Project Summary/Abstract During development of the cerebral cortex, cortical projection neuron subtypes extend axons to innervate distinct targets located at great distances (103-105 cell body diameters) from their nucleus-containing somata. This precise navigation and circuit development is regulated by growth cones (GCs): subcellular compartments at tips of growing axons that rapidly integrate extracellular signals to control development of neural circuits, then mature into synapses. Since GCs respond rapidly to chemical cues, while hours/days are required to transport molecules between GCs and their parent somata, GCs highly likely function semi-autonomously. Prior work has also revealed that local protein synthesis and degradation pathways in GCs are required for responses to some directional cues. However, function of most GC proteins in axon guidance and circuit development have not been identified, and even less is known about subtype-specific roles of GC proteins. This proposal combines recently developed subtype-specific GC purification with ultra-low-input proteomics to 1) investigate subtype-specific GC proteomes at distinct developmental stages (pre- and post-midline crossing) to identify and functionally investigate proteins with stage-specific roles, and 2) investigate subtype-specific proteomes of dysfunctional GCs to identify and functionally investigate dysregulated proteins with critical roles in precise circuit wiring. These rigorous in vivo investigations will deepen understanding of subtype-, and stage-specific mechanisms regulating subcellular proteostasis in GCs, and how these processes control axon pathfinding and formation of synaptic circuitry in cortical projection neuron subtypes. The intersection of circuit formation and subcellular proteostasis has substantial