The goal of this project is to understand how growing neurites turn and branch. During embryogenesis, nerve fibers express highly regulated programs of extension, turning and branching of nerve tips, called growth cones. Our model proposes that growth cone motility expands the neurite tip as filopodia that contact other surfaces and exert tensions to orient the transport and organization of the growing neurite. Extrinsic cues produce local changes in motility to induce turning and branching of nerve tips. This model is tested by in vitro experiments that clarify organization of microtubules, neurofilaments and actin filaments in neurites. Cytoskeletal relationships in neurites are described and contrasted with changes that occur in cytoskeletal associations in growth cones by thin sectioning and whole mount transmission electron microscopy, as well as immunocytochemistry with light and electron microscopy. Cytoskeletal organization in living neurites is analyzed from spatial and temporal distribution of fluorescent tubulin and actin injected into neurons. Cultured neurons are subjected to extrinsic cues that produce neurite turning and branching, and cytoskeletal organization is related to these directive effects. In addition, two drugs are used that profoundly affect turning and branching, taxol and cytochalasin. By examining growth cone turning and branching, this work clarified how complex anatomy of the nervous system is generated and may indicate ways to stimulate and direct regeneration and repair of diseased and injured nervous tissue.
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