The behavioral sophistication and plasticity of animals has been facilitated by evolutionary trends favoring increased central nervous system nervous system complexity and requires precise developmental control of axon and dendrite position within the CMS. The Roundabout (Robo) family of axon guidance receptors performs an ancient role in regulating midline crossing in response to the secreted repellant Slit. In Drosophila, Robo2 and RoboS specify mediolateral position of axons in the CNS, a novel function not shared by Robo. This proposal seeks to understand the molecular and functional bases and evolutionary origins of Robo receptor control of lateral position in the Drosophila CNS. The specific aims are to: 1. Identify the molecular basis for differential activity of Robo and Robo2 in lateral positioning. The Robo2 axon guidance receptor is able to dictate mediolateral position of axons within the Drosophila CNS, while the closely related Robo receptor cannot. To define the minimal sequence elements distinguishing Robo and Robo2 activity in lateral positioning, I will assay a comprehensive series of chimeric Robo receptors for their ability to dictate lateral position in vivo. 2. Determine the biological basis for Robo receptor control of lateral position. To test the hypothesis that differential Slit affinity is responsible for the distinct activities of Robo and Robo2 in lateral positioning, I will (1) test the assumption that a graded distribution of Slit is required for Robo receptor control of lateral position by examining the effects of a membrane-tethered form of Slit on lateral position;(2) use sitedirected mutations to test the effects of disrupted Slit binding on lateral shifting activity of Robo2 and Robo3;and (3) assess whether Robo receptor lateral positioning activity correlates with Slit affinity by comparing the Slit affinities of wild type and chimeric Robo receptors using surface plasmon resonance. 3. Examine the evolutionary origins of Robo receptor control of lateral position. The third aim will test the hypothesis that control of lateral position is a novel function acquired by Robo receptors during the evolution of insects. Robo2 and Robo3 regulate lateral position of CNS axons in Drosophila. Notably, Robo2 and Robo3 orthologs appear restricted to insects, raising the possibility that control of lateral position is a novel activity that first appeared during insect evolution. To gain insight into the evolutionary origins of Robo receptor control of lateral position, I will: (1) assay arthropod and nematode Robo receptors for conserved lateral positioning activity in Drosophila, and (2) investigate the function of Robo orthologs in the flour beetle Tribolium, an insect that retains a primitive set of Robo receptors.