A micropatterned coculture system for axon guidance reveals that Slit promotes axon fasciculation and regulates the expression of L1CAM.

Axon fasciculation is essential for wiring the nervous system during development, but its regulation by guidance cues remains unknown. By using a micropatterned coculture system, we developed a diffused long-term gradient of the Slit protein secreted by cells transfected with the Slit gene. Using hippocampal neurons, we show here that the Slit gradient induces axon fasciculation and that the extent of fasciculation depends on the Slit concentration. This Slit-induced axon fasciculation was abolished by inhibitors of the ROCK-myosin II signaling pathway. Interestingly, the activity of myosin II regulated the level of axon fasciculation. In addition, we showed that neurons with a high level of axon fasciculation express fewer L1 cell adhesion molecules (L1CAMs), and that those with low level of axon fasciculation have more L1CAMs. We suggest that Slit induces axon fasciculation and regulates the ROCK-myosin II signaling pathway and the expression level of L1CAMs. This approach establishes a simple and stable axon guidance model in vitro, and may be broadly applicable for investigating long-term events such as axon fasciculation, neuron migration, and axon regeneration.