GAP-43 dependency defines distinct effects of netrin-1 on cortical and spinal neurite outgrowth and directional guidance.

Growth-associated protein-43 (GAP-43) is a major nervous system protein whose phosphorylation by protein kinase C regulates growth cone responses to extracellular guidance cues via F-actin. GAP-43 is essential for axon pathfinding in both cortical afferents and efferents: when it is genetically deleted, somatosensory, auditory and visual somatotopic maps fail to form, and telencephalic commissural axons fail to cross the midline. Here we investigated whether the midline guidance cue netrin-1 depends on GAP-43 for its functions in neurite growth and guidance. We used 3-dimensional collagen gel co-cultures to show that both endogenous netrin-1, expressed by the spinal cord floor plate, and recombinant netrin-1, expressed by transfected COS7 cells, stimulate neurite outgrowth and chemotropic guidance of neocortical callosal axons. In contrast both were significantly inhibited in GAP-43 (-/-) neocortical callosal axons, mimicking the in vivo phenotype. Conversely, neither netrin-1-stimulated neurite outgrowth nor guidance of dorsal spinal cord commissure axons were affected when GAP-43 was absent, again consistent with in vivo phenotype but suggesting fundamental differences in how neocortical and spinal cord axons respond to netrin-1. In addition, differences in GAP-43 dependency also distinguished how ventrolateral cortical efferents respond to netrin-1: in contrast to callosal neurites, in which netrin-1 required GAP-43 in order to stimulate both outgrowth and guidance, in ventrolateral efferents, netrin-1 required GAP-43 only to stimulate outgrowth, but not guidance. Moreover, netrin-1 increased the numbers of both types of cortical, but not spinal neurites. The results demonstrate previously unappreciated diversity in how different classes of neurons respond to the same guidance cue.