Axon pathfinding and targeting: (R)evolution of insights from in vitro assays

Investigating axonal behaviors while neurons are connecting with each other has been a challenge since the early studies on nervous system development. While molecule-driven axon pathfinding has been theorized by observing neurons at different developmental stages in vivo, direct observation and measurements of axon guidance behaviors required the invention of in vitro systems enabling to test the impact of molecules or cellular extracts on axons growing in vitro. With time, the development of novel in vivo approaches has confirmed the mechanisms highlighted in culture and has led in vitro systems to be adapted for cellular processes that are still inaccessible in intact organisms. We here review the evolution of these in vitro assays, which started with crucial contributions from the Bonhoeffer lab.