Dissection of Synergistic Involvement among Different Guidance Families in Spinal Motor Neuron Development

The assembly of a functional nervous system relies on precisely formed neuronal connections developing as a consequence of guided migration of axonal growth cones. The molecular identity of many cell surface effectors of axon guidance has been uncovered, raising the important question of the ability of relatively few effectors to direct the development of the extraordinarily diverse arrays of axonal pathways. Members of Eph tyrosine kinase receptors and their ephrin ligands have been implicated in many key axon guidance events. The precise understanding of how Ephs and ephrins mediate axonal growth cones guidance has been hindered by the complexity of most in v^-vo models used for the study of axon guidance. A relatively simple in vivo model of axon guidance is the decision to innervate dorsal or ventral limb mesenchyme executed by the axons of spinal motor neurons of the lateral motor column (LMC) arriving at the base of the embryonic limb. We and others have previously demonstrated that LMC axon guidance involves a molecular mirror symmetry of repulsive Eph signaling in two LMC subgroups. However, ours and others’ recent experiments also demonstrate the following: (1) several candidate Eph signaling intermediates are expressed in LMC neurons and (2) Netrin-1, a distinct axon guidance cue present in the limb, could also play a role in LMC axon guidance. These observation suggest the following HYPOTHESIS: In LMC neurons, repulsive signaling by Eph receptors is (A) mediated by specific intracellular molecules, and (B) reinforced by signals from Netrin receptors on LMC axons. We propose to test this hypothesis through the following specific aims and experimental approaches: SA 1: Identify the intracellular effectors relevant to Eph function in LMC axons. 1.1 Use the in vitro stripe assay to study the effects of loss of function of candidate Eph “in trans” signaling intermediates. 1.2 Examine LMC axon proj ection using ⑴ a 2-chimaerin mouse mutant and (2) in vitro stripe assay to determine the role of a 2-chimaerin in Eph signaling in LMC neurons. 1.3 Establish ES cell-derived LMC neuron cultures for biochemical analysis of Eph signaling. SA 2: Determine whether Netrin-1 and Netrin-1 receptors are required for normal LMC neuron development and axon trajectory choice. 2.1 Examine LMC axon proj ection in ⑴ Netrin-1 and Netrin-1 receptor mutant mice embryos and (2) misexpressed chick embryos by in ovo electroporation to determine the role of Netrin1 and Netrin1 receptors in LMC neurons in vivo. 2.2 Study the early development of LMC neurons in mouse and chicken embryos lacking Neogenin1 and Netrin-1 function to determine the role of Neogenin1 in vivo. 2.3 Examine LMC axon proj ection in chick embryos expressing full-length or mutant Netrin repulsive receptors Uc5c constructs to determine how Unc5c function in vivo. SA 3: Determine how Eph and Netrin signals are integrated by LMC growth cones. 3.1 Use the in vitro stripe assay to determine if LMC growth cones can respond to Netrin-1 and ephrin simultaneously. 3.2 Use the in vitro stripe assay and biochemical analysis to determine how Eph and Netrin signaling pathways intersect.