocytes, stained in brown, are present in several areas of the P18 b-raf ^Δ/Δneu but not b-raf ^f/f brain. Subjects were counterstained with hematoxylin. (A) Brain cortex (inset and bottom) and the corresponding quantification of positive cells/brain area. The plot shows means ± SD; *, P < 0.02; **, P < 0.005; and ***, P < 0.0005 comparing three b-raf ^f/f and three b-raf ^Δ/Δneu mice. A quantification of the βIV-tubulin+ cells (premyelinating, early myelinating, and myelinating oligodendrocytes) from the same areas is also shown. (B and C) b-raf ^Δ/Δneu oligodendrocytes fail to differentiate in vitro. Oligodendrocyte precursors from P0 b-raf ^f/f and b-raf ^Δ/Δneu pups were allowed to differentiate for 5 (B) or 6 d (C). Differentiation was analyzed by immunofluorescence staining with α-NG2, α-O4, and α-MBP antibodies. Examples of single+ and double+ cells from a WT culture are shown below in B. In C, cell morphology was visualized by staining with an α-tubulin antibody. The percentage of NG2+ cells (oligodendrocyte progenitors), O4+ cells (pro-oligodendrocytes), and MBP+ cells (terminally differentiated oligodendrocytes) present in the b-raf ^f/f and b-raf ^Δ/Δneu cultures were compared. Each culture consisted of a pool of two WT or KO mice; for the chemical inhibition of MEK, oligodendrocyte cultures were established from pools of four WT mice and kept for 6 d in differentiation medium in the absence (untreated [UT]) or presence of MEK inhibitor (U0126; 10 μM). In all cases, a minimum of 100 cells/culture were counted independently by two investigators. The experiment was repeated three times, the plots show the mean ± SD of the results obtained by assessing three separate experiments (*, P < 0.05 for b-raf ^f/f vs. b-raf ^Δ/Δneu). Bars, 30 μm.