Axons grow over exceedingly long distances to reach their target tissues. The development of the axon involves the formation of interstitial branches along its length as well as elongation at the terminal growth cone. New regions of the axon are highly dynamic, and can rapidly elongate, retract, and change their direction of growth in response to environmental cues. Microtubules are essential architectural elements within the axon that also act as railways for organelle transport. Individual microtubules are coalesced into a dense bundle that traverses the length of the axon. This dense bundle is important for the stability of the axon shaft, but is not conducive to dynamic changes in the morphology of the axon that accompany new growth. In regions of the axon relevant to new growth, the microtubule array must undergo dramatic reorganization into a more plastic form that permits individual microtubules to be rapidly reconfigured. In preparation for a bout of growth, the microtubule bundle locally splays apart and undergoes fragmentation within the growth cone or at a site of interstitial branch formation. The short microtubules are then able to move into lamelliopodia and filopodia that give rise to new regions of axon growth. In some cases, the microtubules continue to move forward, while in other cases, the microtubules change direction and are transported retrogradely. The goals of the present grant application are to better characterize how each of these microtubule behaviors contributes to axon growth, retraction, and branch formation, and to elucidate the molecular cues and mechanisms by which each microtubule behavior is orchestrated. All of the experiments will utilize high-resolution imaging techniques to directly visualize microtubule behaviors in living cortical neurons. One of the specific aims seeks to elucidate the cytoskeletal changes that are induced by various extrinsic factors and by changes in intracellular calcium. The objective is to determine how these factors, known to be relevant to axonal development, induce local changes in actin filaments, which then permit local changes in the microtubule array. Another aim seeks to identify the specific proteins responsible for splaying and fragmenting the microtubules in regions of new growth. The final aim seeks to elucidate the motor-based machinery that transports microtubules anterogradely and retrogradely during bouts of growth and retraction. Together, the proposed experiments will provide new information on the molecules and mechanisms that regulate the development of the axon. This information will be important for understanding axon growth during both normal development and regeneration after injury.