Figure 8. Netrin-1 increases outgrowth of and attracts cultured spinal motor axons.

(A, B) Explants of Isl1-GFP⁺ spinal motor nuclei were dissected to exclude the floor plate at E11. Explants were cultured for 48 hrs with or without Netrin-1 (200 ng/ml) (n=4 different day trials, n=12 explants for each treatment). (C) Graph of average axon length of each explant, showing that axons grow about 30% longer in the presence of Netrin-1 protein. (D) Graph of average number of axons in the explant, showing about 30% enhancement of axon numbers when Netrin-1 protein is added in the media. (E, F) Spinal motor nuclei explants were co-cultured with aggregates of COS-7 cells transfected with no plasmid (mock) or Netrin-1 expression plasmid (n=5 different day trials, n=15 explants for each treatment). (G) Quantification of directional axon growth into quadrants toward and away from the COS cells. The outgrowth ratio was calculated by the average axon length in the quadrant toward the COS cells (proximal) divided by the average axon length in the quadrants away from the cue source (distal). In the presence of Netrin-1, the ratio significantly increased, about 2-fold, showing that more axon outgrowth occurred toward the Netrin-1 source. Scale bars: A, B, 50 μm; C, D,50 μm. *=P < 0.05; **=P < 0.001.