Specialized cells at the midline, which separates the left and right halves of the CNS, have a number of roles in directing growth cone behavior (1). In the vertebrate spinal cord, the insect ventral nerve cord and in C. elegans, midline cells produce guidance cues such as nectins and slit, which act as attractants and repellents, respectively (1). These cells may act as gatekeepers to prevent axons from crossing the midline and to induce a switch in growth cone responsiveness to guidance cues beyond the gateway (1). One such gatekeeper, Robo, is an axon guidance receptor that defines a novel subfamily of Ig superfamily proteins that are conserved from fruit flies to mammals (2,3). Robo acts as a receptor for the repellent Slit and functions in a cell-autonomous fashion (1-3). Non-crossing axons express high levels of Robo, whereas crossing axons express low levels of Robo before reaching the midline and high levels after they cross (1). Robo1 and Robo2 are two human homologs of the Drosophila protein Roundabout (2-4). Robo1 is also homologous to the C. elegans gene sax3, whereas Robo2 is homologous to the zebrafish gene astray (5,6).