How a finite number of genes specify a seemingly infinite number of neuronal connections is a central question in neurobiology. Alternative splicing has been proposed to increase proteome diversity in the brain. Here we show that cell-specific alternative splicing of a cell-surface protein is crucial for neuronal wiring. Down syndrome cell adhesion molecule 2 (Dscam2) is a conserved homophilic binding protein that can induce repulsion between opposing neurons. In the fly visual system, L1 and L2 neurons both require Dscam2 repulsion, but paradoxically, they also physically contact each other. We found that the cell-specific expression of two biochemically distinct alternative isoforms of Dscam2 prevents these cells from repelling each other. Phenotypes were observed in the axon terminals of L1 and L2 when they expressed the incorrect isoform, demonstrating a requirement for distinct isoforms. We conclude that cell-specific alternative splicing is a mechanism for achieving proper connectivity between neurons.

The repulsive guidance molecule A (RGMa) is a contact-mediated axon guidance molecule that has significant roles in central nervous system (CNS) development. Here we have examined whether RGMa has novel roles in cell migration and cell adhesion outside the nervous system. RGMa was found to stimulate cell migration from Xenopus animal cap explants in a neogenindependent and BMP-independent manner. RGMa also stimulated the adhesion of Xenopus animal cap cells, and this adhesion was dependent on neogenin and independent of calcium. To begin to functionally characterize the role of specific domains in RGMa, we assessed the migratory and adhesive activities of deletion mutants. RGMa lacking the partial von Willebrand factor type D (vWF) domain preferentially perturbed cell adhesion, while mutants lacking the RGD motif affected cell migration. We also revealed that manipulating the levels of RGMa in vivo caused major migration defects during Xenopus gastrulation. We have revealed here novel roles of RGMa in cell migration and adhesion and demonstrated that perturbations to the homeostasis of RGMa expression can severely disrupt major morphogenetic events. These results have implications for understanding the role of RGMa in both health and disease. Repulsive guidance molecule (RGM) was first identified in the embryonic chick retinotectal system as a chemorepulsive molecule for retinal axons (35). Subsequently, three different RGMs (RGMa, RGMb, and RGMc) were identified in mouse, and each was found to have a unique spatiotemporal expression pattern (41,48). RGMa is a membrane-bound protein with ϳ430 amino acid (aa) residues that has a glycosylphosphatidylinositol (GPI)-anchored C-terminal domain and a conventional N-terminal signal peptide (35,48). It is cysteine rich and contains a putative autoproteolytic cleavage site, a single tri-amino acid motif, ArgGly-Asp (RGD), a partial von Willebrand factor type D (vWF) domain, and two hydrophobic domains at the N and C termini. These key molecular structures, except for the RGD domain (which is absent in RGMb in most animal models), are shared by all members of the RGM family (5, 35). A conserved RSDSPEI sequence, 5= to the partial von Willebrand type D domain, is present within RGMa from mammals, frogs, and birds. To date, the roles of these different RGM domains remain unknown.The first RGM receptor was identified by cell surface binding of chick RGM to cells expressing neogenin, a member of the immunoglobulin superfamily of transmembrane receptors (31,35,43,(54)(55)(56)(57). RGMa-neogenin interactions are involved in axon guidance in the developing visual system and in axon tract formation in the embryonic brain in vivo (31,35,54,55,57,58). RGMa also has chemorepulsive activity during laminar patterning of the developing mouse dentate gyrus in vitro (4). Interestingly, RGMa knockout mice did not show an abnormality in retinal topography but displayed an exencephalic phenotype in ϳ50% of embryos, suggesting that RGMa plays a role in neural tube closure (37). We and others...

RGMa (repulsive guidance molecule a) was the first identified molecule that possessed the necessary functional activity to repulse and steer growth cones to their target in the brain. By binding to its neogenin receptor, RGMa caused the collapse of growth cones and encouraged axons to grow along specific trajectories in vitro. Although originally characterized in 1990, RGMa was not conclusively shown to mediate axon guidance in vivo for another 12 years. Loss-of-function analysis in mice revealed that RGMa may play a more important role in neural tube morphogenesis. RGMa has now emerged as a molecule with pleiotropic roles involving cell adhesion, cell migration, cell polarity and cell differentiation which together strongly influence early morphogenetic events as well as immune responses. RGMa can be regarded as a molecule for all seasons.

Repulsive guidance molecule A (RGMa) is a glycosylphosphatidylinositol-anchored plasma membrane protein that was originally identified based on its chemorepulsive activity during axon navigation in the developing nervous system. Knock down of RGMa has previously shown to perturb axon navigation in the developing Xenopus forebrain (Wilson and Key, 2006). In order to further understand the in vivo role of RGMa in axon guidance, we have adopted an in vivo gain-of-function approach. RGMa was mosaically overexpressed in the developing Xenopus embryo by the injection of mRNA into single blastomeres. Ectopic expression of RGMa affected the morphology and the topography of developing axon tracts in vivo. Pioneer axons misrouted or aberrantly projected in response to ectopic RGMa in the developing Xenopus forebrain, confirming the in vivo chemorepulsive activity of this ligand. In addition, we show here for the first time that overexpression of RGMa acts cell-autonomously to generate ectopic neurons in the developing embryonic brain. Taken together, the current study reveals a pleiotropic role of RGMa in early vertebrate embryonic brain in the spatial organization of axon tracts, pioneer axon guidance, and neural cell differentiation.