The development of a functional nervous system requires the precise assembly of numerous neural circuits characterized by some critical steps, including (1) the guided migration of axonal growth cones and (2) finely controlled neuronal population size. A high degree of axon guidance specificity through cell surface effectors, followed by programmed cell death (PCD) of inappropriately and/or supernumerary connected neurons are proposed to govern the intricate precision of neural circuit formation. However, our understanding of how complex neuronal circuits are assembled remains rudimentary, thus prompting us to investigate the molecular logic behind these important processes. Members of diffusible Wnt ligands and their receptors have been implicated in several key axon guidance events. The mechanisms of how Wnt signals mediate axonal growth cones guidance has been hindered by the complexity of most in vivo models. A relatively simple in model of axon guidance is the binary decision to innervate dorsal or ventral limb muscles executed by the axons of spinal motor neurons of the lateral motor column (LMC). On the other hand, many neurotrophic factors (NTFs) capable of recusing motor neurons from apoptosis are detected in different limb muscles at the stages when LMC neurons extend axons to their muscle targets, implying that a combination of NTFs is essential to prevent motor neuron PCD. We and other have previously demonstrated that LMC axon guidance involves a molecular symmetry of repulsive ephrin:Eph signaling in two LMC subgroups. In addition, our preliminary results also demonstrate the expression of Wnts and their receptors in the limb and LMC neurons, respectively, which could also play a role in LMC axon guidance. These observation thus suggest the following HYPOTHESIS: In LMC neurons, the proper organization of the neuronal circuit innervating the limb is (A) mediated by bifunctional signals from Wnt receptors on LMC axons, and (B) reinforced by PCD depending on a combinatorial code of NTF/NTF receptor matching motor neurons with their target muscles. We propose to test this hypothesis through the following specific aims and experimental approaches: SA1: Determine whether Wnt and Wnt receptors are required for normal LMC neuron development and axon trajectory choice. 1.1: Examine the role of Wnts and Wnt receptors in LMC neurons using (1) mouse mutants and (2) in ovo electroporation. 1.2: Examine LMC axon projection in chick embryos expressing full-length or mutant Wnt receptor constructs to determine how repulsive and attractive Wnt receptor function. 1.3: Determine whether LMC axonal misrouting persists in adult Wnt and Wnt receptor mutants using (1) retrograde tracing and (2) several locomotor behavioral tasks. SA2: Determine how Wnt and ephrin signals are integrated by LMC growth cones. 2.1: Use the stripe assay to test which Wnt signaling intermediates are required for LMC axon guidance. 2.2: Use the stripe assay to determine if LMC growth cones can respond to Wnt and ephrin simultaneously. 2.3: Use biochemical analysis and the stripe assay to determine how Wnt and ephrin signals intersect. SA3: Test the NTF combinatorial model of motor neurons apoptosis 3.1: Determine the expression profiling of motor neuron NTF receptors and muscle NTFs using the in situ hybridization technique. 3.2: Test the combinatorial model of motor neuron PCD using: (1) retrograde labeling and (2) in ovo electroporation.
|Effective start/end date||8/1/16 → 7/31/17|