Alain Chédotal

Plexins Are a Large Family of Receptors for Transmembrane, Secreted, and GPI-Anchored Semaphorins in Vertebrates

In Drosophila, plexin A is a functional receptor for semaphorin-1a. Here we show that the human plexin gene family comprises at least nine members in four subfamilies. Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D (CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A (Sema-K1). Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed: in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A. The cytoplasmic domain of plexins associates with a tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. We conclude that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and trigger a novel signal transduction pathway controlling cell repulsion.

Analysis of the L1-Deficient Mouse Phenotype Reveals Cross-Talk between Sema3A and L1 Signaling Pathways in Axonal Guidance

In humans, defects of the corticospinal tract have been attributed to mutations in the gene encoding L1 CAM, a phenotype that is reproduced in L1-deficient mice. Using coculture assays, we report that Sema3A secreted from the ventral spinal cord repels cortical axons from wild-type but not from L1-deficient mice. L1 and neuropilin-1 (NP-1) form a stable complex, and their extracellular domains can directly associate. Thus, L1 is a component of the Sema3A receptor complex, and L1 mutations may disrupt Sema3A signaling in the growth cone, leading to guidance errors. Addition of soluble L1Fc chimeric molecules does not restore Sema3A responsiveness of L1-deficient axons; instead, it converts the repulsion of wild-type axons into an attraction, further supporting a function for L1 in the Sema3A transducing pathways within the growth cone.

Neuropilin-2 Regulates the Development of Select Cranial and Sensory Nerves and Hippocampal Mossy Fiber Projections

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

SLIT2-MEDIATED CHEMOREPULSION AND COLLAPSE OF DEVELOPING FOREBRAIN AXONS

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.

The transmembrane semaphorin Sema4D/CD100, an inhibitor of axonal growth, is expressed on oligodendrocytes and upregulated after CNS lesion

Semaphorins are a family of secreted and membrane-bound proteins, known to regulate axonal pathfinding. Sema4D, also called CD100, was first isolated in the immune system where it is involved in B and T cell activation. We found that in the mouse, Sema4D is expressed in cells throughout the CNS white matter, with a peak during the myelination period. Double-labeling experiments with different markers of oligodendrocyte lineage such as olig1, olig2, platelet-derived growth factor receptor α, and proteolipid protein showed that Sema4D was expressed selectively by oligodendrocytes and myelin. The presence of Sema4D in myelin was confirmed using Western blot. Sema4D expression in myelinating oligodendrocytes was further observed using neuron-oligodendrocyte cocultures. Moreover, using stripe assay, we found that Sema4D is strongly inhibitory for postnatal sensory and cerebellar granule cell axons. This prompted us to examine whether Sema4D expression is modified after CNS injury. At 8 d after spinal cord lesions, Sema4D expression was strongly upregulated in oligodendrocytes at the periphery of the lesion. Sema4D-positive cells were not colabeled with the astrocyte marker GFAP, with the microglial and macrophagic marker isolectin B4, or with NG2, a marker of oligodendrocyte precursors. This upregulation was transient because from 1 month after the lesion, Sema4D expression was back to its normal level. These results indicate that Sema4D is a novel inhibitory factor for axonal regeneration expressed in myelin.

Regulation of Cortical Dendrite Development by Slit-Robo Interactions

Slit proteins have previously been shown to regulate axon guidance, branching, and neural migration. Here we report that, in addition to acting as a chemorepellant for cortical axons, Slit1 regulates dendritic development. Slit1 is expressed in the developing cortex, and exposure to Slit1 leads to increased dendritic growth and branching. Conversely, inhibition of Slit-Robo interactions by Robo-Fc fusion proteins or by a dominant-negative Robo attenuates dendritic branching. Stimulation of neurons transfected with a Met-Robo chimeric receptor with Hepatocyte growth factor leads to a robust induction of dendritic growth and branching, suggesting that Robo-mediated signaling is sufficient to induce dendritic remodeling. These experiments indicate that Slit-Robo interactions may exert a significant influence over the specification of cortical neuron morphology by regulating both axon guidance and dendritic patterning.

Spatiotemporal expression patterns of slit and robo genes in the rat brain.

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a repulsive guidance system that prevents inappropriate axons from crossing the central nervous system midline; this repulsive system is mediated by the secreted extracellular matrix protein Slit and its receptors Roundabout (Robo). Three distinct slit genes (slit1, slit2, and slit3) and three distinct robo genes (robo1, robo2, rig‐1) have been cloned in mammals. However, to date, only Robo1 and Robo2 have been shown to be receptors for Slits. In rodents, Slits have been shown to function as chemorepellents for several classes of axons and migrating neurons. In addition, Slit can also stimulate the formation of axonal branches by some sensory axons. To identify Slit‐responsive neurons and to help analyze Slit function, we have studied, by in situ hybridization, the expression pattern of slits and their receptors robo1 and robo2, in the rat central nervous system from embryonic stages to adult age. We found that their expression patterns are very dynamic: in most regions, slit and robo are expressed in a complementary pattern, and their expression is up‐regulated postnatally. Our study confirms the potential role of these molecules in axonal pathfinding and neuronal migration. However, the persistence of robo and slit expression suggests that the couple slit/robo may also have an important function in the adult brain. J. Comp. Neurol. 442:130–155, 2002.

RGM and its receptor neogenin regulate neuronal survival

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.

Neogenin mediates the action of repulsive guidance molecule

Repulsive guidance molecule (RGM) is a recently identified protein implicated in both axonal guidance and neural tube closure. The avoidance of chick RGM in the posterior optic tectum by growing temporal, but not nasal, retinal ganglion cell axons is thought to contribute to visual map formation. In contrast to ephrins, semaphorins, netrins and slits, no receptor mechanism for RGM action has been defined. Here, an expression cloning strategy identified neogenin as a binding site for RGM, with a sub-nanomolar affinity. Consistent with selective axonal responsiveness to RGM, neogenin is expressed in a gradient across the chick retina. Neogenin is known to be one of several netrin-binding proteins but only neogenin interacts with RGM. The avoidance of RGM by temporal retinal axons is blocked by the anti-neogenin antibody and the soluble neogenin ectodomain. Dorsal root ganglion axons are unresponsive to RGM but are converted to a responsive state by neogenin expression. Thus, neogenin functions as an RGM receptor.

Multiple Roles for Slits in the Control of Cell Migration in the Rostral Migratory Stream

The subventricular zone (SVZ) contains undifferentiated cells, which proliferate and generate olfactory bulb (OB) interneurons. Throughout life, these cells leave the SVZ and migrate along the rostral migratory stream (RMS) to the OB where they differentiate. In vitro, the septum and the choroid plexus (CP) secrete repulsive factors that could orient the migration of OB precursors. Slit1 and Slit2, two known chemorepellents for developing axons, can mimic this effect. We show here that the Slit receptors Robo2 and Robo3/Rig-1 are expressed in the SVZ and the RMS and that Slit1 and Slit2 are still present in the adult septum. Using Slit1/2-deficient mice, we found that Slit1 and Slit2 are responsible for both the septum and the CP repulsive activity in vitro. In adult mice lacking Slit1, small chains of SVZ-derived cells migrate caudally into the corpus callosum, supporting a role for Slits in orienting the migration of SVZ cells. Surprisingly, in adult mice, Slit1 was also expressed by type A and type C cells in the SVZ and RMS, suggesting that Slit1 could act cell autonomously. This hypothesis was tested using cultures of SVZ explants or isolated neurospheres from Slit1-/- or Slit1+/- mice. In both types of cultures, the migration of SVZ cells was altered in the absence of Slit1. This suggests that the regulation of the migration of OB precursors by Slit proteins is complex and not limited to repulsion.