Role of Sonic Hedgehog in OPC proliferation and differentiation.

<p><b>A: Proliferation of OPCs in response to Shh.</b> The proliferative capacity of OLIG2+ cells was assessed in the presence or absence of Shh. Images containing pixels labeled for both OLIG2 and BrdU were generated using the “AND” function of Metamorph software. Thus, these labeled cells were counted in presence or absence of Shh. OLIG2+ cell-proliferation rates were higher in the presence of Shh, as illustrated by the histogram. ** P<0.01 (Mann-Whitney test). <b>B: Differentiation of OPCs in response to Shh.</b> Diagram of Shh treatment schedule: P0–10 DIV mature slices cultured in the presence of Shh for the last 3 days of culture, i.e. between the 7th and 10th DIV. Photomicrographs of P0 cerebellar slices maintained for 10 DIV (mature) and labeled with antibodies against MBP. The MBP+ elements are less numerous in the slices treated with Shh for the last 3 days of culture (right) than in the control slice (left). Quantification of the density of MBP immunostaining (representing both oligodendrocytes and internodes) in P0–10 DIV mature control slices (Ctrl) and P0–10 DIV slices treated with Shh (Shh). * P<0.05 (Mann-Whitney test). <b>C: OPC differentiation in response to cyclopamine.</b> Photomicrographs of P0 cerebellar slices maintained for 7 DIV (immature) and labeled with antibodies against MBP. In the control slice (P0–7 DIV), the density of MBP immunostaining is low. In contrast, the density of MBP immunostaining is much higher in P0–7 DIV slices cultured in the presence of cyclopamine (P0–7 DIV Cyclo), a Shh inhibitor. Quantification of MBP immunostaining density in control (Ctrl) and cyclopamine-treated P0–7 DIV (Cyclo) immature slices. The density of MBP immunostaining was determined. ** P<0.01 (Mann-Whitney test). <b>D: OPC differentiation in co-culture experiments in the presence or absence of Shh.</b> Photomicrographs of P0 cerebellar slices labeled with antibodies against MBP at 7 DIV. These slices were co-cultured in the presence of P0 7–14 DIV mature slices treated with a high dose of BrdU during the first 3 days of culture (coc-BrdU) in the presence or absence of Shh. In the presence of Shh, the density of MBP immunostaining present in this immature slice was similar to that of control P0–7 DIV slices (compare C left and D right). In contrast, in the absence of Shh, there were more MBP+ elements in P0–7 DIV slices co-cultured with BrdU treated mature slices (coc-BrdU, left). Quantification of the density of MBP immunostaining in P0–7 DIV immature slices co-cultured in the presence or absence of BrdU treated mature slices and/or Shh. *** P<0.001 (Mann-Whitney tests). Scale bar in A is 70 µm for A and 425 µm for B, C, and D.