Interactive Fly, Drosophila

Netrin-A and Netrin-B


EVOLUTIONARY HOMOLOGS (part 2/3)

Cloning, expression and mutation of vertebrate netrins

The guidance of axons to their targets in the developing nervous system is believed to involve diffusible chemotropic factors secreted by target cells. Floor plate cells at the ventral midline of the spinal cord secrete a diffusible factor or factors that promote the outgrowth of spinal commissural axons and attracts these axons in vitro. Two membrane-associated proteins isolated from brain, Netrin-1 and Netrin-2, possess commissural axon outgrowth-promoting activity. Netrin-1 RNA is expressed by floor plate cells, whereas Netrin-2 RNA is detected at lower levels in the ventral two-thirds of the spinal cord, but not the floor plate. Heterologous cells expressing recombinant Netrin-1 or Netrin-2 secrete diffusible forms of the proteins and can attract commissural axons at a distance. These results show that Netrin-1 is a chemotropic factor expressed by floor plate cells and suggest that the two Netrin proteins guide commissural axons in the developing spinal cord (Kennedy, 1994).

Proteins of the Netrin family have been implicated in axon guidance in both C. elegans and vertebrates. A zebrafish netrin homolog (net1) is expressed in the floor plate and the anterior ventral neural tube. Its expression is ectopically induced by misexpression of sonic hedgehog (shh) (See Drosophila Hedgehog) and a dominant negative mutant of the regulatory subunit of protein kinase A (dnReg). Ectopic activation of net1, however, is restricted to distinct regions in the brain. Upon overexpression of shh or dnReg in cyclops mutants, which have strongly impaired net1 expression in the ventral neural tube, rescue of net1 expression is observed in the brain but not in the spinal cord. Ectopic expression of dnReg and Shh protein can be detected at high levels throughout injected embryos from pre-gastrula stages onwards suggesting that the competence of the neural plate to respond to Shh signaling activity differs regionally. Similar to net1, axial, the zebrafish homolog of mammalian HNF3beta, which is also expressed along the ventral neural tube, is ectopically induced in the brain of embryos injected with dnReg mRNA. Neurons differentiate normally within domains of ectopic net1 and axial expression. Thus, dorsal neuronal differentiation appears to be unaffected despite co-expression of a gene program specific for the ventral neural tube. This also suggests that these ectopically expressing regions have not differentiated into floor plate (Strahle, 1997).

The netrins are laminin-like axon guidance molecules that are conserved among C. elegans, Drosophila, and vertebrates and that have chemoattractive and chemorepellant properties. To study the possible actions of this gene family in the developing and adult mammalian nervous systems, a partial cDNA has been cloned that corresponds to a region conserved among chick netrin-1, netrin-2, and unc-6 and its expression studied, along with the expression of a netrin receptor, dcc, the deleted in colorectal cancer gene, in the developing and adult rat CNS. The localization of cells expressing netrin or dcc suggests that these genes, in addition to their actions in defining the ventral midline, may act in controlling retinal ganglion cell axon guidance in the optic nerve, cell migration in the developing cerebellum and olfactory epithelium, and development and maintenance of connections to the substantia nigra and corpus striatum (Livesey, 1998).

During nervous system development, spinal commissural axons project toward floor plate cells even as trochlear motor axons are extending away from these same floor plate cells. Netrin-1, a diffusible protein made by floor plate cells, can attract spinal commissural axons and repel trochlear axons in vitro, but its role in vivo is unknown. Netrin-1 deficient mice exhibit defects in spinal commissural axon projections that are consistent with the assumption that netrin-1 provides guidance for these axons. Defects in several forebrain commissures are also observed, suggesting additional guidance roles for netrin-1. Trochlear axon projections are largely normal, predicting the existence of additional cues; there is evidence a distinct trochlear axon chemorepellent produced by floor plate cells. These results establish netrin-1 as a guidance cue that likely collaborates with other diffusible cues to guide axons in vivo (Serafini, 1996).

The netrins are a small but highly conserved family of axonal guidance signals found throughout the animal kingdom. Sequence conservation in this family was used to isolate cDNAs for two mouse netrins. Analysis of their expression patterns and functional properties has shown that mouse netrin-1 is in most respects similar to its orthologs in other vertebrates, while the properties of netrin-3 differ markedly from those of other members of this protein family. In contrast to netrin-1, which is widely expressed both in the developing nervous system and in mesodermal tissues, netrin-3 transcripts are largely restricted to dorsal root ganglia and the developing limb buds. Netrin-3 binds with a significantly lower affinity to the netrin receptor DCC (deleted in colorectal cancer) and is also ineffective in eliciting the outgrowth of commissural axons in vitro. These results demonstrate that, although the netrins are highly conserved signals that guide axons to or away from the midline of the developing nervous system, at the same time they show a surprising degree of divergence in vertebrates (Puschel, 1999).

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).

Classical members of the UNC6/netrin family are secreted proteins that play a role as long-range cues for directing growth cones. A novel member netrin-G2 has been identified in mice that along with netrin-G1 constitutes a subfamily within 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; these variants lack 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 has 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 alter shapes of cultured thalamic neurons. It is proposed that netrin-Gs provide short-range cues for axonal and/or dendritic behavior through bi-directional signaling (Nakashiba, 2002).

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

Netrin-1 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; see Drosophila Frazzled) 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).

Netrins interact with integrins

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, Netrin-1 is shown to be expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. α6β4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of α6β4 and α3β1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells (Yebra, 2003).

Integrins α6β4 and α3β1 are laminin receptors primarily expressed in epithelial cells that have been implicated in branching morphogenesis, development, and function of epithelial tissues. In addition to promoting cell adhesion, α6β4 has been involved in signaling that regulates cell survival, proliferation, and migration. Interestingly, α3β1 can cooperate with α6β4 to mediate cell motility, following activation by growth factors such as HGF/SF. The results show that α6β4 plays a major role in epithelial cell adhesion to Netrin-1. This interaction can be mapped to a highly basic region of the Netrin-1 C terminus similar to basic residue-rich domains of laminins, known to support recognition and binding of integrins α3β1, α6β1, and α6β4. The observation that classical Netrin-1 receptors DCC and Neogenin do not mediate adhesion to Netrin-1 may be explained by the extremely low levels of expression of these two receptors detected in the pancreatic epithelium, and supports a primary role of integrins α3β1 and α6β4 in the recognition of Netrin-1 (Yebra, 2003).

Netrin-1 as a survival factor, acting via its receptor

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).

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).

Inhibition of endothelial cell apoptosis by netrin-1 during angiogenesis

Netrin-1 has been proposed to play an important role in embryonic and pathological angiogenesis. However, the data leads to the apparently contradictory conclusions that netrin-1 is either a pro- or an antiangiogenic factor. This study reconciled these opposing observations by demonstrating that netrin-1 acts as a survival factor for endothelial cells, blocking the proapoptotic effect of the dependence receptor UNC5B and its downstream death signaling effector, the serine/threonine kinase DAPK. The netrin-1 effect on blood vessel development is mimicked by caspase inhibitors in ex vivo assays, and the inhibition of caspase activity, the silencing of the UNC5B receptor, and the silencing of DAPK are each sufficient to rescue the vascular sprouting defects induced by netrin-1 silencing in zebrafish. Thus, the proapoptotic effect of unbound UNC5B and the survival effect of netrin-1 on endothelial cells finely tune the angiogenic process (Castets, 2009).

Signaling downstream of netrins

Axon outgrowth is the first step in the formation of neuronal connections, but the pathways that regulate axon extension are still poorly understood. NFAT proteins belong to the Rel/Dorsal family of transcription factors. Mice deficient in calcineurin-NFAT signaling have dramatic defects in axonal outgrowth, yet have little or no defect in neuronal differentiation or survival. In vitro, sensory and commissural neurons lacking calcineurin function or NFATc2, c3, and c4 are unable to respond to neurotrophins or netrin-1 with efficient axonal outgrowth. Neurotrophins and netrins stimulate calcineurin-dependent nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in cultured primary neurons. These data indicate that the ability of these embryonic axons to respond to growth factors with rapid outgrowth requires activation of calcineurin/NFAT signaling by these factors. The precise parsing of signals for elongation, turning and survival could allow independent control of these processes during development (Graef, 2003).

NFAT transcription complexes are appealing candidates for regulating aspects of neuronal morphogenesis because they integrate extracellular signals. Cell membrane signaling results in the assembly of NFAT transcription complexes in the nucleus and the activation of genes that are dependent on the cell type in which the signal is received. A rise in intracellular Ca2+ activates the serine/threonine phosphatase calcineurin and rapidly dephosphorylates the four cytoplasmic subunits NFATc1-4. Dephosphorylation of serines in the amino-termini of NFATc proteins by calcineurin exposes nuclear localization sequences leading to their rapid nuclear import. NFATc cytoplasmic subunits require other transcription factors for DNA binding, including AP-1, MEF2, GATA4, and additional factors generically referred to as nuclear partners (NFATn). The nuclear components of NFAT transcription complexes are often regulated by the PKC and Ras/MAPK pathways. Hence, the assembly of NFAT transcription complexes requires that Ca2+/calcineurin signaling be coincident with other signals. Nuclear import of NFATc family members is opposed by rapid export induced by rephosphorylation mediated by the sequential actions of PKA and GSK3. The rapid export of NFATc proteins from the nucleus can make NFAT signaling responsive to receptor occupancy and/or Ca2+ channel dynamics (Graef, 2003 and references therein).

Evidence is provided for an unexpected role for calcineurin and NFATc family members in controlling the outgrowth of embryonic axons. The results suggest that calcineurin/NFAT signaling is required specifically for axon outgrowth stimulated by growth factors like neurotrophins and netrins and provides a potential regulatory site for controlling axonal elongation independent of neuronal survival (Graef, 2003).

During embryonic development, tangentially migrating precerebellar neurons emit a leading process and then translocate their nuclei inside it (nucleokinesis). Netrin 1 (also known as netrin-1) acts as a chemoattractant factor for neurophilic migration of precerebellar neurons (PCN) both in vivo and in vitro. In the present work, Rho GTPases that could direct axon outgrowth and/or nuclear migration were analyzed. The expression pattern of Rho GTPases in developing PCN is consistent with their involvement in the migration of PCN from the rhombic lips. Pharmacological inhibition of Rho enhances axon outgrowth of PCN and prevents nuclei migration toward a netrin 1 source, whereas inhibition of Rac and Cdc42 sub-families impairs neurite outgrowth of PCN without affecting migration. Through pharmacological inhibition, it has been shown that Rho signaling directs neurophilic migration through Rock activation. Altogether, these results indicate that Rho/Rock acts on signaling pathways favoring nuclear translocation during tangential migration of PCN. Thus, axon extension and nuclear migration of PCN in response to netrin 1 are not strictly dependent processes because: (1) distinct small GTPases are involved; (2) axon extension can occur when migration is blocked, and (3) migration can occur when axon outgrowth is impaired (Causeret, 2004).

The signaling cascades governing neuronal migration and axonal guidance link extracellular signals to cytoskeletal components. MAP1B is a neuron-specific microtubule-associated protein implicated in the crosstalk between microtubules and actin filaments. Netrin 1 regulates, both in vivo and in vitro, mode I MAP1B phosphorylation, which controls MAP1B activity, in a signaling pathway that depends essentially on the kinases GSK3 and CDK5. map1B-deficient neurons from the lower rhombic lip and other brain regions have reduced chemoattractive responses to Netrin 1 in vitro. Furthermore, map1B mutant mice have severe abnormalities, similar to those described in netrin 1-deficient mice, in axonal tracts and in the pontine nuclei. These data indicate that MAP1B phosphorylation is controlled by Netrin 1 and that the lack of MAP1B impairs Netrin 1-mediated chemoattraction in vitro and in vivo. Thus, MAP1B may be a downstream effector in the Netrin 1-signaling pathway (Del Río, 2004).

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).

Ena/VASP proteins play important roles in axon outgrowth and guidance. Ena/VASP activity regulates the assembly and geometry of actin networks within fibroblast lamellipodia. In growth cones, Ena/VASP proteins are concentrated at filopodia tips, yet their role in growth cone responses to guidance signals has not been established. This study finds that Ena/VASP proteins play a pivotal role in formation and elongation of filopodia along neurite shafts and growth cone. Netrin-1-induced filopodia formation is dependent upon Ena/VASP function and directly correlates with Ena/VASP phosphorylation at a regulatory PKA site. Accordingly, Ena/VASP function is required for filopodial formation from the growth cone in response to global PKA activation. It is proposed that Ena/VASP proteins control filopodial dynamics in neurons by remodeling the actin network in response to guidance cues (Lebrand, 2004).

Ion channels formed by the TRP superfamily of proteins act as sensors for temperature, osmolarity, mechanical stress and taste. The growth cones of developing axons are responsible for sensing extracellular guidance factors, many of which trigger Ca2+ influx at the growth cone; however, the identity of the ion channels involved remains to be clarified. TRP-like channel activity exists in the growth cones of cultured Xenopus neurons and can be modulated by exposure to netrin-1 and brain-derived neurotrophic factor, two chemoattractants for axon guidance. Whole-cell recording from growth cones showed that netrin-1 induces a membrane depolarization, part of which remains after all major voltage-dependent channels are blocked. Furthermore, the membrane depolarization is sensitive to blockers of TRP channels. Pharmacological blockade of putative TRP currents or downregulation of Xenopus TRP-1 (xTRPC1) expression with a specific morpholino oligonucleotide abolishes the growth-cone turning and Ca2+ elevation induced by a netrin-1 gradient. Thus, TRPC currents reflect early events in the growth cone's detection of some extracellular guidance signals, resulting in membrane depolarization and cytoplasmic Ca2+ elevation that mediates the turning of growth cones (Wang, 2005).

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).

DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1

During nervous system development, spinal commissural axons project toward and across the ventral midline. They are guided in part by netrin-1, made by midline cells, which attracts the axons by activating the netrin receptor DCC. However, previous studies suggest that additional receptor components are required. This study reports that the Down's syndrome Cell Adhesion Molecule (DSCAM), a candidate gene implicated in the mental retardation phenotype of Down's syndrome, is expressed on spinal commissural axons, binds netrin-1, and is necessary for commissural axons to grow toward and across the midline. DSCAM and DCC can each mediate a turning response of these neurons to netrin-1. Similarly, Xenopus spinal neurons exogenously expressing DSCAM can be attracted by netrin-1 independently of DCC. These results show that DSCAM is a receptor that can mediate turning responses to netrin-1 and support a key role for netrin/DSCAM signaling in commissural axon guidance in vertebrates (Ly, 2008).

Netrins mammary gland and lung morphogenesis

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).

The development of many organs, including the lung, depends upon a process known as branching morphogenesis, in which a simple epithelial bud gives rise to a complex tree-like system of tubes specialized for the transport of gas or fluids. Previous studies on lung development have highlighted a role for fibroblast growth factors (FGFs), made by the mesodermal cells, in promoting the proliferation, budding, and chemotaxis of the epithelial endoderm. By using a three-dimensional culture system, evidence is provided of a novel role for Netrins in modulating the morphogenetic response of lung endoderm to exogenous FGFs. This effect involves inhibition of localized changes in cell shape and phosphorylation of the intracellular mitogen-activated protein kinase(s) (ERK1/2, for extracellular signal-regulated kinase-1 and -2), elicited by exogenous FGFs. The temporal and spatial expression of netrin 1, netrin 4, and Unc5b genes and the localization of Netrin-4 protein in vivo suggest a model in which Netrins in the basal lamina locally modulate and fine-tune the outgrowth and shape of emergent epithelial buds (Liu, 2004).

Taken together with the localization of Netrin RNA and protein during lung development, these in vitro results suggest the following model for the role of Netrins during early branching morphogenesis. A bud is initiated in response to a localized concentration of FGF10 in the mesoderm, probably stabilized by extracellular sulfated proteoglycans, which activate ERK1/2 as part of the downstream signaling cascade. In response to the differential distribution of MAP kinase activity, buds elongate and move away from the original stem. Netrin-1 and -4, secreted by the epithelial cells in the proximal lung and base and necks of buds, are deposited locally in the basal lamina underlying the cells. Here, they act, possibly through Unc5b, to inhibit ERK kinase and thus prevent ectopic budding and fine-tune the size and shape of emerging buds. In support of this hypothesis, activation of Unc5b during axonal guidance has been shown to decrease ERK1/2 activity. However, the involvement of other receptor(s) cannot be ruled out (Liu, 2004).

Netrins as dual function proteins, eliciting attractive and repulsive responses of axons

Extending axons are guided in part by diffusible chemoattractants that lure them to their targets and by diffusible chemorepellents that keep them away from nontarget regions. Floor plate cells at the ventral midline of the neural tube express a diffusible chemoattractant, Netrin-1, that attracts a group of ventrally directed axons. Floor plate cells also have a long-range repulsive effect trochlear motor axons, a set of axons that grow dorsally away from the floor plate in vivo. COS cells secreting recombinant Netrin-1 mimic this effect, suggesting that Netrin-1 is a bifunctional guidance cue that simultaneously attracts some axons to the floor plate while steering others away. This bifunctionality of Netrin-1 in vertebrates mirrors the dual actions of UNC-6, a C. elegans homolog of Netrin-1, involved in guiding both dorsal and ventral migrations in the nematode (Colamarino, 1995).

Netrin-1 can attract ventrally migrating axons and repel a subset of dorsally migrating axons in the spinal cord and rostral hindbrain in vitro. It is not know, however, whether Netrin-1 can act as a global cue to guide all circumferentially migrating axons. Netrin-1 can attract alar plate axons that cross the floor plate along its entire rostrocaudal axis. However, dorsally directed axons forming the posterior commissure are repelled at the floor plate by a netrin-independent mechanism. These results suggest that Netrin-1 functions as a global guidance cue for attraction to the midline. Moreover, floor plate­mediated chemorepulsion may also operate generally to direct dorsal migrations, but its molecular basis may involve both netrin-dependent and -independent mechanisms (Shirasaki, 1996).

Netrin-1 is known to function as a chemoattractant for several classes of developing axons and as a chemorepellent for other classes of axons, apparently dependent on the receptor type expressed by responsive cells. In culture, growth cones of embryonic Xenopus spinal neurons exhibit chemoattractive turning toward the source of netrin-1 but show chemorepulsive responses in the presence of a competitive analog of cAMP or an inhibitor of protein kinase A. Both attractive and repulsive responses are abolished by depleting extracellular calcium and by adding a blocking antibody against the netrin-1 receptor Deleted in Colorectal Cancer. Thus, nerve growth cones may respond to the same guidance cue with opposite turning behavior, dependent on other coincident signals that set the level of cytosolic cAMP (Ming, 1997).

Previous studies have shown that BDNF-induced turning of growth cones also exhibits either attraction or repulsion, depending on differences in cyclic-AMP-dependent activity in neurons. The currient studies suggest that Ca2+ signaling (acting downstream from the BDNF receptor known as TrkB) lies upstream from the cAMP-dependent step in the cascade of events, since attractive turning induced by a forskolin gradient is not affected by removal of extracellular CA2+ (Song, 1997). A netrin-induced Ca2+ influx may trigger a rise in cAMP through activation of Ca2+-dependent adenylate cyclases, thus creating a cAMP gradient within the growth cone, a condition known to result in an attractive response of Xenopus growth cones. It is possible that a gradient of cytosolic Ca2+ induced by netrin-1 is responsible for triggering the repulsive response of the growth cone, but the effect is normally overridden by the attractive response due to a cAMP gradient generated by the Ca2+ gradient. Inhibition of cAMP-dependent processes using competitive cAMP analogs may thus unmask the repulsive action of the cytosolic Ca2+ gradient. In principle, cAMP and the cAMP-dependent protein kinase pathway could regulate either the receptors for different diffusible guidance cues or the activity of the downstream effector molecules activated by these receptors. The cAMP-dependent protein kinase may thereby act as a gating mechanism, being differentially permissive for a receptor-induced signaling cascade, depending on the cascade's functional status. One group of potential downstream targets of PKA (see Drosophila PKA)is small GTP-binding proteins of the rho family, e.g., rhoA, rac1 and cdc42, which are known to mediate morphological changes by regulating the actin cytoskeleton and to play a role in growth cone turning. It is known that PKA can phosphorylate rhoA (see Drosophila Rho1), leading to the translocation of membrane-associated rhoA to the cytoplasm. thus providing an additional mechanism for its inactivation. The observation that lowering PKA activity converts netrin-1-induced attraction into repulsion suggests the intriguing possibility that activation of an UNC-5-like protein may down-regulate PKA. UNC-5 appears to be either a receptor or a component of a receptor complex, involved in netrin-mediated repulsion. For example, UNC-5 may inhibit adenylate cyclase activity or stimulate phosphodiesterase, which lowers the cAMP level and consequently PKA activity (Ming, 1997).

Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Deleted in Colorectal Cancer (DCC) family are implicated in attraction and those of the UNC5 family in repulsion, but genetic evidence also suggests involvement of the DCC protein UNC-40 in some cases of repulsion. To test whether these proteins form a receptor complex for repulsion, the attractive responses, mediated by DCC, of Xenopus spinal axons to netrin-1 were studied. Attraction is converted to repulsion by expression of UNC5 proteins in these cells. This repulsion requires DCC function; the UNC5 cytoplasmic domain is sufficient to effect the conversion, and repulsion can be initiated by netrin-1 binding to either UNC5 or DCC. The isolated cytoplasmic domains of DCC and UNC5 proteins interact directly, but this interaction is repressed in the context of the full-length proteins. Evidence is presented that netrin-1 triggers the formation of a receptor complex of DCC and UNC5 proteins and simultaneously derepresses the interaction between their cytoplasmic domains, thereby converting DCC-mediated attraction to UNC5/DCC-mediated repulsion (Hong, 1999).

To test whether the ectodomain of UNC5 proteins is required for repulsion, an examination was made of the effect of expressing a chimeric receptor in which the transmembrane and cytoplasmic domains of UNC5H2 (a human UNC5 homolog) were fused to the extracellular domain of DCC. Neurons expressing this DCC/UNC5H2 chimera show the same repulsive response to netrin-1 as do neurons expressing UNC5H2. To determine whether the transmembrane and cytoplasmic domains of UNC5H2 need to be fused to a netrin-binding ectodomain (as is the case for DCC), a chimeric receptor was examined in which the transmembrane and cytoplasmic domains of UNC5H2 were fused to the ectodomain of the NGF receptor TrkA, which does not bind netrin-1. Xenopus spinal neurons do not express TrkA endogenously and do not respond to an NGF gradient with either attraction or repulsion. Neurons expressing the TrkA/UNC5H2 chimera are repelled by netrin-1, a response that is blocked by the anti-DCC antibody; NGF has no effect on these neurons. These results suggested that the cytoplasmic domain of UNC5H2 might be sufficient for repulsion. This possibility was tested by generating a cDNA coding for the cytoplasmic domain of UNC5H2 preceded by a myristoylation sequence that targets cytoplasmic proteins to the inner leaflet of the plasma membrane. Neurons expressing this myristoylated UNC5H2 cytoplasmic domain construct exhibit marked repulsive responses to netrin-1. Thus, expression of the cytoplasmic domain of UNC5H2 is sufficient to convert netrin-mediated attraction to repulsion. It was then shown that netrin-1 triggers the formation of a heterodimeric or heteromultimeric complex involving DCC and UNC5H2 (Hong, 1999).

To further dissect the interaction between DCC and UNC5H2, attempts were made to identify regions in the DCC cytoplasmic domain required for the interaction. The first 46 amino acids are both necessary and sufficient for the interaction. Deletion of the juxtamembrane (JM) region (aas 1120-1148) does not abolish the interaction when performed in the context of the full-length cytoplasmic domain, and conversely, a construct comprising the JM domain alone does not suffice for the interaction. This shows that the JM domain is neither necessary nor sufficient for the interaction and identifies amino acids 1149-1166 as a key stretch required for the interaction. These 18 amino acids comprise the P1 domain, previously identified as a conserved domain among members of the DCC family. However, a construct comprising the P1 domain alone (aas 1149-1466) is not sufficient for the interaction. It is possible that the P1 domain does not fold properly in the absence of some adjacent sequences on either its amino- or carboxy-terminal ends; alternatively, the juxtamembrane region may be redundant with some other region of the cytoplasmic domain, with either one being sufficient but at least one being necessary (Hong, 1999).

Attempts were then made to identify the regions of UNC5 cytoplasmic domains required for DCC binding. Whereas a construct comprising UNC5H2 residues 707-946 is functional, a construct comprising residues 724-946 is not functional. Thus, residues 707-724 are required for binding the DCC cytoplasmic domain. These 18 residues are highly conserved among all previously described UNC5 proteins, and this domain has been termed the DB domain (since it is required for DCC binding). The DB domain is not the only domain required for repulsion, however. Deleting both the C-teminal Death Domain and 113 amino acids between the DB and the DD domains, but leaving the rest intact, including the DB domain, also results in a dominant-negative construct. Thus, sequences between the DD and DB domains are also important for repulsion, as could arise if these sequences are important for binding adaptor proteins. Deletion of the DB domain and all sequences carboxy terminal to it or deletion of all cytoplasmic domain sequences also results in the generation of dominant-negative constructs (Hong, 1999).

A paradox was raised by the finding that the isolated cytoplasmic domains of DCC and UNC5 proteins can interact, yet the full-length proteins do not coprecipitate in the absence of netrin-1. This raises the possibility that the interaction between cytoplasmic domains might be repressed in the context of the full-length proteins. To explore this possibility, a myristoylated cytoplasmic domain of one of the receptors (DCC or UNC5H2) was coexpressed with the full-length version of the other to see if they would coprecipitate. Full-length DCC coprecipitates with the myristoylated UNC5H2 cytoplasmic domain, but only in the presence of netrin-1. Similarly, only a low level of interaction of full-length UNC5H2 with the myristoylated DCC cytoplasmic domain is observed constitutively, and addition of netrin-1 dramatically increases the interaction. These results imply that in the absence of ligand, the UNC5H2 and DCC cytoplasmic domains are largely inaccessible to one another and that addition of netrin-1 causes some change in UNC5H2 and DCC that enables association of their cytoplasmic domains (Hong, 1999).

Why have a mechanism that switches from attraction to repulsion? The answer presumably lies in the fact that growth cones, as they navigate to their targets, change their responsiveness to guidance cues as they progress. Once a growth cone has reached a particular intermediate target, it must change its priorities in order to be able to move on to the next target. For example, commissural axons are initially attracted to the floor plate using netrin-1, but upon crossing the midline, they lose responsiveness to netrin-1. Since the axons continue to express DCC, the switching off must involve some other change. Another switch in growth cone sensitivity at the midline is the acquisition of Slit responsiveness by upregulation of expression of the Robo receptor in Drosophila. Although not yet demonstrated in vivo, it seems likely that there are circumstances where it is desirable not just to switch on or off responsiveness to a particular cue, but rather to convert the responsiveness from attraction to repulsion, to help move the growth cone along. The ability of one receptor to switch responses mediated by another receptor provides an economical means to achieve this end and avoid confusing the growth cone with simultaneous conflicting signals for attraction and repulsion (Hong, 1999 and references).

Inferior olivary neurons (ION) migrate circumferentially around the caudal rhombencephalon starting from the alar plate to locate ventrally close to the floor-plate, ipsilaterally to their site of proliferation. The floor-plate constitutes a source of diffusible factors. Among them, netrin-1 is implied in the survival and attraction of migrating ION in vivo and in vitro. A possible involvement of slit-1/2 during ION migration has been explored. slit-1 and slit-2 are coexpressed in the floor-plate of the rhombencephalon throughout ION development. robo-2, a slit receptor, is expressed in migrating ION, in particular when they reach the vicinity of the floor-plate, Using in vitro assays in collagen matrix, netrin-1 exerts an attractive effect on ION leading processes and nuclei, Slit has a weak repulsive effect on ION axon outgrowth and no effect on migration by itself, but when combined with netrin-1, it antagonizes part of or all of the effects of netrin-1 in a dose-dependent manner, inhibiting the attraction of axons and the migration of cell nuclei. The results indicate that slit silences the attractive effects of netrin-1 and participates in the correct ventral positioning of ION, stopping the migration when cell bodies reach the floor-plate (Causeret, 2002).

Netrins regulate migration of neurons

Netrin 1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. Tissue explants have been used to study the action of netrin 1 in the migration of several cerebellar and precerebellar cell progenitors. Netrin 1 exerts a strong chemoattractive effect on migrating neurons from the embryonic lower rhombic lip at E12-E14; these neurons give rise to precerebellar nuclei. Netrin 1 promotes the exit of postmitotic migrating neurons from the embryonic lower rhombic lip and upregulates the expression of adhesion protein TAG-1 in these neurons. In addition, in the presence of netrin 1, the migrating neurons are not isolated but are associated with thick fascicles of neurites, typical of the neurophilic way of migration. In contrast, the embryonic upper rhombic lip, which contains tangentially migrating granule cell progenitors, does not respond to netrin 1. Finally, in the postnatal cerebellum, netrin 1 repels both the parallel fibers and migrating granule cells growing out from explants taken from the external germinal layer. The developmental patterns of expression in vivo of netrin 1 and its receptors are consistent with the notion that netrin 1 secreted in the midline acts as a chemoattractive cue for precerebellar neurons migrating circumferentially along the extramural stream. Similarly, the pattern of expression in the postnatal cerebellum suggests that netrin 1 could regulate the tangential migration of postmitotic premigratory granule cells. Thus, molecular mechanisms considered as primarily involved in axonal guidance appear also to steer neuronal cell migration (Alcantara, 2000).

During their circumferential migration, the nuclei of inferior olivary neurons translocate within their axons until they reach the floor plate where they stop, although their axons have already crossed the midline to project to the contralateral cerebellum. Signals released by the floor plate, including netrin-1, have been implicated in promoting axonal growth and chemoattraction during axonal pathfinding in different midline crossing systems. Experiments that strongly suggest that the floor plate could also be involved in the migration of inferior olivary neurons. The expression pattern of netrin receptors DCC (for deleted in colorectal cancer), neogenin (a DCC-related protein), and members of the Unc5 family in wild-type mice is consistent with a possible role for netrins in directing the migration of precerebellar neurons from the rhombic lips. In mice deficient in netrin-1 production the number of inferior olivary neurons is markedly decreased. Some of these neurons are located ectopically along the migration stream, whereas the others are located medioventrally and form an atrophic inferior olivary complex: most subnuclei are missing. However, axons of the remaining olivary cell bodies located in the vicinity of the floor plate still succeed in entering their target, the cerebellum, but they establish an ipsilateral projection instead of the normal contralateral projection. In vitro experiments involving ablations of the midline show a fusion of the two olivary masses normally located on either side of the ventral midline, suggesting that the floor plate may function as a specific stop signal for inferior olivary neurons. These results establish a requirement for netrin-1 in the migration of inferior olivary neurons and suggest that it may function as a specific guidance cue for the initial steps of the migration from the rhombic lips and then later in the development of the normal crossed projection of the inferior olivary neurons. They also establish a requirement for netrin-1, either directly or indirectly, for the survival of inferior olivary neurons (Bloch-Gallego, 1999).

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).

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).

Motoneurons are essential for vascular pathfinding

The neural and vascular systems share common guidance cues that have direct and independent signaling effects on nerves and endothelial cells. This study shows that zebrafish Netrin 1a directs Dcc-mediated axon guidance of motoneurons and that this neural guidance function is essential for lymphangiogenesis. Specifically, Netrin 1a secreted by the muscle pioneers at the horizontal myoseptum (HMS) is required for the sprouting of dcc-expressing rostral primary motoneuron (RoP) axons and neighboring axons along the HMS, adjacent to the future trajectory of the parachordal chain (PAC). These axons are required for the formation of the PAC and, subsequently, the thoracic duct. The failure to form the PAC in netrin 1a or dcc morphants is phenocopied by laser ablation of motoneurons and is rescued both by cellular transplants and overexpression of dcc mRNA. These results provide a definitive example of the requirement of axons in endothelial guidance leading to the parallel patterning of nerves and vessels in vivo (Lim, 2011).

Regulation of Netrin expression

Netrins, a family of growth cone guidance molecules, are expressed both in the ventral neural tube and in subsets of mesodermal cells. In an effort to better understand the regulation of netrins, the expression of netrin-1a was examined in mutant cyclops, no tail, and floating head zebrafish embryos; such mutants show perterbances in their axial midline structures. Netrin-1a expression requires signals present in notochord and floor plate cells. In the myotome, but not the neural tube, netrin-1a expression requires sonic hedgehog. In embryos lacking sonic hedgehog (the sonic-you locus) netrin-1a expression is reduced or absent in the myotomes but present in the neural tube. Embryos lacking sonic hedgehog express tiggy-winkle hedgehog in the floor plate, suggesting that, in the neural tube, tiggy-winkle hedgehog can compensate for the lack of sonic hedgehog by inducing netrin-1a expression. Ectopic expression of sonic hedgehog, tiggy-winkle hedgehog, or echidna hedgehog induces ectopic netrin-1a expression in the neural tube, and ectopic expression of either sonic hedgehog or tiggy-winkle hedgehog (but not echidna hedgehog) induces ectopic netrin-1a expression in somites. These data demonstrate that in vertebrates netrin expression is regulated by Hedgehog signaling (Lauderdale, 1998).

Evidence for an interaction between mammalian Sina homologs (termed Siah proteins) and DCC (deleted in colorectal cancer) suggests that Sina/Siah functions to regulate protein concentrations via the ubiquitin-protease pathway, and draws into quiestion the classification of Sina as a transcripiton factor. DCC is postulated to function as transmembrane receptor for the axon and cell guidance factor netrin-1. DCC cytoplasmic domain binds to proteins encoded by mammalian homologs of the Drosophila seven in absentia (sina) gene, as well as to Drosophila Sina protein. Immunofluorescence studies suggest the Sina/Siah proteins localize predominantly in the cytoplasm and in association with DCC. DCC is found to be ubiquitinated (chemically modified to promote degradation); the Sina/Siah proteins regulate DCC's level of expression. Proteasome inhibitors block the effects of Sina/Siah on DCC, and the Sina/Siah proteins interact with ubiquitin-conjugating enzymes (Ubcs). A mutant Siah protein, lacking the amino-terminal Ubc-binding sequences, complexes with DCC, but does not degrade it. The in vivo interaction between Sina/Siah and DCC was confirmed through studies of transgenic Drosophila lines in which DCC and Sina were ectopically expressed in the eye. Ectopic expression of human DCC in the developing eye interfers with Sina function and cause R7 defects. Taken together, the data imply that the Sina/Siah proteins regulate DCC (and perhaps other proteins) via the ubiquitin-proteasome pathway (Hu, 1997).

The floor plate is an organizing center that controls neural differentiation and axonogenesis in the neural tube. The axon guidance molecule Netrin1 is expressed in the floor plate of zebrafish embryos. To elucidate the regulatory mechanisms underlying expression in the floor plate, the netrin1 locus was scanned for regulatory regions and an enhancer was identified that drives expression in the floor plate and hypochord of transgenic embryos. The expression of the transgene is ectopically activated by Cyclops (Nodal) signals but does not respond to Hedgehog signals. Other netrin1 enhancers, which have yet to be identified, control the observed ectopic expression of endogenous netrin1 in response to shh injection. The winged-helix transcription factor foxA2 (also HNF3ß, axial: Drosophila homolog Forkhead) is expressed in the notochord and floor plate. Knock-down of FoxA2 leads to loss of floor plate, while notochord and hypochord development is unaffected, suggesting a specific requirement of FoxA2 in the floor plate. The transgene is ectopically activated by FoxA2, and expression of FoxA2 leads to rescue of floor plate differentiation in mutant embryos that are deficient in Cyclops signalling. Zebrafish and mouse use different signalling systems to specify floor plate. The zebrafish netrin1 regulatory region also drives expression in the floor plate of mouse and chicken embryos. This suggests that components of the regulatory circuits controlling expression in the floor plate are conserved and that FoxA2 -- given its importance for midline development also in the mouse -- may be one such component (Rastegar, 2003).

FLRT3 is a Robo1-interacting protein that determines Netrin-1 attraction in developing axons

Guidance molecules are normally presented to cells in an overlapping fashion; however, little is known about how their signals are integrated to control the formation of neural circuits. In the thalamocortical system, the topographical sorting of distinct axonal subpopulations relies on the emergent cooperation between Slit1 and Netrin-1 guidance cues presented by intermediate cellular targets. However, the mechanism by which both cues interact to drive distinct axonal responses remains unknown. This study shows that the attractive response to the guidance cue Netrin-1 is controlled by Slit/Robo1 signaling and by FLRT3, a novel coreceptor for Robo1. While thalamic axons lacking FLRT3 are insensitive to Netrin-1, thalamic axons containing FLRT3 can modulate their Netrin-1 responsiveness in a context-dependent manner. In the presence of Slit1, both Robo1 and FLRT3 receptors are required to induce Netrin-1 attraction by the upregulation of surface DCC through the activation of protein kinase A. Finally, the absence of FLRT3 produces defects in axon guidance in vivo. These results highlight a novel mechanism by which interactions between limited numbers of axon guidance cues can multiply the responses in developing axons, as required for proper axonal tract formation in the mammalian brain (Leyva-Diaz, 2014).

Role of netrins in brain and spinal cord axonal guidance

Continued: Netrins Evolutionary homologs  part 3/3 | back to  part 1/3


Netrin-A and Netrin-B: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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