Cloning, expression, and function of laminin in neuronal guidance

The proper guidance of migrating growth cones during development relies on the balance of multiple guidance cues in the embryonic environment. In addition to guidance cues, growth cones are in contact with other substrates that may contribute to the pathfinding of neurons. For example, in the developing insect peripheral nervous system, pioneer neurons migrate on and between layers of the basal lamina, which consists of a network of many different proteins. Previous studies have demonstrated that one basal lamina molecule, laminin, promotes outgrowth of many classes of neurons in vitro. Laminin is a major component of the basal lamina and is a potent promoter of neurite outgrowth in vitro. In order to determine the role of laminin in neuronal development, grasshopper laminin subunit genes were cloned. Laminin was found to be expressed by hemocytes and laminin protein was abundant in the basal lamina throughout the embryo. Laminin expression was coincident with the outgrowth and guidance of the Tibial (Ti1) pioneer neurons in the developing limb bud. Laminin deposition in the basal lamina was conferred by hemocytes that migrate within the lumen of the limb with no apparent trajectory. In spite of this, laminin immunoreactivity in the basal lamina was uniform andis available as a substrate for axonal outgrowth. In this study, the simple grasshopper nervous system was used to investigate the role of laminin in neuronal pathfinding. The role of two sites on laminin was investigated using subunit specific antibodies and peptides as blocking reagents in vivo. While reagents aimed at a cell adhesion motif on the β chain did not affect Ti1 pathfinding, antibodies and peptides that block the nidogen binding site on laminin resulted in stalled Ti1 axon migration, predominantly at the precise location in the trochanter, where they make a stereotyped ventral turn. After prolonged culturing, Ti1 axons remained stalled at the same location. The basal lamina in blocked embryos appeared to suffer no obviousstructural defects, as assessed with immunofluorescence. Therefore, while Ti1 axons were capable of some outgrowth in the presence of blocking reagents, they were not able to navigate an essential turn and complete the trajectory to the CNS. This data suggests that the Ti growth cone's interaction with the nidogen binding site on laminin is vital for neuronal pathfinding in vivo.Medicine, Faculty ofGraduat