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The majority of excitatory synapses in the mammalian CNS (central nervous system) are formed on dendritic spines (1), and spine morphology and distribution are critical for synaptic transmission (2-6), synaptic integration and plasticity (7). Here, we show that a secreted semaphorin, Sema3F, is a negative regulator of spine development and synaptic structure. Mice with null mutations in genes encoding Sema3F, and its holoreceptor components neuropilin-2 (Npn-2, also known as Nrp2) and plexin A3 (PlexA3, also known as Plxna3), exhibit increased dentate gyrus (DG) granule cell (GC) and cortical layer V pyramidal neuron spine number and size, and also aberrant spine distribution. Moreover, Sema3F promotes loss of spines and excitatory synapses in dissociated neurons in vitro, and in [Npn-2.sup.-/-] brain slices cortical layer V and DG GCs exhibit increased mEPSC (miniature excitatory postsynaptic current) frequency. In contrast, a distinct Sema3A-Npn-1/PlexA4 signalling cascade controls basal dendritic arborization in layer V cortical neurons, but does not influence spine morphogenesis or distribution. These disparate effects of secreted semaphorins are reflected in the restricted dendritic localization of Npn-2 to apical dendrites and of Npn-1 (also known as Nrpl) to all dendrites of cortical pyramidal neurons. Therefore, Sema3F signalling controls spine distribution along select dendritic processes, and distinct secreted semaphorin signalling events orchestrate CNS connectivity through the differential control of spine morphogenesis, synapse formation, and the elaboration of dendritic morphology., Several axon guidance cues, including class 3 semaphorins (Sema3s), have key roles in synapse formation and function (8-11). For example, Sema3A promotes the elaboration of dendrite complexity in vitro (12,13), [...]

Naturally occurring axonal pruning and neuronal cell death help to sculpt neuronal connections during development, but their mechanistic basis remains poorly understood. Here we report that R-amyloid precursor protein (APP) and death receptor 6 (DR6, also known as TNFRSF21) activate a widespread caspase-dependent self-destruction program. DR6 is broadly expressed by developing neurons, and is required for normal cell body death and axonal pruning both in vivo and after trophic-factor deprivation in vitro. Unlike neuronal cell body apoptosis, which requires caspase 3, we show that axonal degeneration requires caspase 6, which is activated in a punctate pattern that parallels the pattern of axonal fragmentation. DR6 is activated locally by an inactive surface ligand(s) that is released in an active form after trophic-factor deprivation, and we identify APP as a DR6 ligand. Trophic-factor deprivation triggers the shedding of surface APP in a β-secretase (BACE)-dependent manner. Loss- and gain-of-function studies support a model in which a cleaved amino-terminal fragment of APP (N-APP) binds DR6 and triggers degeneration. Genetic support is provided by a common neuromuscular junction phenotype in mutant mice. Our results indicate that APP and DR6 are components of a neuronal self-destruction pathway, and suggest that an extracellular fragment of APP, acting via DR6 and caspase 6, contributes to Alzheimer's disease., The initial formative phase of nervous system development, involving the generation of neurons and extension of axons, is followed by a regressive phase in which inappropriate axonal branches are pruned [...]

Adult neural progenitor cells (aNPCs) exhibit limited migration in vivo with the exception of the rostral migratory stream and injury-induced movement. Surprisingly little is known regarding those signals regulating attraction or inhibition of the aNPC. These studies demonstrate that aNPCs respond principally to a repulsive cue expressed at the embryonic floor plate (FP) and also the injured adult CNS. Adult spinal cord progenitor cells (aSCPs) were seeded onto organotypic slice preparations of the intact embryonic or injured adult spinal cord. Cell migration assays combined with genetic and molecular perturbation of FP-derived migration cues or aSCP receptors establish netrin-1 (Ntn-1) but not Slit-2, Shh, or Ephrin-B3 as the primary FP-derived repellant. When slices were prepared from injured spinal cord, aSCP migration away from the injury core was Ntn-1-dependent. These studies establish Ntn-1 as a critical regulator of aSCP migration in the intact and injured CNS. adult stem cells | floor plate | migration | guidance

Inhibitory molecules associated with myelin and the glial scar limit axon regeneration in the adult central nervous system (CNS), but the underlying signaling mechanisms of regeneration inhibition are not fully understood. Here, we show that suppressing the kinase function of the epidermal growth factor receptor (EGFR) blocks the activities of both myelin inhibitors and chondroitin sulfate proteoglycans in inhibiting neurite outgrowth. In addition, regeneration inhibitors trigger the phosphorylation of EGFR in a calcium-dependent manner. Local administration of EGFR inhibitors promotes significant regeneration of injured optic nerve fibers, pointing to a promising therapeutic avenue for enhancing axon regeneration after CNS injury., Failure of successful axon regeneration in the CNS is attributed not only to the intrinsic regenerative incompetence of mature neurons but also to the environment encountered by injured axons (1-7). [...]

A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector ('targeting trapping') yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be [approximately equal to] 1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike. ES cells | gene trapping

Author(s): Peter Carmeliet (corresponding author) [1]; Marc Tessier-Lavigne [2] During evolution, organisms have come to perform more specialized tasks, requiring an increased degree of information processing by neurons and supply [...]

Axon regeneration failure in the adult mammalian CNS is attributed in part to the inhibitory nature of CNS myelin. Three myelin-associated, structurally distinct proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, have been implicated in this inhibition. Neuronal Nogo receptor (NgR) binds to each of the three inhibitors and has been proposed to mediate their inhibitory signals by complexing with a signal-transducing coreceptor, the neurotrophin receptor p[75.sup.NTR]. To assess the contribution of NgR to mediating myelin inhibitory signals and regeneration failure in vivo, we generated and characterized NgR-deficient mice. Nogo transcripts are up-regulated in NgR mutants, indicating that NgR regulates Nogo in vivo. However, neurite outgrowth from NgR-deficient postnatal dorsal root ganglion or cerebellar granule neurons is inhibited by myelin and by a Nogo-66 substrate to the same extent as is from wild-type neurons, whereas p[75.sup.NTR]-deficient neurons are less inhibited. The NgR ligand-binding domain promotes neurite outgrowth on Nogo-66, regardless of the genotype of the neurons, indicating that the NgR ligand-binding domain can act independent of NgR. Thus, NgR is not essential for mediating inhibitory signals from CNS myelin, at least in the neurons tested, whereas p[75.sup.NTR] plays a central role in this response. Neither NgR- nor p[75.sup.NTR]-deficient mice showed enhanced regeneration of corticospinal tract axons in comparison with wild-type controls after spinal dorsal hemisection. Our results thus fail to support a central role for NgR in axonal growth inhibition in vitro or in corticospinal tract regeneration block in vivo. p75 | myelin inhibitors | spinal cord injury

During axon guidance, the ventral guidance of the Caenorhabditis elegans anterior ventral microtubule axon is controlled by two cues, the UNC-6/netrin attractant recognized by the UNC-40/DCC receptor and the SLT-1/slit repellent recognized by the SAX-3/robo receptor, We show here that loss-of-function mutations in clr-1 enhance netrin-dependent attraction, suppressing ventral guidance defects in slt-1 mutants, clr-1 encodes a transmembrane receptor protein tyrosine phosphatase (RPTP) that functions in AVM to inhibit signaling through the DCC family receptor UNC-40 and its effector, UNC-34/enabled. The known effects of other RPTPs in axon guidance could result from modulation of guidance receptors like UNC-40/DCC., Axons in the developing nervous system respond to attractive and repulsive guidance cues of the netrin, slit, semaphorin, and ephrin families (1-3). The interpretation of a guidance signal as a [...]

Author(s): Xiaowei Lu [1]; Annette G. M. Borchers [2]; Christine Jolicoeur [1]; Helen Rayburn [1]; Julie C. Baker [2]; Marc Tessier-Lavigne (corresponding author) [1, 3] In addition to the apical-basal [...]

Commissural neurons in the mammalian dorsal spinal cord send axons ventrally toward the floor plate, where they cross the midline and turn anteriorly toward the brain; a gradient of chemoattractant(s) inside the spinal cord controls this turning. In rodents, several Wnt proteins stimulate the extension of commissural axons after midline crossing (postcrossing). We found that Wnt4 messenger RNA is expressed in a decreasing anterior-to-posterior gradient in the floor plate, and that a directed source of Wnt4 protein attracted postcrossing commissural axons. Commissural axons in mice lacking the Wnt receptor Frizzled3 displayed anterior-posterior guidance defects after midline crossing. Thus, Wnt-Frizzled signaling guides commissural axons along the anterior-posterior axis of the spinal cord., Axonal connections are patterned along the anterior-posterior (A-P) and dorsal-ventral (D-V) neuraxes. Guidance molecules that play essential roles in the D-V guidance of axons have been identified, whereas the nature [...]

Author(s): Guido Serini (corresponding author) [1]; Donatella Valdembri [1, 2]; Sara Zanivan [1, 2]; Giulia Morterra [1, 2]; Constanze Burkhardt [3]; Francesca Caccavari [1]; Luca Zammataro [1]; Luca Primo [1]; [...]

Author(s): Makoto Nishiyama [1]; Akemi Hoshino [1]; Lily Tsai [1]; John R. Henley [2]; Yoshio Goshima [3]; Marc Tessier-Lavigne [4]; Mu-ming Poo [2]; Kyonsoo Hong (corresponding author) [1] Signalling by [...]

In the visual system, differential gene expression underlies development of the anterior-posterior and dorsal-ventral axes. Here we present the results of a microarray screen to identify genes differentially expressed in the developing retina. We assayed gene expression in nasal (anterior), temporal (posterior), dorsal, and ventral embryonic mouse retina. We used a statistical method to estimate gene expression between different retina regions. Genes were clustered according to their expression pattern and were ranked within each cluster. We identified groups of genes expressed in gradients or with restricted patterns of expression as verified by in situ hybridization. A common theme for the identified genes is the differential expression in the dorsal-ventral axis. By analyzing gene expression patterns, we provide insight into the molecular organization of the developing retina.

Author(s): Philip A. Leighton [1, 2]; Kevin J. Mitchell [1, 2, 3]; Lisa V. Goodrich [1, 2]; Xiaowei Lu [2]; Kathy Pinson [3]; Paul Scherz [3]; William C. Skarnes [3]; [...]

Author(s): Kyonsoo Hong [1, 2]; Makoto Nishiyama [1, 2]; John Henley [1]; Marc Tessier-Lavigne [3]; Mu-ming Poo (corresponding author) [1] Pathfinding by growing axons in the developing nervous system is [...]

Spinal commissural neurons rely on trophic support from the floor plate of the spinal cord. The neurons develop extra trophic requirements after sending their axons along the floor plate for several days. This helps faulty neurons to be eliminated quickly and could stop straying neuronal circuits from being formed when the nervous system is being developed.

Extracellular matrix (ECM) molecules modulate the response of growing axons to secreted guidance cues, as well as acting in substrate-mediated adhesion. It has been established that, in Xenopus, laminin-1 from the ECM converts netrin-mediated attraction into repulsion. The netrin-1-induced rise in cyclic AMP could assist in promoting a polymerization-based attractive response, while laminin-1 could encourage a repulsive response by favouring netrin-1-mediated depolymerization.

Author(s): Dominik Paquet [1]; Dylan Kwart [1]; Antonia Chen [1]; Andrew Sproul [2]; Samson Jacob [2]; Shaun Teo [1]; Kimberly Moore Olsen [1]; Andrew Gregg [1, 3]; Scott Noggle [2]; [...]

Cues in the extracellular environment of the nervous system guide migrating cells and axons to their targets. Netrins are cues that can function as attractants and repellents. In Caenorhabditis elegans, UNC-5, a transmembrane protein, has been implicated in certain responses, with the loss of unc-5 function leading to migration defects. A report identifies two UNC-5 vertebrate homologues, defining a new immunoglobulin subfamily. This indicates that the UNC-5 homologues and the mouse rostral cerebellar malformation gene are netrin-binding proteins.

The Deleted in colorectal cancer (DCC) gene was identified as a potential tumour-suppressor gene on human chromosome 18q, although it has not been conclusively implicated as the colorectal tumour-suppressor gene. Investigations indicate that DCC is an axonal chemoattractant netrin-1 receptor. The effects of inactivation of the mouse homologue of DCC are similar to those in netrin-1-deficient mice.

Neuronal growth cones navigate over long distances along specific pathways to find their correct targets. The mechanisms and molecules that direct this pathfinding are the topics of this review. Growth cones appear to be guided by at least four different mechanisms: contact attraction, chemoattraction, contact repulsion, and chemorepulsion. Evidence is accumulating that these mechanisms act simultaneously and in a coordinated manner to direct pathfinding and that they are mediated by mechanistically and evolutionarily conserved ligand-receptor systems., The remarkable feats of information-processing performed by the brain are determined to a large extent by the intricate network of connections between nerve cells (or neurons). The magnitude of the [...]

The floor plate is a specialized group of midline neuroepithelial cells that appears to regulate cell differential and axonal growth in the developing vertebrate nervous system. A floor plate-specific chemorattractant [...]

Heparan sulfate (HS) is required for morphogen signaling during Drosophila pattern formation, but little is known about its physiological importance in mammalian development. To define the developmental role of HS in mammalian species, we conditionally disrupted the HS-polymerizing enzyme EXT1 in the embryonic mouse brain. The EXT1-null brain exhibited patterning defects that are composites of those caused by mutations of multiple HS-binding morphogens. Furthermore, the EXT1-null brain displayed severe guidance errors in major commissural tracts, revealing a pivotal role of HS in midline axon guidance. These findings demonstrate that HS is essential for mammalian brain development., During development, a number of secreted factors play critical roles in morphogenesis, growth regulation, and differentiation. Many of these morphogens and mitogens, including fibroblast growth factors (FGFs), Wingless/ Wnt, and [...]

Author(s): Ami Okada (corresponding author) [1, 5, 6]; Frédéric Charron [1, 2, 3, 5]; Steves Morin [2]; David S. Shin [1]; Karen Wong [4]; Pierre J. Fabre [2, 3]; Marc [...]

Author(s): Xiaowei Lu [1, 6]; Ferdinand le Noble [2, 6]; Li Yuan [2, 6]; Quingjan Jiang [2, 7]; Benjamin de Lafarge [2]; Daisuke Sugiyama [3]; Christiane Bréant [2]; Filip Claes [...]

Most striatal and cortical interneurons arise from the basal telencephalon, later segregating to their respective targets. Here, we show that migrating cortical interneurons avoid entering the striatum because of a chemorepulsive signal composed at least in part of semaphorin 3A and semaphorin 3F. Migrating interneurons expressing neuropilins, receptors for semaphorins, are directed to the cortex; those lacking them go to the striatum. Loss of neuropilin function increases the number of inteneurons that migrate into the striatum. These observations reveal a mechanism by which neuropilins mediate sorting of distinct neuronal populations into different brain structures, and provide evidence that, in addition to guiding axons, these receptors also control neuronal migration in the central nervous system., Most striatal and cortical interneurons derive from a distant region in the basal telencephalon, the medial ganglionic eminence (MGE) (1-3). During development, interneurons migrate tangentially along stereotypical pathways to reach [...]

Axonal growth cones that cross the nervous system midline change their responsiveness to midline guidance cues: They become repelled by the repellent Slit and simultaneously lose responsiveness to the attractant [...]

Netrins stimulate and orient axon growth through a mechanism requiring receptors of the DCC family. It has been unclear, however, whether DCC proteins are involved directly in signaling or are [...]

The axonal chemoattractant netrin-1 guides spinal commissural axons by activating its receptor DCC (Deleted in Colorectal Cancer). We have found that chemical inhibitors of metalloproteases potentiate netrin-mediated axon outgrowth in vitro. We have also found that DCC is a substrate for metalloprotease-dependent ectodomain shedding, and that the inhibitors block proteolytic processing of DCC and cause an increase in DCC protein levels on axons within spinal cord explants. Thus, potentiation of netrin activity by inhibitors may result from stabilization of DCC on the axons, and proteolytic activity may regulate axon migration by controlling the number of functional extracellular axon guidance receptors., There is evidence that proteolytic activity present on neuronal growth cones may regulate their migratory activity (1). This possibility is supported by the presence of axonal stalling defects in flies [...]

Neurons make connections with other neurons or muscles through axons that transmit electrical impulses (see the figure, this page). Traumatic injuries to the spinal cord cause paralysis because axons of [...]

Author(s): Guldo Serini; Donatella Valdembri; Sara Zanivan; Giulia Morterra; Constanze Burkhardt; Francesca Caccavarl; Luca Zammataro; Luca Primo; Luca Tamagnone; Malcolm Logan; Marc Tessier-Lavigne; Masahiko Taniguchl; Andreas W. Püschel; Federico Bussolino [...]

Author(s): Christelle Forcet [1, 2]; Elke Stein [2, 3]; Laurent Pays [1]; Véronique Corset [1]; Fabien Llambi [1]; Marc Tessier-Lavigne [3]; Patrick Mehlen (corresponding author) [1] Neuronal growth cones are [...]