Interactive Fly, Drosophila



Mammalian DCC: Homolog of Drosophila Frazzled

Mouse DCC and mouse Neogenin share the same degree of amino acid similarity with the Drosophila ortholog, Frazzled (24% and 26% across the entire molecule, respectively). To gain a better understanding of the role of DCC and Neogenin in neural and nonneural tissues during vertebrate development, in situ hybridization studies have been carried out to determine the expression patterns of these proteins throughout the mid to late stages of mouse embryogenesis. This analysis reveals striking contrasts in both the spatial and temporal expression patterns of these closely related molecules. While DCC mRNA expression is predominately restricted to the developing central nervous system (CNS), Neogenin mRNA is detected in a broad spectrum of embryonic tissues. Outside the CNS, Neogenin expression is observed mainly in mesodermal derivatives, such as organ primordia and cartilage condensations of many developing embryonic structures. Within the CNS, initiation of DCC expression correlates with the onset of neurogenesis and is maintained at high levels in all regions of the developing CNS actively undergoing neurogenesis. By E18.5, DCC expression is detected only in structures such as the olfactory bulb, the hippocampus, and the cerebellum, all tissues known to sustain active neurogenesis well into postnatal life. In contrast, Neogenin expression is weak in the early developing CNS but broadened and intensified as neurogenesis proceeds. In summary, these observations indicate that Neogenin is the predominant member of this subfamily in mesodermal tissues, while DCC and Neogenin may play complementary roles in the generation of the fully functional CNS (Gad, 1997).

Mice homozygous for the spontaneous rostral cerebellar malformation mutation (rcm) 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 mutant rcm mice. The cerebellum of mutants is smaller and has fewer folia than in the wild type; ectopic cerebellar cells are present in the midbrain regions by three days after birth, and there are abnormalites in postnatal cerebellar neuronal migration. rcm encodes a transmembrane receptor of the immunoglobulin superfamily. The sequence of Rcm is highly similar to that of UNC-5, a C. 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. Analysis of the brain in lateral regions where the inferior colliculus adjoins the cerebellum reveals an abnormal band of granule cells extending from the cerebellum and continuing into the inferior colliculus. Ectopic Purkinje cells are found in the inferior colliculus and tegmentum of the midbrain (Ackerman, 1997).

Inactivation of the murine Dcc gene causes defects in axonal projections that are similar to those observed in netrin-1-deficient mice, but these defects do not affect growth, differentiation, morphogenesis or tumorigenesis in mouse intestine. Within the dorsal spinal cord of embryos homozygous for the null Dcc allele there is a reduction in the number of commissural axons, although those that do extend appear to adopt a normal dorsal-to-ventral trajectory. There appears to be a misrouting of many axons that project into the ventral spinal cord, with some projecting more medially and others more laterally. The corpus callosum and hippocampal commissure are completely absent in Dcc deficient mice. The axons that normally form these commissures appear to be present but fail to cross the midline and remain ipsilateral, projecting to aberrant locations and form tangles. The anterior commissure is severly reduced: the large anterior and posterior limbs of the anterior commissure do not form and join near the midline. These observations fail to support a tumour-suppressor function for Dcc, but are consistent with the hypothesis that DCC is a component of a receptor for netrin-1. It is hypothesized that Dcc is deleted in colorectal cancer because a linked gene might function as a tumor suppressor (Fazell, 1997).

At embryonic day 11, when the first neuronal populations are differentiating, rat DCC is found in developing motor columns. DCC is also found in the dorsal spinal cord over the cell bodies of commissural neurons. At embryonic day 13, intense DCC expression is found in the cell bodies of commissural neurons and in a subpopulation of cells in the developing motor columns. Neurogenin is found in ventricular neuroepithelial cells at E11, with the highest expression midway along the dorsal-ventral axis. Neogenin is widespread in the E13 spinal cord, with highest levels in the ventral third of the ventricular zone, but it is almost absent in commissural neurons. Thus expression patterns of DCC and Neogenin are strikingly complementary. DCC immunoreactivity is detected not just in commissural axons that grow out in response to Netrin-1, but also on their growth cones. DCC is seen as well in neurons in the hindbrain (cerebral plate neurons), and neurons in the midbrain and forebrain. The DCC-positive fibers in the midbrain and diencephalon appear to include the axons in the first tracts that develop, such as the mesencephalic tract of the trigeminal nerve, the circumferential descending axons (or tecto-bulbar tract), the posterior commissure, and the medial longitudianl fasciculus. The DCC-positive cells in the superficial layer of the cerebral cortex appear to be Cajal-Retzius cells. In addition DCC is expressed on axons in the region where the anterior commissure and optic chiasm are forming, and on axons in the tract of the postoptic commissure (Keino-Masu, 1996).

Perturbing DCC function with monoclonal antibody against the extracellular domain of DCC results in a dose-dependent reduction in the extent of commissural axon outgrowth, evoked by Netrin-1 from explants of E11 or E13 rat dorsal spinal cord. Anti-DCC antibody also interferes with the outgrowth of commissural axons from E13 dorsal explants that is evoked by floor plate cells (Keino-Masu, 1996).

The DCC protein is found in axons of the central and peripheral nervous system and in differentiated cell types of the intestine. Colorectal tumors that have lost their capacity to differentiate into mucus producing cells uniformly lack DCC expression; the loss of a chromosome 18q allele is often accompanied by loss of DCC expression in colon tumors. These results provide evidence that DCC encodes a cell surface-localized protein and emphasize the inverse relationship between differentiation and tumorigenesis (Hendrick, 1994).

Mouse and human DCC share 96% identity at the amino acid level. Analysis of DCC mRNA expression throughout the mid and late stages of gestation in the mouse, demonstrated that DCC mRNA is expressed at significantly higher levels in the developing mouse embryo than in any adult tissue. In addition, an embryo-specific alternatively spliced form of DCC is expressed from day 9.5 through day 18.5. The expression of both the alternatively spliced forms of DCC is developmentally regulated such that the embryonic form of DCC predominates in day 9.5 and 10.5 embryos. In later stage embryos the expression of this alternatively spliced form of DCC is down-regulated with respect to that of the adult form. Whole-mount in situ hybridization of day 11.5 mouse embryos reveals that DCC mRNA is expressed at high levels in the developing brain and the neural tube. However, no DCC mRNA was detected in any other embryonic tissue at this stage of development. These observations suggest that during embryogenesis DCC may play a pivotal role in the development of the central nervous system (Cooper, 1995).

DCC (deleted in colorectal carcinoma) is a broadly expressed cell-surface receptor. Netrin-1 was recently identified as a DCC ligand in brain, but the possibility of other DCC ligands was suggested by the finding that an anti-DCC antibody (clone AF5) neutralizes netrin-1-dependent commissural axon outgrowth without blocking DCC/netrin-1 interactions. A DCC-Ig fusion protein binds to neural and epithelial derived cell lines, indicating that these lines express ligand(s) for DCC. The cell-surface binding activity is mediated by the loop between beta-strands F and G of the fifth fibronectin type III repeat FNIII-D5. The loop includes the sequence KNRR, which resembles heparin-binding motifs in other proteins. Heparinase and heparitinase treatment of cells reduces binding of DCC-Ig, suggesting that heparan sulfate proteoglycans are cell-surface DCC ligand(s). This is further supported by heparin blocking experiments and by binding of DCC-Ig to immobilized heparan sulfate. The interaction between DCC-Ig and heparan sulfate/heparin, both on the surface of cells and immobilized on plastic, is blocked by the same anti-DCC antibody that blocks netrin-1-dependent commissural axon outgrowth. Taken together, these findings suggest that the DCC-Ig/heparin interaction may contribute to the biological activity of DCC (Bennett, 1997).

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. 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 the more caudal CNS, guidance about the hypothalamic ventral midline does not require midline expression of netrin (Deiner, 1999).

Human chromosome 18q is among the chromosomal regions thought to harbor a tumor suppressor gene(s), frequently inactivated during the development of several cancer types, particularly those of the gastrointestinal tract. A candidate tumor suppressor gene from 18q (DCC) is markedly decreased or absent in colorectal cancers and cell lines, and a subset of colorectal cancers have been shown to have somatic mutations within the DCC gene. Although studies of 18q LOH and DCC gene expression in other cancer types suggest that DCC inactivation may contribute to the pathogenesis of other tumor types, few studies have provided definitive data to demonstrate that DCC inactivation is a critical genetic event in these tumors. Little is known about the function of DCC in the regulation of normal cell growth and tumor suppression. The predicted structural similarity of DCC to the N-CAM family of cell-surface proteins suggests that it may function through cell-cell and/or cell-extracellular matrix interactions (Fearon, 1994)

The Drosophila seven in absentia (sina) gene was initially discovered because its inactivation leads to R7 photoreceptor defects. Recent data indicate that Sina binds to the Sevenless pathway protein Phyllopod, and together they mediate degradation of Tramtrack, a transcriptional repressor of R7 cell fate. Independent studies have shown that Sina and its highly related mammalian homologs Siah-1 and Siah-2 bind to the DCC (deleted in colorectal cancer) protein and promote its proteolysis via the ubiquitin-proteasome pathway. To determine the roles of mammalian Siahs in proteolysis and their interactions with target proteins, Siah-1 domains critical for regulation of DCC were determined. Mutant Siah-1 proteins, harboring missense mutations in the carboxy (C)-terminal domain analogous to those present in Drosophila sina loss-of-function alleles, fail to promote DCC degradation. Point mutations and deletion of the amino (N)-terminal RING finger domain of Siah-1 abrogates its ability to promote DCC proteolysis. In the course of defining Siah-1 sequences required for DCC degradation, it was found that Siah-1 is itself rapidly degraded via the proteasome pathway, and RING domain mutations stabilize the Siah-1 protein. Siah-1 oligomerizes with itself and other Sina and Siah proteins via C-terminal sequences. Finally, evidence that endogenous Siah-1 regulates DCC proteolysis in cells was obtained through studies of an apparent dominant negative mutant of Siah-1, as well as via an antisense approach. The data indicate that the Siah-1 N-terminal RING domain is required for its proteolysis function, while the C-terminal sequences regulate oligomerization and binding to target proteins, such as DCC (Hu, 1999).

Cell and growth cone migrations along the dorsoventral axis of C. elegans are mediated by the UNC-5 and UNC-40 receptor subtypes for the secreted UNC-6 guidance cue. To characterize UNC-6 receptor function in vivo, genetic interactions between unc-5 and unc-40 were examined in the migrations of the hermaphrodite distal tip cells. Cell migration defects as severe as those associated with a null mutation in unc-6 are produced only by null mutations in both unc-5 and unc-40, indicating that either receptor retains some partial function in the absence of the other. Hypomorphic unc-5 alleles exhibit two distinct types of interallelic genetic interactions. In an unc-40 wild-type genetic background, some pairs of hypomorphic unc-5 alleles exhibit a partial allelic complementation. In an unc-40 null background, however, unc-5 hypomorphs exhibit dominant negative effects. It is proposed that the UNC-5 and UNC-40 netrin receptors can function to mediate chemorepulsion in DTC migrations either independently or together, and the observed genetic interactions suggest that this flexibility in modes of signaling results from the formation of a variety of oligomeric receptor complexes (Merz, 2001).

The idea that UNC-5 and UNC-40 cannot substitute for one another despite having the same general role in the second DTC migration phase suggests that they may have distinct downstream targets. Although signaling pathways linking transmembrane guidance receptors like UNC-5 and UNC-40 to the cytoskeleton are not well understood, there are in principle several ways in which cell adhesion and cytoskeletal dynamics may be regulated in cell migration. Since motility requires the comprehensive and coordinated function of the cytoskeleton, guidance systems may have to send several parallel signals to various targets to effectively direct the cytoskeleton. It is also possible that the UNC-5 and UNC-40 receptor subtypes regulate distinct subsets of the motility apparatus. For example, one receptor subtype may be more important for filopodial extension and the other for filopodial retraction. Although evidence that both receptor subtypes act within the DTCs at the same time has been provided, their subcellular localization during signaling has not been examined. A combination of precise localization and functional studies in combination with biochemical assays will be necessary to address these issues (Merz, 2001).

Migrating axons require the correct presentation of guidance molecules, often at multiple choice points, to find their target. Netrin 1, a bifunctional cue involved in both attracting and repelling axons, is involved in many cell migration and axon pathfinding processes in the CNS. The netrin 1 receptor DCC and its Caenorhabditis elegans homolog UNC-40 have been implicated in directing the guidance of axons toward netrin sources, whereas the C. elegans UNC-6 receptor, UNC-5, is necessary for migrations away from UNC-6. However, a role of vertebrate UNC-5 homologs in axonal migration has not been demonstrated. The Unc5h3 gene product, shown previously to regulate cerebellar granule cell migrations, also controls the guidance of the corticospinal tract, the major tract responsible for coordination of limb movements. Furthermore, corticospinal tract fibers respond differently to loss of UNC5H3. In addition, corticospinal tract defects are observed in mice homozygous for a spontaneous mutation that truncates the Dcc transcript. Postnatal day 0 netrin 1 mutant mice also demonstrate corticospinal tract abnormalities. Last, interactions between the Dcc and Unc5h3 mutations were observed in gene dosage experiments. This is the first evidence of an involvement in axon guidance for any member of the vertebrate unc-5 family and confirms that both the cellular and axonal guidance functions of C. elegans unc-5 have been conserved in vertebrates (Finger, 2002).

Vagal neural crest-derived precursors of the enteric nervous system colonize the bowel by descending within the enteric mesenchyme. Perpendicular secondary migration, toward the mucosa and into the pancreas, result, respectively, in the formation of submucosal and pancreatic ganglia. The hypothesis was tested that netrins guide these secondary migrations. Studies using RT-PCR, in situ hybridization, and immunocytochemistry indicate that netrins (netrins-1 and -3 in mice and netrin-2 in chicks) and netrin receptors [deleted in colorectal cancer (DCC), neogenin, and the adenosine A2b receptor] are expressed by the fetal mucosal epithelium and pancreas. Crest-derived cells express DCC, which is developmentally regulated. Crest-derived cells migrate out of explants of gut toward cocultured cells expressing netrin-1 or toward cocultured explants of pancreas. Crest-derived cells also migrate inwardly toward the mucosa of cultured rings of bowel. These migrations are specifically blocked by antibodies to DCC and by inhibition of protein kinase A, which interferes with DCC signaling. Submucosal and pancreatic ganglia are absent at E12.5, E15, and P0 in transgenic mice lacking DCC. Netrins also promoted the survival/development of enteric crest-derived cells. The formation of submucosal and pancreatic ganglia thus involves the attraction of DCC-expressing crest-derived cells by netrins (Jiang, 2003).

Neural migration. Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism

Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive responses. Evidence is provided that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. This study determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes (Xu, 2014).

The crystal structure of netrin-1 in complex with DCC reveals the bifunctionality of netrin-1 as a guidance cue

Netrin-1 (see Drosophila Netrins) is a guidance cue that can trigger either attraction or repulsion effects on migrating axons of neurons, depending on the repertoire of receptors available on the growth cone. How a single chemotropic molecule can act in such contradictory ways has long been a puzzle at the molecular level. This study presents the crystal structure of netrin-1 in complex with the Deleted in Colorectal Cancer (DCC) receptor.One netrin-1 molecule can simultaneously bind to two DCC molecules through a DCC-specific site and through a unique generic receptor binding site, where sulfate ions staple together positively charged patches on both DCC and netrin-1. Furthermore, this study demonstrates that UNC5A (See Drosophila Unc5) can replace DCC on the generic receptor binding site to switch the response from attraction to repulsion. It is proposed that the modularity of binding allows for the association of other netrin receptors at the generic binding site, eliciting alternative turning responses (Finci, 2014).

The UNC-6/Netrin receptors UNC-40/DCC and UNC-5 inhibit growth cone filopodial protrusion via UNC-73/Trio, Rac-like GTPases and UNC-33/CRMP

UNC-6/Netrin is a conserved axon guidance cue that can mediate both attraction and repulsion. Previous studies have discovered that attractive UNC-40/DCC receptor signaling (Drosophila Frazzled) stimulates growth cone filopodial protrusion and that repulsive UNC-40-UNC-5 heterodimers (see Drosophila Unc5) inhibit filopodial protrusion in C. elegans. This study identified cytoplasmic signaling molecules required for UNC-6-mediated inhibition of filopodial protrusion involved in axon repulsion. The Rac-like GTPases CED-10 and MIG-2, the Rac GTP exchange factor UNC-73/Trio, UNC-44/Ankyrin and UNC-33/CRMP act in inhibitory UNC-6 signaling. These molecules were required for the normal limitation of filopodial protrusion in developing growth cones and for inhibition of growth cone filopodial protrusion caused by activated MYR::UNC-40 and MYR::UNC-5 receptor signaling. Epistasis studies using activated CED-10 and MIG-2 indicated that UNC-44 and UNC-33 act downstream of the Rac-like GTPases in filopodial inhibition. UNC-73, UNC-33 and UNC-44 did not affect the accumulation of full-length UNC-5::GFP and UNC-40::GFP in growth cones, consistent with a model in which UNC-73, UNC-33 and UNC-44 influence cytoskeletal function during growth cone filopodial inhibition (Norris, 2014).

Netrin receptors and mammary gland development

Netrin-1 and its receptors play an essential role patterning the nervous system by guiding neurons and axons to their targets. To explore whether netrin-1 organizes nonneural tissues, its role in mammary gland morphogenesis was examined. Netrin-1 is expressed in prelumenal cells, and its receptor neogenin is expressed in a complementary pattern in adjacent cap cells of terminal end buds (TEBs). Loss of either gene results in disorganized TEBs characterized by exaggerated subcapsular spaces, breaks in basal lamina, dissociated cap cells, and an increased influx of cap cells into the prelumenal compartment. Cell aggregation assays demonstrate that neogenin mediates netrin-1-dependent cell clustering. Thus, netrin-1 appears to act locally through neogenin to stabilize the multipotent progenitor (cap) cell layer during mammary gland development. These results suggest that netrin-1 and its receptor neogenin provide an adhesive, rather than a guidance, function during nonneural organogenesis (Srinivasan, 2003).

Mammalian DCC: Interaction with Netrins

The netrins comprise a small phylogenetically conserved family of guidance cues important for guiding particular axonal growth cones to their targets. Two netrin genes, netrin-1 and netrin-2, have been described in chicken, but in mouse, so far a single netrin gene, an ortholog of chick netrin-1, has been reported. A second mouse netrin gene, which has been named netrin-3, is reported here. Netrin-3 does not appear to be the ortholog of chick netrin-2 but is the ortholog of a recently identified human netrin gene termed NTN2L ('netrin-2-like'), as evidenced by a high degree of sequence conservation and by chromosomal localization. Netrin-3 is expressed in sensory ganglia, mesenchymal cells, and muscles during the time of peripheral nerve development but is largely excluded from the CNS at early stages of its development. The murine netrin-3 protein binds to netrin receptors of the DCC (deleted in colorectal cancer) family [DCC and neogenin] and the UNC5 family (UNC5H1, UNC5H2 and UNC5H3). Unlike chick netrin-1, however, murine netrin-3 binds to DCC with lower affinity than to the other four receptors. Consistent with this finding, although murine netrin-3 can mimic the outgrowth-promoting activity of netrin-1 on commissural axons, it has lower specific activity than netrin-1. Thus, like netrin-1, netrin-3 may also function in axon guidance during development but may function predominantly in the development of the peripheral nervous system and may act primarily through netrin receptors other than DCC (Wang, 1999).

Signaling downstream of Netrin receptors

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. Dcc is shown to activate mitogen-activated protein kinase (MAPK) signalling, by means of extracellular signal-regulated kinase (ERK)-1 and -2, upon 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 (Forcet, 2002).

These results support a role for the MAPK pathway in responses to the chemoattractant netrin-1. This pathway has been implicated in neurite extension stimulated by neurotrophic factors activating Trk receptors and by cell adhesion molecules like N-cadherin, laminin and L1. Conversely, activation of some repulsive receptors of the Eph family can repress MAPK signalling, although a causal role in repulsion has not been established. How does ERK activation affect axonal growth and guidance? Some effects probably result from phosphorylation of cytoskeletal ERK targets such as microtubule-associated proteins and neurofilaments. Interestingly, a MAPK-dependent mechanism drives internalization of the cell adhesion molecules ApCAM and L1 at the rear of the growth cone, which may allow protein cycling from the rear to the leading edge necessary for growth cone advance. Dcc-stimulated MAPK activation leads to activation of the transcription factor Elk-1 and SRE-regulated gene expression, providing a mechanism for transcriptional control by netrin-1. Elk-1 is present not just in neuronal cell bodies but also in axon terminals, but whether axonal Elk-1 participates in axon guidance is unknown. Other targets of ERK-1/2 involved in axon guidance may be translation regulators. New protein translation is stimulated by netrin-1 and required for netrin-mediated attraction of Xenopus retinal growth cones in vitro. Principal factors for translation initiation like eIF4E and eIF4E-BP1 are phosphorylated by an ERK-1/2-dependent pathway, providing a potential mechanism linking netrin-1 to protein synthesis for axon growth and guidance (Forcet, 2002).

The axon guidance cue netrin is importantly involved in neuronal development. DCC (deleted in colorectal cancer) is a functional receptor for netrin and mediates axon outgrowth and the steering response. Different regions of the intracellular domain of DCC directly interact with the tyrosine kinases Src and focal adhesion kinase (FAK). Netrin activates both FAK and Src and stimulates tyrosine phosphorylation of DCC. Inhibition of Src family kinases reduces DCC tyrosine phosphorylation and blocks both axon attraction and outgrowth of neurons in response to netrin. Mutation of the tyrosine phosphorylation residue in DCC abolishes its function of mediating netrin-induced axon attraction. On the basis of these observations, a model is suggested in which DCC functions as a kinase-coupled receptor, and FAK and Src act immediately downstream of DCC in netrin signaling (Li, 2004).

Although netrins are an important family of neuronal guidance proteins, intracellular mechanisms that mediate netrin function are not well understood. This study shows that netrin-1 induces tyrosine phosphorylation of proteins including focal adhesion kinase (FAK) and the Src family kinase Fyn. Blockers of Src family kinases inhibit FAK phosphorylation and axon outgrowth and attraction by netrin. Dominant-negative FAK and Fyn mutants inhibit the attractive turning response to netrin. Axon outgrowth and attraction induced by netrin-1 are significantly reduced in neurons lacking the FAK gene. These results show the biochemical and functional links between netrin, a prototypical neuronal guidance cue, and FAK, a central player in intracellular signaling that is crucial for cell migration (Liu, 2004).

Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1-DCC signaling are still poorly characterized. This study shows that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1-dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1; this suggests an essential role of the Src kinases in Netrin-1-dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and Y1418 has been demonstrated to be crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants. It is proposed that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1 (Meriane, 2004).

The signaling mechanisms that lie immediately downstream of netrin receptors remain poorly understood. This study reports that the netrin receptor DCC interacts with the focal adhesion kinase (FAK), a kinase implicated in regulating cell adhesion and migration. FAK is expressed in developing brains and is localized with DCC in cultured neurons. Netrin-1 induces FAK and DCC tyrosine phosphorylation. Disruption of FAK signaling abolishes netrin-1-induced neurite outgrowth and attractive growth cone turning. Taken together, these results indicate a new signaling mechanism for DCC, in which FAK is activated upon netrin-1 stimulation and mediates netrin-1 function; they also identify a critical role for FAK in axon navigation (Ren, 2004).

Netrins are a family of secreted proteins that guide the migration of cells and axonal growth cones during development. DCC (deleted in colorectal cancer) is a receptor for netrin-1 implicated in mediating these responses. DCC interacts constitutively with the SH3/SH2 adaptor Nck in commissural neurons. This interaction is direct and requires the SH3 but not SH2 domains of Nck-1. Moreover, both DCC and Nck-1 associate with the actin cytoskeleton, and this association is mediated by DCC. A dominant negative Nck-1 inhibits the ability of DCC to induce neurite outgrowth in N1E-115 cells and to activate Rac1 in fibroblasts in response to netrin-1. These studies provide evidence for an important role of mammalian Nck-1 in a novel signaling pathway from an extracellular guidance cue to changes in the actin-based cytoskeleton responsible for axonal guidance (Li, 2002a).

Netrins are chemotropic guidance cues that attract or repel growing axons during development. DCC (deleted in colorectal cancer), a transmembrane protein that is a receptor for netrin-1, is implicated in mediating both responses. However, the mechanism by which this is achieved remains unclear. This study reports that Rho GTPases are required for embryonic spinal commissural axon outgrowth induced by netrin-1. Using N1E-115 neuroblastoma cells, it was found that both Rac1 and Cdc42 activities are required for DCC-induced neurite outgrowth. In contrast, down-regulation of RhoA and its effector Rho kinase stimulates the ability of DCC to induce neurite outgrowth. In Swiss 3T3 fibroblasts, DCC was found to trigger actin reorganization through activation of Rac1 but not Cdc42 or RhoA. Stimulation of DCC receptors with netrin-1 results in a 4-fold increase in Rac1 activation. These results implicate the small GTPases Rac1, Cdc42, and RhoA as essential components that participate in signaling the response of axons to netrin-1 during neural development (Li, 2002b).

The molecular mechanisms underlying the elaboration of branched processes during the later stages of oligodendrocyte maturation are not well understood. This study describes a novel role for the chemotropic guidance cue netrin 1 and its receptor deleted in colorectal carcinoma (Dcc) in the remodeling of oligodendrocyte processes. Postmigratory, premyelinating oligodendrocytes express Dcc but not netrin 1, whereas mature myelinating oligodendrocytes express both. Netrin 1 promotes process extension by premyelinating oligodendrocytes in vitro and in vivo. Addition of netrin 1 to mature oligodendrocytes in vitro evoked a Dcc-dependent increase in process branching. Furthermore, expression of netrin 1 and Dcc by mature oligodendrocytes was required for the elaboration of myelin-like membrane sheets. Maturation of oligodendrocyte processes requires intracellular signaling mechanisms involving Fyn, focal adhesion kinase (FAK), neuronal Wiscott-Aldrich syndrome protein (N-WASP) and RhoA; however, the extracellular cues upstream of these proteins in oligodendrocytes are poorly defined. A requirement was identified for Src family kinase activity downstream of netrin-1-dependent process extension and branching. Using oligodendrocytes derived from Fyn knockout mice, Fyn was shown to be essential for netrin-1-induced increases in process branching. Netrin 1 binding to Dcc on mature oligodendrocytes recruits Fyn to a complex with the Dcc intracellular domain that includes FAK and N-WASP, resulting in the inhibition of RhoA and inducing process remodeling. These findings support a novel role for netrin 1 in promoting oligodendrocyte process branching and myelin-like membrane sheet formation. These essential steps in oligodendroglial maturation facilitate the detection of target axons, a key step towards myelination (Rajasekharan, 2009).

Netrin receptors, cell survival and apoptosis

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 tumor-suppressor gene, and encodes a receptor for netrin-1, a molecule involved in axon guidance. Loss of DCC expression in tumors is not restricted to colon carcinoma, and, although there is no increase in the frequency of tumor formation in DCC hemizygous mice, reestablishment of DCC expression suppresses tumorigenicity. However, the mechanism for DCC is unknown. DCC is found to induce 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 tumor-suppressor protein by inducing apoptosis in settings in which ligand is unavailable (for example, during metastasis or tumor growth beyond local blood supply) through functional caspase cascades by a mechanism that requires cleavage of DCC at Asp 1,290 (Mehlen, 1998).

Acting as receptors for netrin-1, the membrane receptors DCC and UNC5H have been shown to be crucial for axon guidance and neuronal migration. DCC has also been proposed as a dependence receptor inducing apoptosis in cells that are beyond netrin-1 availability. Dependence receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand, but inhibiting apoptosis in the presence of ligand. The netrin-1 receptors UNC5H (UNC5H1, UNC5H2, UNC5H3) also act as dependence receptors. UNC5H receptors induce apoptosis, but this effect is blocked in the presence of netrin-1. Moreover, UNC5H receptors are cleaved in vitro by caspase in their intracellular domains. This cleavage may lead to the exposure of a fragment encompassing a death domain required for cell death induction in vivo. Evidence is presented that during development of the nervous system, the presence of netrin-1 is crucial to maintain survival of UNC5H- and DCC-expressing neurons, especially in the ventricular zone of the brainstem. Altogether, these results argue for a role of netrin-1 during the development of the nervous system, not only as a guidance cue but as a survival factor via its receptors DCC and UNC5H (Llambi, 2001).

Since UNC5H proteins are cleaved by protease and more specifically by caspase, an interesting model suggests that this cleavage allows the release or the exposure of a fragment that induces cell death. However, while expression of cleavage fragments issued from DCC, RET and AR allow cell death induction, expression of the UNC5H2 C-terminal fragment lying after Asp412 has no pro-apoptotic activity unless a myristoylation signal peptide is added. This observation then suggests the requirement of a sub-membrane localization of this fragment for cell death induction. Interestingly, both DCC and UNC5H proteins show oligomeric properties, which may explain heterodimeric binding of full-length UNC5H molecules with caspase-cleaved C-terminal fragments. One hypothesis then is that a heterodimeric complex allows, within membrane proximity, the exposure of the pro-apoptotic fragment lying downstream of the caspase cleavage site (Llambi, 2001).

It is of interest that this pro-apoptotic fragment contains a death domain. Such death domains have been found in various receptors including death receptors Fas and tumor necrosis factor receptor (TNFR) and the dependence receptor p75NTR. They are considered as 'adaptor' domains, allowing interaction of these receptors with 'adaptor' proteins. Death domains can be divided into two types (i.e. I or II) depending on their ability to homodimerize. Sequence alignment reveals that the UNC5H2 death domain is more related to the type II death domain of p75NTR than to the type I death domain of Fas, suggesting that the death domain of UNC5H probably displays no ability to homodimerize. In any case, both Fas and p75NTR death domains have been reported to be crucial for cell death induction. Remarkably, the deletion of the UNC5H2 death domain totally abrogates UNC5H2 pro-apoptotic activity. Taken together these results suggest that in the absence of netrin-1, UNC5H proteins induce cell death via the requirement of their death domain, which is probably exposed via the caspase cleavage. The role of this death domain is, however, completely unknown. The death domain of Fas allows, via the recruitment of the 'adaptor' molecule FADD, the formation of a caspase-activating complex that drives caspase-8 activation. It is also interesting to note that DCC, while without a death domain, recruits a caspase-activating complex allowing caspase-3 activation via the interaction of DCC with caspase-9. Whether the UNC5H death domain is involved in another caspase-activating complex through the recruitment of 'adaptor' molecules via its death domain needs now to be analysed further (Llambi, 2001).

A novel Netrin-1-sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. This study demonstrates that SNARE-mediated exocytosis is a prerequisite for axon branching and identifies the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. A direct interaction was identified between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1-sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. These data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion (Winkle, 2014).

Ventral migration of early-born neurons requires Dcc and is essential for the projections of primary afferents in the spinal cord

Neuronal migration and lamina-specific primary afferent projections are crucial for establishing spinal cord circuits, but the underlying mechanisms are poorly understood. In mice lacking the netrin receptor Dcc, some early-born neurons can not migrate ventrally in the spinal cord. Conversely, forced expression of Dcc causes ventral migration and prevents dorsolateral migration of late-born spinal neurons. In the superficial layer of the spinal cord of Dcc–/– mutants, mislocalized neurons are followed by proprioceptive afferents, while their presence repels nociceptive afferents through Sema3a. Thus, this study has shown that Dcc is a key molecule required for ventral migration of early-born neurons, and that appropriate neuronal migration is a prerequisite for, and coupled to, normal projections of primary afferents in the developing spinal cord (Ding, 2005).

Molecular marker analysis indicates that migration of the dorsal neurons is temporally regulated in the developing spinal cord: in contrast to neurons born at E11.5 or afterwards, neurons born prior to E11.5 in the dorsal neural tube make minimal contributions to the formation of the dorsal horn. Nevertheless, the issue of whether early-born neurons ever contribute to the superficial dorsal horn has remained unsolved because using molecular markers to trace cell migration is complicated by the possibility that gene expression may change in migrating neurons. Using BrdU labeling, it has been demonstrated that neurons born prior to E11.5 do not contribute to the formation of the superficial dorsal horn. Consistent with previous studies, it is found that these early-born neurons undergo ventral migration and contribute to the formation of the intermediate region of the spinal cord. Using both loss- and gain-of-function studies, evidence is provided that Dcc is required for ventral migration of early-born neurons, and can re-direct the migration of late-born neurons, indicating that Dcc is both necessary and sufficient for directing the migration of developing spinal neurons towards the ventral spinal cord. Signaling through Dcc appears to play an instructive role in ventral migration of early-born neurons. In the absence of Dcc, many dI1-3 and dI5 neurons fail to migrate ventrally or ventrolaterally. Intriguingly, some dI5 (Lmx1b) neurons even migrate dorsally in Dcc–/– mutants. This suggests that the presence of Dcc is sufficient to override the dorsal-driving force exerted by some unknown molecules (Ding, 2005).

In Dcc–/– mutants, some early-born neurons appear to undergo normal ventral migration. Several factors may account for this observation: (1) not all early-born neurons express Dcc; (2) Dcc is not the only receptor for netrins, and it may be functionally redundant with other Dcc family members such as neogenin genes or Unc5h3 receptors. Indeed, the observation that a similar defect found in Unc5h3 mutant mice supports such a possibility. Thus, multiple receptors for netrins may guide the migration of early-born neurons synergistically. In Dcc–/– mutants, the migration of late-born neurons appears to be normal, despite their proximity to early-born neurons during their migration from the VZ to the superficial dorsal horn, consistent with weak or no expression of Dcc in these neurons. Furthermore, although late-born neurons that take up exogenous Dcc fail to settle in the superficial layer of the dorsal horn, other non-electroporated late-born neurons exhibit no apparent aberrant migration. Thus, Dcc appears to function in a cell autonomous manner (Ding, 2005).

The simplest explanation for how Dcc controls ventral migration is that it allows early-born neurons to respond to Ntn1, which is expressed in the floorplate and perhaps also as a ventral-to-dorsal gradient in the spinal cord. In this aspect, the function of Dcc is reminiscent of its role in ventral projection of commissural neurons in the spinal cord. Moreover, the fact that overexpression of Dcc in late-born neurons altered their migratory behavior suggests that an intracellular signal transduction pathway that can be activated by Dcc may also be present in these neurons (Ding, 2005).

This study reveals an unexpected role of early-born neurons in the patterning of primary afferents in the spinal cord. Interestingly, not all early-born neurons are required for the projection of proprioceptive afferents. For example, no obvious defects were found in the projections of proprioceptive afferents in mice lacking Math1. In Lmx1b mutants, the specification of dI5 neurons is impaired, but proprioceptive afferent do project to the ventral horn. Therefore, at least dI1 and dI5 neurons are dispensable for the projections of proprioceptive afferents (Ding, 2005).

In Dcc–/– mutants, early-born neurons are mispositioned in the medial superficial dorsal horn to which proprioceptive afferents aberrantly project. This suggests that early-born neurons contain a chemoattractant necessary for directing the projection of proprioceptive afferents. Although Sema3a is most notable for its chemorepellent function, it may also function as a chemoattractant in the nervous system. However, the previous finding that Sema3a has little influence on NT3-responsive axons raises the possibility that other unknown chemoattractant(s) may be involved. However, the observation that TrkA+ afferents are excluded from the region where early-born neurons are present argues that early-born neurons have a repulsive function for cutaneous afferents. Several lines of evidence suggest that early-born neurons may exert this function through Sema3a. Sema3a expression is abnormally detected in the region where early-born neurons are ectopically located in the medial superficial dorsal horn. In addition, forced expression of Sema3a in the dorsal horn by in utero electroporation repels TrkA+ primary afferents. Therefore, this study indicates that Sema3a repels cutaneous afferents in vivo, which is consistent with studies performed in chicken and rat spinal cord. It further offers an explanation for why TrkA+ afferents avoid the midline region of the dorsal horn where early-born neurons are present as their entry route. In both chick and mouse spinal cord, the entry of cutaneous primary afferents into the dorsal side of the spinal cord occurs only after a waiting period that is coincident with the abundant presence of early-born neurons and Sema3a at the dorsal side of the spinal cord. Thus, a spatial regulated distribution of Sema3a+ in early-born neurons may have a key role in preventing TrkA+ primary afferents from entering the dorsal spinal cord prematurely. Together, these results demonstrate that early-born neurons possess dual roles in guiding the projections of primary afferents: (1) to steer the initial ingrowth of proprioceptive afferents towards the ventral horn, and (2) to repel the nociceptive afferents from the midline region and from the deep dorsal horn (Ding, 2005).

The results suggest that an early-born neurons-derived signal is crucial for the initial ingrowth of proprioceptive afferent along the midline and toward the intermediate region within the spinal cord. Since Dcc is not expressed in DRG neurons, this result is in contrast with previous studies that showed that factors guiding the ventral projection of proprioceptive afferents are most likely to reside in the proprioceptive neurons in the DRG. Interestingly, in the absence of Dcc, some proprioceptive afferents do manage to project to the intermediate region as well as the ventral horn of the spinal cord at later stages, albeit in much reduced number. Whether a delay of ventral projection of proprioceptive afferents reflects a partial defect of early-born neurons or a compensation mechanism remains unknown. Nevertheless, because a majority of early-born neurons no longer migrate further once they reach the intermediate region, other signals may come into play for further elongation of those afferents. Together with the findings that transcription factors such as Er81 and Runx3 are required in DRG neurons for proprioceptive afferent projection, the data suggest that coordinated signals derived from both primary afferents and their central target neurons within the spinal cord are essential for the projections of the primary afferents (Ding, 2005).

Netrin 1 and Dcc signalling are required for confinement of central axons within the central nervous system

The establishment of anatomically stereotyped axonal projections is fundamental to neuronal function. While most neurons project their axons within the central nervous system (CNS), only axons of centrally born motoneurons and peripherally born sensory neurons link the CNS and peripheral nervous system (PNS) together by navigating through specialized CNS/PNS transition zones. Such selective restriction is of importance because inappropriate CNS axonal exit could lead to loss of correct connectivity and also to gain of erroneous functions. However, to date, surprisingly little is known about the molecular-genetic mechanisms that regulate how central axons are confined within the CNS during development. This study shows that netrin 1/Dcc/Unc5 chemotropism contributes to axonal confinement within the CNS. In both Ntn1 and Dcc mutant mouse embryos, some spinal interneuronal axons exit the CNS by traversing the CNS/PNS transition zones normally reserved for motor and sensory axons. Evidence that netrin 1 signalling preserves CNS/PNS axonal integrity in three ways: (1) netrin 1/Dcc ventral attraction diverts axons away from potential exit points; (2) a Dcc/Unc5c-dependent netrin 1 chemoinhibitory barrier in the dorsolateral spinal cord prevents interneurons from being close to the dorsal CNS/PNS transition zone; and (3) a netrin 1/Dcc-dependent, Unc5c-independent mechanism that actively prevents exit from the CNS. Together, these findings provide insights into the molecular mechanisms that maintain CNS/PNS integrity and present the first evidence that chemotropic signalling regulates interneuronal CNS axonal confinement in vertebrates (Laumonnerie, 2014).

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