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A series of molecular cues guide axons as they extend in the embryonic environment. In her Perspective, Pasquale discusses new findings that reveal how signals originating from adhesive contacts between axons and the cells they encounter can repel the axon, causing it to break the adhesive contacts and move away (Hattori et al. and Galko and Tessier-Lavigne).
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DCC (deleted in colorectal cancer)-the receptor of the netrin-1 neuronal guidance factor-is expressed and is active in the central nervous system (CNS) during development, but is down-regulated during maturation. The substantia nigra contains the highest level of netrin-1 mRNA in the adult rodent brain, and corresponding mRNA for DCC has also been detected in this region but has not been localized to any particular neuron type. In this study, an antibody raised against DCC was used to determine if the protein was expressed by adult dopamine neurons, and identify their distribution and projections. Significant DCC-immunoreactivity was detected in midbrain, where it was localized to ventrally displaced A9 dopamine neurons in the substantia nigra, and ventromedial A10 dopamine neurons predominantly situated in and around the interfascicular nucleus. Strong immunoreactivity was not detected in dopamine neurons found elsewhere, or in non-dopamine-containing neurons in the midbrain. Terminal fields selectively labeled with DCC antibody corresponded to known nigrostriatal projections to the dorsolateral striatal patches and dorsomedial shell of the accumbens, and were also detected in prefrontal cortex, septum, lateral habenular and ventral pallidum. The unique distribution of DCC-immunoreactivity in adult ventral midbrain dopamine neurons suggests that netrin-1/DCC signaling could function in plasticity and remodeling previously identified in dopamine projection pathways. In particular, a recent report that DCC is regulated through the ubiquitin-proteosome system via Siah/Sina proteins, is consistent with a potential involvement in genetic and sporadic forms of Parkinson's disease.
Netrins and their classical receptors--DCC and neogenin--play key roles in neuronal guidance. Recent developments identify new roles for netrins in epithelial and vascular morphogenesis. Netrins accomplish these effects, at least in part, through binding to integrins and/or activating integrin-associated kinases such as FAK and Fyn. Here we discuss these recent findings and propose that integrins and classical netrin receptors cooperate to regulate multiple aspects of development.
Migration of neurons from proliferative zones to their functional sites is fundamental to the normal development of the central nervous system. Mice homozygous for the spontaneous rostral cerebellar malformation mutation (rcm(s)) or a newly identified transgenic insertion allele (rcm(tg)) exhibit cerebellar and midbrain defects, apparently as a result of abnormal neuronal migration. Laminar structure abnormalities in lateral regions of the rostral cerebellar cortex have been described in homozygous rcm(s) mice. We now demonstrate that the cerebellum of both rcm(s) and rcm(tg) homozygotes is smaller and has fewer folia than in the wild-type, ectopic cerebellar cells are present in midbrain regions by three days after birth, and there are abnormalities in postnatal cerebellar neuronal migration. We have cloned the rcm complementary DNA, which encodes a transmembrane receptor of the immunoglobulin superfamily. The sequence of the rcm protein (Rcm) is highly similar to that of UNC-5, a Caenorhabditis elegans protein that is essential for dorsal guidance of pioneer axons and for the movement of cells away from the netrin ligand, which is encoded by the unc-6 gene. As Rcm is a member of a newly described family of vertebrate homologues of UNC-5 which are netrin-binding proteins, our results indicate that UNC-5-like proteins may have a conserved function in mediating netrin-guided migration.
Like axons, dendrites need guidance for proper orientation and positioning within the brain. Guidance determines synaptic connectivity as well as the strength of transmission. Recent in vivo studies have demonstrated that several cell-surface receptors, previously known as axon guidance molecules, are also responsible for the directed outgrowth of dendrites. Collectively, these studies reveal that the function of guidance molecules in individual neurons and individual processes is diverse and likely to be specifically regulated. Here, these studies are reviewed and emerging issues and implications are discussed.
The physical restoration of dopamine circuits damaged or lost in Parkinson disease by implanting embryonic stem (ES)-derived cells may become a treatment. It is critical to understand responses of ES-derived dopamine (DA) neurons to guidance signals that determine axonal path and targeting. Using a collagen gel culture system, we examined effects of secreted molecules Netrin-1 and Slits on neurite outgrowth of fetal DA neurons and murine ES-differentiated DA neurons. We have previously shown that fetal DA neurons express DCC and Robo1/2 receptors and that Netrin-1 and Slit2 function as an attractant and a repellent for DA neurite outgrowth. In the present study, we observe that both Slit1 and Slit3 repel and inhibit neurite growth of fetal DA neurons. Here, we also demonstrate that ES-differentiated neurons including DA neurons express the Netrin receptor DCC and Slit receptor Robo proteins. In the gel culture system of ES cells, Netrin-1 promoted neurite outgrowth mediated by DCC receptor, and Slit1 and Slit3 were inhibitory for neurite outgrowth through Robo receptors. Slit2 appeared to exert inhibitory as well as repulsive effects in the coculture assay. However, unlike fetal DA neurites, no directed neurite outgrowth was observed in the cocultures of ES-derived DA neurons with Netrin-1-, Slit1-, and Slit3-producing cells. The findings suggest that ES-derived DA neurons generated by current protocols can respond to guidance cues in vitro in a similar manner to fetal cells but also exhibit distinct responses. This may result from developmental differences generated by present in vitro methods of cell patterning or conditioning during ES cell differentiation.
Optic nerve formation in mouse involves interactions between netrin-1 at the optic disk and the netrin-1 receptor DCC (deleted in colorectal cancer) expressed on retinal ganglion cell (RGC) axons. Deficiency in either protein causes RGC pathfinding defects at the disk leading to optic nerve hypoplasia (). Here we show that further along the visual pathway, RGC axons in netrin-1- or DCC-deficient mice grow in unusually angular trajectories within the ventral hypothalamus. In heterozygous Sey(neu) mice that also have a small optic nerve, RGC axon trajectories appear normal, indicating that the altered RGC axon trajectories in netrin-1 and DCC mutants are not secondarily caused by optic nerve hypoplasia. Intrinsic hypothalamic patterning is also affected in netrin-1 and DCC mutants, including a severe reduction in the posterior axon projections of gonadotropin-releasing hormone neurons. In addition to axon pathway defects, antidiuretic hormone and oxytocin neurons are found ectopically in the ventromedial hypothalamus, apparently no longer confined to the supraoptic nucleus in mutants. In summary, netrin-1 and DCC, presumably via direct interactions, govern both axon pathway formation and neuronal position during hypothalamic development, and loss of netrin-1 or DCC function affects both visual and neuroendocrine systems. Netrin protein localization also indicates that unlike in more caudal CNS, guidance about the hypothalamic ventral midline does not require midline expression of netrin.
Classical members of the UNC6/netrin family are secreted proteins which play a role as long-range cues for directing growth cones. We here identified in mice a novel member netrin-G2 which constitute a subfamily with netrin-G1 among the UNC6/netrin family. Both of these netrin-Gs are characterized by glycosyl phosphatidyl-inositol linkage onto cells, molecular variants presumably generated by alternative splicing and lack of any appreciable affinity to receptors for classical netrins. These genes are preferentially expressed in the central nervous system with complementary distribution in most brain areas, that is netrin-G1 in the dorsal thalamus, olfactory bulb and inferior colliculus, and netrin-G2 in the cerebral cortex, habenular nucleus and superior colliculus. Consistently, immunohistochemical analysis revealed that netrin-G1 molecules are present on thalamocortical but not corticothalamic axons. Thalamic and neocortical neurons extended long neurites on immobilized recombinant netrin-G1 or netrin-G2 in vitro. Immobilized anti-netrin-G1 antibodies altered shapes of cultured thalamic neurons. We propose that netrin-Gs provide short-range cues for axonal and/or dendritic behavior through bi-directional signaling.
The UNC5Hs are axon guidance receptors that mediate netrin-1-dependent chemorepulsion, and dependence receptors that mediate netrin-1-independent apoptosis. Here, we report an interaction between UNC5H1 and NRAGE. Our experiments show that this interaction is responsible for apoptosis induced by UNC5H1, and this level of apoptosis is greater than the amount induced by either UNC5H2 or UNC5H3. We mapped the NRAGE binding domain of UNC5H1 to its ZU-5 domain and show that this region, in addition to an adjacent PEST sequence, is required for UNC5H1-mediated apoptosis. Chimeric UNC5H2 and UNC5H3 receptors, containing the NRAGE binding domain and PEST sequence of UNC5H1, bind NRAGE and cause increased levels of apoptosis. UNC5H1 expression does not induce apoptosis in differentiated PC12 cells, which down-regulate NRAGE, but induces apoptosis in native PC12 cells that endogenously express high levels of NRAGE and in differentiated PC12 cells when NRAGE is overexpressed. Together, these results demonstrate a mechanism for UNC5H1-mediated apoptosis that requires an interaction with the MAGE protein NRAGE.
Netrins, a family of laminin-related secreted proteins, have critical roles in axon guidance and cell migration during development. The deleted in colorectal cancer (DCC) protein has been implicated as a netrin-1 receptor component. The expression and function of netrins in adult tissues remain unknown, and direct interaction of netrin-1 with DCC has not been demonstrated. We cloned the human netrin-1 (NTN1L) gene, mapped it to chromosome 17p12-13, and found that it encodes a 604 amino acid protein with 98% identity to mouse netrin-1 and 50% identity with the Caenorhabditis elegans UNC-6 protein. NTN1L transcripts were detected in essentially all normal adult tissues studied, and markedly reduced or absent NTN1L expression was seen in approximately 50% of brain tumors and neuroblastomas. In one neuroblastoma, missense mutations at highly conserved NTN1L codons were found. Netrin-1 protein could be cross-linked to DCC protein on the cell surface, but it did not immunoprecipitate with DCC in the absence of cross-linking and it failed to bind to a soluble fusion protein containing the entire DCC extracellular domain. Our findings demonstrating NTN1L loss of expression and mutations suggest that NTN1L alterations may contribute to the development of some cancers. Furthermore, the binding of netrin-1 to DCC appears to depend on the presence of a coreceptor or accessory proteins.
Netrins are a family of secreted proteins that direct the migration of cells and axonal growth cones during neural development. They are bifunctional cues, attracting some cell types and repelling others. Netrins function as either short- or long-range cues, in some circumstances acting close to the surface of the cells that produce them and in other cases at a distance. Two classes of receptors mediate the response to netrin-1, the deleted in colorectal cancer family and the UNC-5 homolog family. Although netrin function has been extensively studied in the embryonic nervous system, netrin-1 is expressed in the adult mammalian spinal cord at a level similar to that in the embryonic CNS. In the adult and embryonic CNS, the majority of netrin-1 protein is not freely soluble but is associated with membranes and extracellular matrix. This distribution is consistent with netrin-1 acting as a short-range cue. Here we present a model whereby netrin-1 in the embryonic neural epithelium could act as a membrane-associated long-range cue. Netrin-1 is expressed in the adult by multiple types of neurons and by myelinating glia: oligodendrocytes in the CNS and Schwann cells in the PNS. In the white matter of the adult CNS, netrin-1 protein is absent from compact myelin but enriched in periaxonal myelin at the interface between axons and oligodendrocytes. This distribution suggests that in the adult nervous system netrin-1 may function to mediate cell-cell interactions. Furthermore, netrin receptor expression persists in neurons following injury, raising the possibility that netrin-1 may influence axonal regeneration.
Cells depend for their survival on stimulation by trophic factors and other prosurvival signals, the withdrawal of which induces apoptosis, both via the loss of antiapoptotic signaling and the activation of proapoptotic signaling via specific receptors. These receptors, dubbed dependence receptors, activate apoptotic pathways following the withdrawal of trophic factors and other supportive stimuli. Such receptors may feature in developmental cell death, carcinogenesis (including metastasis), neurodegeneration, and possibly subapoptotic events such as neurite retraction and somal atrophy. Mechanistic studies of dependence receptors suggest that these receptors form ligand-dependent complexes that include specific caspases. Complex formation in the absence of ligand leads to caspase activation by a mechanism that is typically dependent on caspase cleavage of the receptor itself, releasing proapoptotic peptides. Cellular dependence receptors, considered in the aggregate, may thus form a system of molecular integration, analogous to the electrical integration system provided by dendritic arbors in the nervous system.
Neuronal growth cones are guided to their targets by attractive and repulsive guidance cues. In mammals, netrin-1 is a bifunctional cue, attracting some axons and repelling others. Deleted in colorectal cancer (Dcc) is a receptor for netrin-1 that mediates its chemoattractive effect on commissural axons, but the signalling mechanisms that transduce this effect are poorly understood. Here we show that Dcc activates mitogen-activated protein kinase (MAPK) signalling, by means of extracellular signal-regulated kinase (ERK)-1 and -2, on netrin-1 binding in both transfected cells and commissural neurons. This activation is associated with recruitment of ERK-1/2 to a Dcc receptor complex. Inhibition of ERK-1/2 antagonizes netrin-dependent axon outgrowth and orientation. Thus, activation of MAPK signalling through Dcc contributes to netrin signalling in axon growth and guidance.
The proper regulation of temporal and spatial expression of the axon guidance cues and their receptors is critical for the normal wiring of nervous system during development. Netrins, a family of secreted guidance cues, are involved in the midline crossing of spinal commissural axons and in the guidance of cortical efferents. Axons normally lose the responsiveness to their attractants when they arrive at their targets, where the attractant is produced. However the molecular mechanism is still unknown. We investigated the molecular mechanism of down-regulation of netrin-1 signaling in the embryonic cortical neurons. Netrin-1 induced the ubiquitination and proteolytic cleavage of Deleted in Colorectal Cancer (DCC), a transmembrane receptor for netrin, in dissociated cortical neurons. A dramatic decrease of DCC level particularly on the cell surface was also observed after netrin-1 stimulation. Specific ubiquitin-proteasome inhibitors prevented the netrin-induced DCC cleavage and decrease of cell surface DCC. We suggest that the ligand-mediated down-regulation of DCC might participate in the loss of netrin-responsiveness in the developing nervous system.
Significant progress has been made recently in understanding axon guidance to and from choice points. Netrins have been shown to function as conserved midline chemoattractants in vertebrates and insects, and receptors for netrins and semaphorins/collapsins have been identified. More evidence has accumulated that repulsion plays a key role in guidance, including the involvement of the ephrin/Eph receptor system in contact repulsion.
The development of colonic carcinoma is associated with the mutation of a specific set of genes. One of these, DCC (deleted in colorectal cancer), is a candidate tumour-suppressor gene, and encodes a receptor for netrin-1, a molecule involved in axon guidance. Loss of DCC expression in tumours is not restricted to colon carcinoma, and, although there is no increase in the frequency of tumour formation in DCC hemizygous mice, reestablishment of DCC expression suppresses tumorigenicity. However, the mechanism of action of DCC is unknown. Here we show that DCC induces apoptosis in the absence of ligand binding, but blocks apoptosis when engaged by netrin-1. Furthermore, DCC is a caspase substrate, and mutation of the site at which caspase-3 cleaves DCC suppresses the pro-apoptotic effect of DCC completely. These results indicate that DCC may function as a tumour-suppressor protein by inducing apoptosis in settings in which ligand is unavailable (for example, during metastasis or tumour growth beyond local blood supply) through functional caspase cascades by a mechanism that requires cleavage of DCC at Asp 1,290.
The C. elegans SAX-3/Robo receptor acts in anterior-posterior, dorsal-ventral and midline guidance decisions. Here we show that SAX-3 signaling involves the C. elegans Enabled protein UNC-34 and an unexpected Netrin-independent function of the Netrin receptor UNC-40/DCC. Genetic interactions with gain- and loss-of-function mutations suggest that unc-34 and unc-40 act together with sax-3 in several guidance decisions, but the C. elegans Netrin gene unc-6 does not act in the same genetic pathways. Within the migrating axon, sax-3, unc-34 and unc-40 all act cell-autonomously. Our results support a role for UNC-34/Enabled proteins in SAX-3-mediated repulsion, and show that UNC-40/DCC can potentiate SAX-3/Robo signaling via a mechanism that may involve direct binding of the two guidance receptors. A combinatorial logic dictates alternative functions for UNC-40/DCC, which can act in attraction to UNC-6/Netrin, repulsion from Netrin (with UNC-5), or repulsion from Slit (with SAX-3).