Figure 6

Netrin1 and Slit2 have opposing effects on direction, and synergistic effects on growth, of explanted medial longitudinal fasciculus axons. Tissue was dissected from ventral midbrain of embryonic day 11.5 mouse embryos, placed in collagen gel, and cultured for 48 hours with guidance cues. (A-D) Ventral midbrain explants were co-cultured with aggregates of (COS) cells transfected with no plasmid (mock, n = 10), Netrin1 (n = 10), or Slit2 (n = 6) expression plasmids. To combine Netrin and Slit signals, equal amounts of transfected cells were mixed to form aggregates (n = 12). Each explant is shown with the transfected COS aggregate at the top of the photograph (dashed lines). Axons were labeled with anti-deleted in colorectal cancer (DCC) antibodies, to avoid other nearby neuron types that may have been inadvertently included in the explanted tissue (for example, the DCC-negative oculomotor neurons). (E) Quantification of directional axon growth into quadrants toward or away from the COS cells. An outgrowth ratio was calculated by dividing the average axon length in pixels in the quadrant toward the cue source, divided by the quadrant away from the cue source. Note the neutral effects of untransfected COS cells, attraction by Netrin1 cells, and repulsion by Slit2 cells. Mixed aggregates secreting both Netrin1 and Slit2 had neutral directional effects, but caused a prominent increase in overall axon growth. The number of explants analyzed is shown on each bar, and also applies to graphs (F) and (G). (F) Graph of average axon length in the quadrants toward and away from the cue source. (G) Graph of overall axon outgrowth, showing the sum from both quadrants. Note the significantly increased growth when Netrin1 and Slit2 signals were combined. Errors bars show ± SEM. Statistical comparison by t-test: *P < 0.05; **P < 0.01. Scale bar in (D) also applies to all: 100 μm.