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Fig. 4. Netrin 1 induces Dcc-dependent process extension by immature
oligodendrocytes, and Dcc-dependent process branching and myelin-like membrane
sheet extension by mature oligodendrocytes, in vitro. (A) Addition
of netrin 1 (100 ng/ml, 24 hours) to cultured rat oligodendrocytes results in
a 4% decrease in the ratio of immature cells expressing Cnp only (arrowheads)
as compared with more mature cells expressing Cnp and Mbp. Netrin 1 did not
change the ratio of cells expressing Mbp only versus Mbp and Mag. (B)
Cnp-positive immature oligodendrocytes are multipolar cells with one major
process. Dashed red lines indicate examples of the processes measured.
(C) The length of the major process increased following the addition of
netrin 1 (100 ng/ml) for 24 hours. Application of Dcc function-blocking
antibody (Dccfb) to immature oligodendrocytes blocks the netrin-1-induced
increase in process extension, whereas Dccfb alone does not have a significant
effect on process extension. (D) A mature Mbp- and RIP-positive
oligodendrocyte extending both myelin-like membrane sheets and branched
processes. Sheet area was quantified by tracing the Mbp-positive myelin-like
membrane sheets (left, red outlines). Branching was quantified by measuring
the number of intersections that processes made with concentric circles
(right, red), which are numbered 1-5 to reflect increasing distance from the
cell body (and as labeled on the x-axis of bar charts displaying
branching complexity). (E) Netrin 1 (100 ng/ml, 24 hours) increased the
area of Mbp-positive sheets compared with the control. This effect was blocked
by Dccfb. (F) Processes of mature oligodendrocytes exposed to netrin 1
for 24 hours exhibited an increase in branching. Dose-response analysis
indicated maximal branching at 100 ng/ml netrin 1. (G) Addition of Dcc
function-blocking antibody together with netrin 1 prevented the
netrin-1-dependent increase in branching, and decreased branching compared
with controls. A, 20x0.5 n.a. objective; B,D,F, 40x0.75 n.a.
objective. Scale bars: 40 µm in A; 20 µm in B,D,F. A.U., arbitrary
units. *P<0.05, **P<0.005,
versus control. Error bars indicate s.e.m.
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