Project Summary/Abstract The function of the nervous system is dependent on the correct formation of neural circuits during development. Circuits are generated when growth cones at the tips of axons use molecular cues in the environment to guide axon extension. Netrin1 is an axon guidance cue first thought to acts as a long-range, diffusible, chemoattractant that emanates from the floor plate (FP). However, recent work by the Butler lab and other groups, has suggested this model is incorrect. In the mouse spinal cord, netrin1 is expressed by both FP cells and neural progenitor cells (NPCs) in the ventricular zone (VZ). In the absence of either netrin1 or its receptor Dcc, axons innervate the VZ and commissural axons either stall or defasciculate. These phenotypes are only observed when netrin1 is removed from NPCs and not the FP cells, suggesting NPC-derived netrin1 is responsible for guiding axon extension. Our studies suggest that NPC-derived netrin1 is deposited on the pial surface (margin) of the spinal cord, where it acts as an adhesive substrate that promotes commissural axon outgrowth. My objective is to define the mechanisms that allow netrin1 to be transported to the pial surface of the spinal cord. In the visual system, netrin1 can be cleaved into fragments, isoforms, with unique spatial and biological properties. This led me to believe that cleavage of netrin1 facilitates its transport to the pial surface. My preliminary data suggests that netrin1 isoforms exist in the spinal cord, and that netrin1 is differentially cleaved to permit its localization to different regions of the spinal cord. Furthermore, my data suggests that netrin1 transport is mediated by the motor protein Kif1a. In Aim 1, I will track the path of netrin1 from the VZ onto the pial surface using high resolution microscopy and characterize the sequence of netrin1 isoforms, using mass spectrometry. In Aim 2, I will use SiRNA knockdown and gain-of-function studies to investigate if KiF1a is necessary for the transport of netrin1. Investigating these mechanisms is critical to 1) gaining a better understanding of the basis of neurodevelopmental disorders and 2) the repair and regeneration of damaged circuits.