Microtubules (MTs) are one of the major cytoskeletal components of the cell, essential for many fundamental cellular and developmental processes, such as cell division, motility and polarity. In large and highly polarized cells like neurons, MTs have been regarded as essential structures for stable neuronal morphology and serve as tracks for long-distance transport; however, fundamental new insights into the role of neural MTs have emerged. New findings demonstrate that the MT cytoskeleton plays an active role during different phases of neuronal development: MTs determine axon formation, control polarized cargo trafficking, and regulate the dynamics of dendritic spines, the major sites of excitatory synaptic input. Failures in MT function have been linked to various neurological and neurodegenerative diseases and recent studies highlight MTs as a potential target for therapeutic intervention.How neuronal MTs are formed and stabilized during neuronal polarity and differentiation is largely unknown, and whether this requires the centrosome is under debate. The overall aim of this proposal is to investigate basic mechanisms responsible for organizing the microtubule cytoskeleton during neuronal development. Here, we will take a multidisciplinary approach and combine biochemistry, neurobiology, molecular engineering, and advanced microscopy to study the role of MTs at three stages of neuronal differentiation. We propose to determine: i) the role of (non-)centrosomal MT nucleation during early development, ii) the mechanism by which dendrites organize MTs into anti-parallel arrays, iii) the relation between MTs spine entry and cargo transport in mature neurons. We anticipate that these studies will uncover how the MT cytoskeleton is built and organized at different phases of neuronal development, which will be relevant for understanding polarized transport, synaptic processes and associated neurodegenerative disorders.