The long-term goal of this project is to learn how to manipulate axon growth in vivo. There are many instances when this would be an essential part of a therapeutic treatment after injury to the nervous system. In addition, the possible use of transplanted neurons to treat various CNS diseases may depend on these neurons sending axons to appropriate targets. The path growing axons follow during development is regulated by guidance mechanisms consisting of molecular cues that are non- uniformly distributed along the path. A particular role for gradients of attracting and repelling guidance cues has been proposed. A detailed understanding of how gradients of guidance cues direct the growth of axons is necessary to achieve this long-term goal. Recent progress has resulted in the identification and demonstration of in vivo function of 3 non-diffusible gradient molecules involved in the guidance of growth of the Til pioneer axons in the legs of the cockroach embryo. These molecules, localized to the substrate over which the axons grow, include 2 in the basement membrane and l on the surface of the epidermal epithelial cells. The complexity of the interactions between these guidance cues and the growth cone is greater than had been expected. This complexity is expressed in the fact that multiple and functionally redundant cues exist and in that these cues may interact with one another. The cockroach embryo is an ideal system in which to study the complexity of this developmental event. Its anatomical simplicity enables the proximal growth of identified pioneer axons in the leg to be observed in living preparations. In this proposal each of these cues will be molecularly characterized, the details of their ability to guide axon growth both in vivo and in vitro will be examined, as will be the in vivo interactions among them. A fourth molecule distributed in a gradient along the path of axon growth has been identified and experiments are proposed to examine its role in axon guidance. Like many developmental events, axon guidance is also likely to be phylogenetically conserved. The information obtained from studies of this insect are likely to be very relevant to understanding these processes in mammals.