Figure 12.

Behavior of growth cones at a substrate border of neurons depleted of DCX and DCLK. Neurons were co-transfected with control siRNA (CTRL) or DCX+DCLK siRNA (siRNAs) and either cDNAs to EGFP alone or EGFP tagged DCX, plated on a poly-d-lysine substrate for 3 days and then re-plated on a discrete area of laminin on the surface of a uniform poly-d-lysine substrate as described in the Materials and Methods section. Upon reaching the border between the laminin and poly-d-lysine, the growth cones of axons originating on the laminin side can either cross the border (‘ignore) or remain on the laminin side (‘not ignore’). After fixation, the neurons were stained to reveal laminin, actin filaments and tubulin as described in Materials and Methods. (A) An image showing both laminin and actin staining, whereas (B) shows the same field stained for tubulin (the scale bar = 9 μm). The border between the laminin and poly-d-lysine is indicated by the arrows; we note that the staining for laminin was somewhat greater at the border than at regions internal to the border. Within the images are examples of axons that either ignored (double arrowhead) or did not ignore (single arrowhead) the border. The focal plane of axons commonly changed as they approached the border. We counted axons that ignored or did not ignore the border under the various conditions and the results are shown in the graph (a minimum of 120 axons were counted for each condition). In cultures of control neurons, ∼50% of the axons either ignored or did not ignore the border. Overexpression of human DCX increased the percentage of axons ignoring the border, although the difference was not statistically significant (P > 0.05, χ²-test). Depletion of both DCX and DCLK reduced the percentage of axons that ignored the border compared with controls (from ∼50 to ∼30%, P < 0.0001, χ²-test, CTRL-EGFP versus DCX+DCLK siRNA with EGFP). In neurons treated with DCX+DCLK siRNAs, overexpression of human DCX reversed the effects of siRNA treatment alone such that the percentage of growth cones ignoring the borders resembled that in control neurons over-expressing EGFP-DCX.