Fig. 8. SFK activity is required for netrin-1-induced changes in oligodendrocyte morphology. (A) Oligodendrocytes were obtained from mice lacking Fyn (Fyn KO) and treated with netrin 1 (100 ng/ml, 24 hours). Cells obtained from wild-type mice of a matched genetic background were used as controls. (B) Netrin 1 increased branching in control oligodendrocytes (F2 hybrid), but not in Fyn KO oligodendrocytes. (C,C') Phospho-SFK [P-SFK (pY416); red] immunoreactivity colocalizes (yellow) with Dcc (green) within the oligodendrocyte cell body and proximal branches. The confocal section shown is adjacent to the substrate and the appearance of staining throughout the cell body is not indicative of staining in the nucleus. (D,D') Portions of the images in C and C' have been magnified to illustrate punctate staining present within distal branches. Arrows indicate colocalized enrichment of phospho-SFK (red) and DCC (green) at puncta. (E) Binary images of phospho-SFK immunostaining were used to quantify the number of phospho-SFK-positive puncta. (F) The relative number of phospho-SFK-positive puncta increased in the presence of netrin 1 (100 ng/ml, 24 hour treatment). PP2 (2 µM) blocked the netrin-dependent increase in puncta, and PP2 alone showed a significant decrease in puncta compared with the control. PP3 (2 µM) did not block the effects of netrin 1 and had no independent effect on the cells. (G) The number of Fyn-positive puncta per unit area was not significantly (n.s.) changed upon addition of netrin 1. (H) The netrin-1-dependent increase in process length of Cnp-positive immature oligodendrocytes was blocked by the addition of PP2 (2 µM). Addition of PP3 did not disrupt the effect of netrin 1 on the cells. Oligodendrocytes treated with PP2 alone (2 µM), but not PP3 alone, exhibit a small but significant decrease in process extension compared with the control. (I) Disruption of SFK activity in mature Mbp-positive oligodendrocytes prevented the increased branching observed in the presence of netrin 1. PP2 and PP3 did not independently affect oligodendrocyte morphology. ^*P<0.05, ^**P<0.005, ^***P<0.001, versus control. Error bars indicate s.e.m. A,E, 40x0.75 n.a. objective; C-D', confocal microscopy, 40x0.75 n.a. objective. Scale bars: 20 µm in A,E; 40 µm in C',D'.