During development, precerebellar neurons migrate dorsoventrally from the rhombic lip to the floor plate. Some of these neurons cross the midline while others stop. We have identified a role for the slit receptor Rig-1/Robo3 in directing this process. During their tangential migration, neurons of all major hindbrain precerebellar nuclei express high levels of Rig-1 mRNA. Rig-1 expression is rapidly downregulated as their leading process crosses the floor plate. Interestingly, most precerebellar nuclei do not develop normally in Rig-1-deficient mice, as they fail to cross the midline. In addition, inferior olivary neurons, which normally send axons into the contralateral cerebellum, project ipsilaterally in Rig-1 mutant mice. Similarly, neurons of the lateral reticular nucleus and basilar pons are unable to migrate across the floor plate and instead remain ipsilateral. These results demonstrate that Rig-1 controls the ability of both precerebellar neuron cell bodies and their axons to cross the midline.

Repulsive guidance molecule (RGM) is an axon guidance protein that repels retinal axons upon activation of the neogenin receptor. To understand the functions of RGM-neogenin complexes in vivo, we used gene transfer technology to perturb their expression in the developing neural tube of chick embryos. Surprisingly, neogenin over-expression or RGM down-expression in the neural tube induces apoptosis. Neogenin pro-apoptotic activity in immortalized neuronal cells and in the neural tube is associated with the cleavage of its cytoplasmic domain by caspases. Thus neogenin is a dependence receptor inducing cell death in the absence of RGM, whereas the presence of RGM inhibits this effect.

Repulsive guidance molecule (RGM) is a membrane-bound protein originally isolated as a guidance molecule for retinal axons. Three RGM isoforms (RGMa-RGMc) exist in vertebrates. We showed previously that RGMa is a cell-survival factor in the neuroepithelium of chick embryos that suppresses the proapoptotic activity of its receptor neogenin. In the present study, we performed gain-and loss-offunction analysis of RGMa in chick embryos to further investigate RGMa function. We found that RGMa overexpression promotes neuronal differentiation, whereas RGMa small interference RNA represses it. Similar experiments conducted at later developmental stages using retroviral vectors reveal that perturbation of RGMa expression disturbs the retinotectal projection. Our work provides the first evidence for a role for RGMs in axon guidance in vivo. In addition, these results suggest that RGMa exerts multiple functions during neural development.