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Fig. 2. Rac and Cdc42 GTPases play an essential role in axon outgrowth of PCN
toward a netrin 1 source. E11 rhombic lip explants were faced with netrin
1-secreting cells (Net) and cultured for 3 days. Axon outgrowth and nuclear
migration were then analyzed after Tuj1 and DAPI staining, respectively. In
control conditions, axon growth developed mainly toward the netrin 1 source
(A) and nuclear migration occurred almost exclusively within the netrin
1-attracted neurites (C). Addition of 1 ng/ml of lethal toxin, to specifically
inhibit Rac and Cdc42, resulted in a severe impairment of axon growth (B) but
did not affect nuclear migration (D). Measurement of the surface covered by
migrating nuclei (E), or quantification of their number (F), represented as
cumulative distributions and histograms (mean±s.e.m.), revealed no
significant difference between control (n=24) and drug-treated
(n=24) explants. (G) Axon outgrowth toward (proximal) and away
(distal) from the netrin 1 source (proximal and distal quadrants are
represented in A; *P<0.001 compared with control; error
bars represent s.e.m.). (H) Migration/outgrowth ratio in control and
drug-treated explants (*P<0.001). (I,J) High
magnification of axons stained with rhodamine-conjugated phalloidin to
visualize F-actin structures. Whereas control growth cones showed F-actin
enrichment (arrows in I), lethal toxin-treated axons lacked phalloidin
staining at their distal tip (arrows in J). Scale bars: A, 500 µm (for
A,B); C, 200 µm (for C,D); I,J, 10 µm.
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