The long-range goal of this research is to understand how axons choose the correct pathways to grow along during development. Appropriate pathfinding is crucial for the developing individual because if axons do not find their correct targets, they will not establish the appropriate synaptic connections and the nervous system will not function properly. This research focuses on sensory neurons innervating the chick hindlimb because using such an easily accessible system greatly facilitates experimental manipulations at virtually any point during embryonic development. Previous experiments have shown that, as sensory axons grow into the limb, they respond differentially to cues in the surrounding environment and to other axons, associating with some and separating from others. Preferential adhesivity, mediated by cell adhesion molecules (CAMs), contributes to the ability of sensory axons to fasciculate along appropriate axons and form nerve-specific bundles as they grow distally. The proposed studies will extend previous work examining the roles of fasciculation, CAMs, and environmental cues in sensory axon pathfinding. Retrograde labeling, antibody staining to distinguish between sensory and motoneuron (MN) axons, and confocal microscopy will be combined to examine the trajectories of specific subtypes of sensory axons and their spatial relationships with other axons in a variety of experimental situations. Specifically, some of the proposed studies will use injections of function-blocking antibodies to determine how several CAMs (G4/L1, N-cadherin, DM-GRASP/SC1, axonin-1, NrCAM, neurofascin, and F11) affect sensory/MN axon intermixing, fasciculation, the formation of nerve-specific bundles, and pathfinding by both cutaneous and muscle sensory axons. EM will be used to determine whether sensory axons fasciculate at early stages and after CAM perturbations. Other studies determine whether certain specific limb-associated cues are required for the correct sorting of sensory axons normally destined to project along different peripheral nerves. An important implication of previous findings was that sensory neurons projecting along different peripheral nerves must be different from one another at early stages, as their axons first extend toward the limb. Accordingly, some of the proposed studies will examine differences in gene expression among the different types of sensory neurons.
