The Synergistic Function of Netrin-1 and Ephrin Signals in Spinal Motor Axon Guidance

Our bodies interact with the external world through voluntary movement exemplified by the computational tasks coordinated by precisely formed neuronal connections. Neuronal connectivity defects caused by axon guidance errors during development can induce drastic consequences that are frequently incompatible with life, such as mental retardation. However, how complex neuronal circuits are assembled still remains poorly understood. To address this question, we study the molecular mechanism controlling the specification of connections between spinal motor neurons and their muscle targets. A relatively simple in vivo system suitable for our study is the axonal projections of spinal motor neurons that innervate limb muscles and resides in the lateral motor column (LMC) of the ventral spinal cord. LMC neurons are comprised of medial and lateral divisions and grow towards the base of the limb to diverge to form, respectively, ventral and dorsal limb nerves. Members of Eph tyrosine kinase receptors and their ephrin ligands have been suggested to modulate the dorso-ventral selection of LMC motor axons. In addition, our preliminary studies implicated the involvement of another guidance systems, Netrin-1 and its receptors, in LMC pathfindings. To better understand the functions of Netrin system in spinal motor neurons and the mechanisms of how multiple guidance systems cooperate, we investigate the following HYPOTHESIS: Netrin can (1) elicits growth cone attraction and repulsion by binding its receptors that are modulated by electrical activity, and (2) integrate with ephrin signals to regulate trajectories of spinal motor axons into the limb. We propose to test this hypothesis through the following specific aims and experimental approaches: SA1: Determine the role of Netrin-1 receptors in LMC neuron development. 1.1 Determine how attractive Netrin receptors function using (1) Netrin, Dee, and Neogenin mutant mice embryos and (2) missexpressed chick embryos by in ovo electroporation. 1.2 Determine how repulsive Netrin receptors function using (1) Une5e mutant mice embryos and (2) biochemical approaches to screen for proteins interacting with Unc5c upon exposure to Netrin-1 SA2: Investigate whether electrical activity is required for Netrin-1-mediated axon guidance. 2.1 Study the requirement of electrical activity for Netrin receptor expression and LMC axon guidance by (1) generating a loss of LMC neurons spontaneous activity using the GABA blocker in ovo and (2) a genetic mean through the knockout of Munc18. 2.2 Study the Netrin receptor localization dependence on electrical activity by measuring the presence of surface Netrin-1 receptors following (1) electroporating light-gated activity agents and (2) drug treatment to stimulate or block electrical activity. SA3: Determine how Netrin and ephrin signals are integrated by LMC growth cones. 3.1 Investigate the mode of integration between Netrin and ephrin signaling using (1) the in vitro stripe assay and (2) co-electroporation of receptors in ovo. 3.2 Investigate the underlying mechanisms of cooperative Netrin and ephrin signals by (1) observations of growth cone dynamics, (2) co-immunoprecipitation, and (3) in vitro stripe assays.