The long-range goal of this research is to understand how growing sensory axons choose the correct pathways to follow in the developing chick hindlimb. Previous work has shown that, as they enter the limb, axons that will project along one peripheral nerve become segregated from axons that will project along other peripheral nerves. The proposed studies will examine how various cell adhesion molecules (CAMs), expressed on the surfaces of sensory and motoneuron axons, influence the interactions that occur between growing axons and thereby contribute to the ability of sensory axons to sort out correctly into nerve-specific bundles. To do this, antibodies that block the function of various CAMs (e.g. G4/L1, N- cadmerin, NCAM, SCI/DM-GRASP) or an enzyme, endoneuraminidase N, that removes the polysialic acid from NCAM and consequently enhances CAM- mediated adhesivity, will be injected directly into the hindlimb in ovo.
The aims are: l) to determine the consequences for sensory axons of perturbing CAM function, starting at a time when motoneuron axons have already sorted out into nerve-specific bundles at the base of the limb but most sensory axons are still entering the plexus (i.e. at St.25). Retrograde labeling will be used to assess the effects of such perturbations on the segmental pattern of projections. Confocal laser scanning microscopy and various combinations of axonal tracing (retrograde and anterograde) together with immunofluorescence procedures to distinguish between sensory and motoneuron axons will be used to ascertain how axonal trajectories, the rate of axonal outgrowth, the extent of fasciculation between axons, and the spatial relationships among different types of axons are altered in the experimental embryos in comparison to normal and control embryos. 2) to determine the consequences of perturbing CAM function, starting at a time when motoneuron axons and the first sensory axons are entering the spinal nerves (i.e. at St.20-21). The intent of these studies is to alter the initial associations between motoneuron axons and some sensory axons and to thereby test whether the initial position a sensory axon occupies in the spinal nerves determines which peripheral nerve it later grows along. 3) to assess the roles of CAMs in mediating interactions between sensory and motoneuron axons, by using similar types of perturbations, in a simplified tissue culture system in which the two types of axons are forced to-row together. Together, these studies will increase our understanding of the mechanisms underlying sensory axon guidance in the chick hindlimb, of the ways in which interactions among axons can affect pathfinding decisions, and of the roles of CAMs in mediating interactions among axons. The latter is of particular importance since relatively little is known about the role of CAMs in the development of the central nervous system and yet it is clear, based on the consequences of exposure to certain toxic agents and the neurological manifestations of several genetic disorders, that proper CAM functioning is essential for the normal development of the nervous system.
