In vitro studies of growth cone behavior support a role for fasciculation mediated by cell adhesion molecules in sensory axon guidance during development

Abstract

Axonal interactions, which are mediated by cell adhesion molecules (CAMs) as well as other types of membrane proteins, are important for sensory axon pathfinding in the developing chick hindlimb. We have previously shown that injection of antibodies that block the function of either G4/L1 or N- cadherin into the limb, starting when the first sensory axons reach the plexus, alters the segmental pattern of projections along cutaneous nerves. Specific removal of polysialic acid from NCAM using the enzyme endoneuraminidase N (Endo N) also resulted in significant changes in cutaneous projection patterns, while injection of antibodies against NCAM itself had no obvious effect (M. G. Honig and U.S. Rutishauser, 1996, Der. Biol. 175, 325-337). To help understand the cellular basis for these findings, we developed a tissue culture system in which the axons from dorsal root ganglion explants grow within defined laminin lanes and examined whether the same treatments increased or decreased a growth cone's tendency to be closely associated with neighboring axons. After 2 days in culture, images of the cultures were recorded, antibodies or Endo N was added, and images of the same fields were recaptured an hour later. To quantify the results, growth cones located in defined regions of the laminin lanes were classified, before and after the perturbation, as 'free' (i.e., growing primarily on the laminin substratum), 'fasciculated' (i.e., growing tightly along other neurites), or 'intermediate' (i.e., growing both on the laminin substratum and in contact with other neurites). We found that anti-G4/L1 and anti-N-cadherin, but not anti-NCAM, caused an increase in defasciculated growth cones, whereas Endo N resulted in an increase in fasciculated growth cones. These changes in fasciculation are consistent with the changes in cutaneous projections seen in our previous in ovo perturbations. The results from these tissue culture experiments thus provide strong support for the idea that one mechanism by which CAMs affect sensory axon pathfinding in vivo is by regulating the affinity of sensory growth cones for neighboring axons, which in turn can modulate the growth cone's ability to navigate through the surrounding environment.