Fig. 4. Myelin membrane sheet formation is reduced by dystroglycan-blocking antibodies. (A) Oligodendrocytes were differentiated for 2 or 4 days on PDL or laminin (Lm) in the presence of dystroglycan-blocking antibodies (anti-DG) or control antibodies. Indirect immunofluorescence was used to detect myelin basic protein (MBP) expression. The percentage of MBP-positive cells was determined using DAPI nuclear stain to obtain total cell counts per field. In the presence of blocking antibodies, no change in the percentage of MBP-positive cells was observed at either time point or on either substrate. (B) The average percentage of cells differentiated on laminin that express galactocerebroside C (GalC) following treatment with dystroglycan-blocking antibodies (anti-DG) or control antibodies. No significant change was found. (C) Typical morphology at day 2. Oligodendrocytes were differentiated for 2 days on laminin in the presence of dystroglycan-blocking or control antibodies. MBP immunofluorescence was used to visualize differentiated cells. Nuclei were detected using DAPI nuclear stain. (D) Typical morphology at day 4. Oligodendrocytes were differentiated for 4 days in the presence of dystroglycan (DG) or control antibodies. MBP immunofluorescence was used to visualize differentiated cells. Nuclei were detected using DAPI nuclear stain. (E) Myelin membrane sheet complexity is reduced by DG-blocking antibodies. A morphology classification scheme (bottom) was used to evaluate the degree of myelin membrane in MBP-positive cells. Examples of MBP-expressing cells: stages 1, 2 and 3 show increasing levels of process outgrowth and branching, without myelin membrane, whereas stages 4, 5 and 6 show increasing levels of complexity and myelin membrane. The percentage of cells within each category is shown for oligodendrocytes differentiated for 2 or 4 days in the presence or absence of DG-blocking antibodies (control, black squares; anti-DG, gray triangles). In the presence of DG-blocking antibodies, no difference in morphology profile is observed at day 2; however, at day 4, a significant shift away from more-complex myelin membrane structures is observed (^*P<0.05, ^**P<0.01, error bars represent s.d.). Scale bars: 100 µm.