We are interested in how circuitry that drives locomotory behavior is constructed during development. During embryogenesis, many cells navigate extensively to their final destinations, where they form precise connections with their neighbors. We intend to define the molecules and mechanisms that guide three cell types (mouse ES cells, motor neurons, and neural crest) to their targets. Mouse ES cells have been stably transfected to express EphA4 and we are examining their fate prior to and after stimulations with ephrins, in vitro and in vivo. Motor neurons originate in the ventral neural tube and extend their axons to innervate particular muscles in the limb. Our previous studies showed that these cells and their migratory pathways expressed unique combinations of Eph receptor tyrosine kinases (RTKs) and their ligands, the ephrins. We also showed that distinct subsets of motor neurons that express EphA4 RTK respond differently to ephrin-A5. Previously, we also demonstrated that chick ephexin, a downstream intracellular signaling molecule that is phosphorylated by EphA4, is required for axon stalling at the base of the limb and that EphA4 works with Ret, a signaling receptor that interacts with Glial Derived Neurotrophic Factor (GDNF). Our current research focuses on unraveling the mechanisms that account for precise axon pathfinding to the limb and defining the exact role of Ret/GDNF in this process. Neural crest cells, a stem cell-like population, emanate from the dorsal neural tube and migrate along stereotypical pathways to their target regions to form various derivatives including sensory/sympathetic ganglia, components of the heart and craniofacial skeleton, and pigment cells. We are interested in how this stem cell-like population generates such a diverse array of derivatives and are examining the function of Hox genes in this process. Furthermore, we are analyzing how various molecules contribute to neural crest motility, directed movement, and settling patterns in cardiac, trunk, and cranialfacial regions. Together, these studies will yield important insights about the positive and negative cues that sculpt precise patterns of cellular architecture during development.