Netrin, Slit and Wnt receptors allow axons to choose the axis of migration

One of the challenges to understanding nervous system development has been to establish how a fairly limited number of axon guidance cues can set up the patterning of very complex nervous systems. Studies on organisms with relatively simple nervous systems such as Drosophila melanogaster and C. elegans have provided many insights into axon guidance mechanisms. The axons of many neurons migrate along both the dorsal-ventral (DV) and the anterior-posterior (AP) axes at different phases of development, and in addition they may also cross the midline. Axon migration in the dorsal-ventral (DV) direction is mainly controlled by Netrins with their receptors; UNC-40/DCC and UNC-5, and the Slits with their receptors; Robo/SAX-3. Axon guidance in the anterior-posterior (AP) axis is mainly controlled by Wnts with their receptors; the Frizzleds/Fz. An individual axon may be subjected to opposing attractive and repulsive forces coming from opposite sides in the same axis but there may also be opposing cues in the other axis of migration. All the information from the cues has to be integrated within the growth cone at the leading edge of the migrating axon to elicit a response. Recent studies have provided insight into how this is achieved.Evidence suggests that the axis of axon migration is determined by the manner in which Netrin, Slit and Wnt receptors are polarized (localized) within the neuron prior to axon outgrowth. The same molecules are involved in both axon outgrowth and axon guidance, for at least some neurons in C. elegans, whether the cue is the attractive cue UNC-6/Netrin working though UNC-40/DCC or the repulsive cue SLT-1/Slit working though the receptor SAX-3/Robo (Adler et al., 2006, Chang et al., 2006, Quinn et al., 2006, 2008). The molecules involved in cell signaling in this case are polarized within the cell body of the neuron before process outgrowth and direct the axon outgrowth. Expression of the Netrin receptor UNC-40/DCC or the Slit receptor SAX-3/Robo in axons that normally migrate in the AP direction causes neuronal polarity reversal in a Netrin and Slit independent manner (Levy-Strumpf and Culotti 2007, Watari-Goshima et al., 2007). Localization of the receptors in this case is caused by the kinesin-related VAB-8L which appears to govern the site of axon outgrowth in these neurons by causing receptor localization. Therefore, asymmetric localization of axon guidance receptors is followed by axon outgrowth in vivo using the receptor's normal cue, either attractive, repulsive or unknown cues.     

The Rac GTP exchange factor TIAM-1 acts with CDC-42 and the guidance receptor UNC-40/DCC in neuronal protrusion and axon guidance

The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions.     

Dcc regulates asymmetric outgrowth of forebrain neurons in zebrafish

The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates.     

Localization mechanisms of the axon guidance molecule UNC-6/Netrin and its receptors, UNC-5 and UNC-40, in Caenorhabditis elegans

Netrin is an evolutionarily conserved, secretory axon guidance molecule. Netrin's receptors, UNC-5 and UNC-40/DCC, are single trans-membrane proteins with immunoglobulin domains at their extra-cellular regions. Netrin is thought to provide its positional information by establishing a concentration gradient. UNC-5 and UNC-40 act at growth cones, which are specialized axonal tip structures that are generally located at a long distance from the neural cell body. Thus, the proper localization of both Netrin and its receptors is critical for their function. This review addresses the localization mechanisms of UNC-6/Netrin and its receptors in Caenorhabditis elegans, focusing on our recent reports. These findings include novel insights on cytoplasmic proteins that function upstream of the receptors.     

UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.     

The Tripartite Motif Protein MADD-2 Functions with the Receptor UNC-40 (DCC) in Netrin-Mediated Axon Attraction and Branching

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Highlights? UNC-6 (Netrin), its receptor UNC-40 (DCC), and the TRIM protein MADD-2 promote axon branching ? MADD-2 and UNC-40 proteins are localized to the affected axon branch ? MADD-2 stimulates axon attraction to Netrin by acting as an UNC-40 cofactor ? MADD-2 enables UNC-40 to recruit MIG-10, an actin-binding effector protein     

Glypican Is a Modulator of Netrin-Mediated Axon Guidance

Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor–expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.     

Migration of neuronal cells along the anterior-posterior body axis of C. elegans: Wnts are in control

Migrating neuronal cells are directed to their final positions by an array of guidance cues. It has been shown that guidance molecules such as UNC-6/Netrin and SLT-1/Slit play a major role in controlling cell and axon migrations along the dorsal-ventral body axis. Much less is known, however, about the mechanisms that mediate migration along the anterior-posterior (AP) body axis. Recent research in Caenorhabditis elegans has uncovered an important role of the Wnt family of signalling molecules in controlling AP-directed neuronal cell migration and polarity. A common theme that emerges from these studies is that multiple Wnt proteins function in parallel as instructive cues or permissive signals to control neuronal patterning along this major body axis.     

The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and,in parallel,Rac and UNC-115/AbLIM

Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, we generated a gain-of-function UNC-40 molecule, MYR::UNC-40. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. Our results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton.     

The netrin protein family

The name netrin is derived from the Sanskrit Netr, meaning ''guide''. Netrins are a family of extracellular proteins that direct cell and axon migration during embryogenesis. Three secreted netrins (netrins 1, 3 and 4), and two glycosylphosphatidylinositol (GPI)-anchored membrane proteins, netrins G1 and G2, have been identified in mammals. The secreted netrins are bifunctional, acting as attractants for some cell types and repellents for others. Receptors for the secreted netrins include the Deleted in Colorectal Cancer (DCC) family, the Down''s syndrome cell adhesion molecule (DSCAM), and the UNC-5 homolog family: Unc5A, B, C and D in mammals. Netrin Gs do not appear to interact with these receptors, but regulate synaptic interactions between neurons by binding to the transmembrane netrin G ligands NGL1 and 2. The chemotropic function of secreted netrins has been best characterized with regard to axon guidance during the development of the nervous system. Extending axons are tipped by a flattened, membranous structure called the growth cone. Multiple extracellular guidance cues direct axonal growth cones to their ultimate targets where synapses form. Such cues can be locally derived (short-range), or can be secreted diffusible cues that allow target cells to signal axons from a distance (long-range). The secreted netrins function as short-range and long-range guidance cues in different circumstances. In addition to directing cell migration, functional roles for netrins have been identified in the regulation of cell adhesion, the maturation of cell morphology, cell survival and tumorigenesis.     

UNC-6/netrin and its receptors UNC-5 and UNC-40/DCC modulate growth cone protrusion in vivo in C. elegans

The UNC-6/netrin guidance cue functions in axon guidance in vertebrates and invertebrates, mediating attraction via UNC-40/DCC family receptors and repulsion via by UNC-5 family receptors. The growth cone reads guidance cues and extends lamellipodia and filopodia, actin-based structures that sense the extracellular environment and power the forward motion of the growth cone. We show that UNC-6/netrin, UNC-5 and UNC-40/DCC modulated the extent of growth cone protrusion that correlated with attraction versus repulsion. Loss-of-function unc-5 mutants displayed increased protrusion in repelled growth cones, whereas loss-of-function unc-6 or unc-40 mutants caused decreased protrusion. In contrast to previous studies, our work suggests that the severe guidance defects in unc-5 mutants may be due to latent UNC-40 attractive signaling that steers the growth cone back towards the ventral source of UNC-6. UNC-6/Netrin signaling also controlled polarity of growth cone protrusion and F-actin accumulation that correlated with attraction versus repulsion. However, filopodial dynamics were affected independently of polarity of protrusion, indicating that the extent versus polarity of protrusion are at least in part separate mechanisms. In summary, we show here that growth cone guidance in response to UNC-6/netrin involves a combination of polarized growth cone protrusion as well as a balance between stimulation and inhibition of growth cone (e.g. filopodial) protrusion.     

Netrin instructs synaptic vesicle clustering through Rac GTPase, MIG-10, and the actin cytoskeleton

Netrin is a chemotrophic factor known to regulate a number of neurodevelopmental processes, including cell migration, axon guidance, and synaptogenesis. Although the role of Netrin in synaptogenesis is conserved throughout evolution, the mechanisms by which it instructs synapse assembly are not understood. Here we identify a mechanism by which the Netrin receptor UNC-40/DCC instructs synaptic vesicle clustering in vivo. UNC-40 localized to presynaptic regions in response to Netrin. We show that UNC-40 interacted with CED-5/DOCK180 and instructed CED-5 presynaptic localization. CED-5 in turn signaled through CED-10/Rac1 and MIG-10/Lamellipodin to organize the actin cytoskeleton in presynaptic regions. Localization of this signaling pathway to presynaptic regions was necessary for synaptic vesicle clustering during synapse assembly but not for the subcellular localization of active zone proteins. Thus, vesicle clustering and localization of active zone proteins are instructed by separate pathways downstream of Netrin. Our data indicate that signaling modules known to organize the actin cytoskeleton during guidance can be co-opted to instruct synaptic vesicle clustering.     

Role of netrin UNC-6 in patterning the longitudinal nerves of Caenorhabditis elegans

The nervous system of Caenorhabditis elegans comprises circumferential and longitudinal axon tracts. Netrin UNC-6 is required for the guidance of circumferential axon migrations and is expressed by ventral neuroglia and neurons in temporally and spatially regulated patterns. Migrating axons mediate the UNC-6 signal through the UNC-5 and UNC-40 receptors. It is thought that UNC-6 is secreted and becomes associated with basement membranes and cell surfaces to form gradients that direct circumferentially migrating axons toward or away from the ventral UNC-6 sources. Little is known about the effects of UNC-6 on longitudinally migrating axons. In unc-6, unc-5, and unc-40 null mutants, some longitudinal nerves are dorsally or ventrally misdirected. Furthermore, the organization of axons are disrupted within nerves. We show that cells ectopically expressing UNC-6 can redirect the migrations of some neighboring longitudinal axons, suggesting that the gradients postulated to direct circumferential migration also help specify the dorsoventral positions of these longitudinal nerves. We also manipulated the temporal and spatial expression pattern of UNC-6 by two different means. First, we removed the PVT midline neuron which expresses UNC-6 for a short time during axon outgrowths. Second, we expressed UNC-6 uniformly in the nervous system throughout development. The results suggest that changing UNC-6 expression patterns modify the distribution of the cue by providing new localized sources. This new guidance information is critical for organizing the axons of longitudinal nerves.     

The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans

UNC-51 and UNC-14 are required for the axon guidance of many neurons in Caenorhabditis elegans. UNC-51 is a serine/threonine kinase homologous to yeast Atg1, which is required for autophagy. The binding partner of UNC-51, UNC-14, contains a RUN domain that is predicted to play an important role in multiple Ras-like GTPase signaling pathways. How these molecules function in axon guidance is largely unknown. Here we observed that, in unc-51 and unc-14 mutants, UNC-5, the receptor for axon-guidance protein Netrin/UNC-6, abnormally localized in neuronal cell bodies. By contrast, the localization of many other proteins required for axon guidance was undisturbed. Moreover, UNC-5 localization was normal in animals with mutations in the genes for axon guidance proteins, several motor proteins, vesicle components and autophagy-related proteins. We also found that unc-5 and unc-6 interacted genetically with unc-51 and unc-14 to affect axon guidance, and that UNC-5 co-localized with UNC-51 and UNC-14 in neurons. These results suggest that UNC-51 and UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5, and that UNC-5 uses a unique mechanism for its localization; the functionality of UNC-5 is probably regulated by this localization.     

The dual role of the ligand UNC-6/Netrin in both axon guidance and synaptogenesis in C. elegans

The extracellular cue UNC-6/Netrin is a well-known axon guidance molecule and recently it has also been shown to be involved with localization of pre-synaptic complexes. Working through the UNC-40/DCC/Fra receptor, UNC-6/Netrin promotes the formation of pre-synaptic terminals between the pre-synaptic AIY interneuron and its post-synaptic partner, the RIA interneuron. In the DA9 motor neuron, UNC-6/Netrin has an alternate role promoting the exclusion of pre-synaptic components from the dendrite via its UNC-5-receptor. Surprisingly, the requirement for UNC-5 persists even after DA9 axon migration is complete, because synapses become mis-localized after it is depleted. This observation provides at least a partial explanation for the persistence of UNC-6/Netrin and UNC-5 in the adult nervous system. These activities parallel the previously known bi-functional axon guidance effects of UNC-6/Netrin, since it can attract cells and axons expressing UNC-40/DCC/Fra and repel those expressing UNC-5 alone or in combination with UNC-40. UNC-6/Netrin cooperates with the Wnt family members to exclude synapses from compartments within the DA9 axon, so that they only occur in regions free of the influence of both UNC-6/Netrin and the Wnts. Regulation of both axon guidance and synapse formation by axon guidance cues permits coordination in circuit assembly between pre- and post-synaptic cells.Key words: nervous system development, axon guidance, synaptogenesis, Netrin/UNC-6, UNC-40/DCC/Fra, UNC-5, LIN-44/Wnt, EGL-20/Wnt, LIN-17/FrizzledDuring development of the nervous system, differentiated pro-neural cells become polarized and send out processes from the cell body that later become dendrites and axons. The pro-neural cells themselves and later their axons, often migrate long distances to their eventual targets using guidance cues.¹ Once the destination is reached, the axon usually selects among several available targets and establishes synapses with the correct post-synaptic partner. The synapse is the site of communication between the pre- and post-synaptic cell and many of the molecules involved in synapse formation are known.² Development of both the pre- and post-synaptic cells needs to be orchestrated to ensure that they are available to form synapses with each other and this process can be directed by guidepost cells. In organisms such as vertebrates, the guidepost cells are often glia, which guide two neurons to ensure that the correct synapse is formed.³ A case is presented here where glial cells secrete a cue to control the localization of pre-synaptic complexes in C. elegans. One notable aspect of this process is that the glial-secreted cue is a well-known axon guidance molecule, namely Netrin/UNC-6, but here it plays an additional and surprising role in selecting the site for the construction of a pre-synaptic complex.⁴UNC-6/Netrin, is a well-known bi-functional axon guidance cue that can attract some axons and repel others. It is a laminin-related molecule, originally isolated from C. elegans, with homologues in higher organisms.^5–7 UNC-6 has two receptors in C. elegans: UNC-40 and UNC-5.^8,9 Both have homologues in higher organisms: UNC-40/DCC/Fra (Deleted in Colorectal Cancer in vertebrates/Frazzled in Drosophila) and UNC-5/Unc5.^6,7,10 UNC-6/Netrin is expressed by cells mostly located in the ventral regions of C. elegans where it attracts many cells and axons expressing the receptor UNC-40.^8,11 Conversely, UNC-6/Netrin repulses axons and cells expressing UNC-5 alone, or in combination with UNC-40.^9,12 One aspect of this developmental process, however, that has been somewhat puzzling has been the observation that expression of both UNC-6 and UNC-5 persist into adulthood.^11,13 A partial explanation for the persistence of UNC-6 and UNC-5 is provided by Poon et al.¹⁴ who found that UNC-5 is required for both the initial polarized localization and maintenance of the pre-synaptic complexes in the DA9 motor neuron axon in C. elegans. The mechanisms used by the proteins in these new roles have not been established, but the localization of both UNC-5 and UNC-40 in the axons is controlled by their normal ligand, UNC-6/Netrin.UNC-40/DCC/Fra plays two independent roles in establishing the connection between the pre-synaptic AIY amphid inter-neuron and its post-synaptic partner, the RIA inter-neuron in the nerve ring of C. elegans, since it is involved in both axon guidance of RIA and synapse localization in AIY.⁴ The two neurons are located in the head region, close to the nerve ring and can be visualized using cell-specific markers (Fig. 1). UNC-6/Netrin plays a conventional guidance role in directing migration of the post-synaptic inter-neuron RIA, since the ventral trajectory of its axon is altered in the absence of UNC-40. The axon of the AIY inter-neuron migrates anteriorly from its cell body, then dorsally and synapses onto three other interneurons: RIA, AIZ and RIB. The AIY axon usually migrates normally without the UNC-40 receptor, which is not surprising as it does not make a ventral migration.Open in a separate windowFigure 1A schematic of the region close to the head of C. elegans is shown where the synapses between the pre-synaptic AIY interneuron (red) and the post-synaptic RIA interneuron (blue) occur. The pre-synaptic regions are shown as black dots. The glial cell (sheath cell) that is the source of UNC-6/Netrin is shown in green. The insert below the schematic shows the regions of the AIY axon divided into zones 1, 2 and 3 that are discussed in the text. Redrawn from Colon-Ramos et al.⁴ and WormAtlas²⁴ (with permission).Pre-synaptic complexes in AIY were detected by expression of fluorescently-tagged synaptic vesicle associated RAB-3.⁴ They were found mainly in the “elbow” region (Fig. 1, zone 2) and about eight more complex-containing areas were found in the region most distant from the cell body within the nerve ring in wild-type animals (Fig. 1, zone 3). A hypomorphic allele of unc-40, wy81, was found in a genetic screen for mutants exhibiting altered localization of pre-synaptic complexes. In the absence of fully functional UNC-40, the pre-synaptic markers were not observed in zone 2, but were present in the more distal region, zone 3. In addition, the pre-synaptic region (zone 2) of AIY did not have an expanded diameter in the manner characteristic of this particular synapse, as detected by electron microscopy. The synapses between AIY and RIA in the absence of UNC-40 were abnormal in several other respects. There was a severe reduction in the active zone proteins ELKS-1/ERC/CAST and SYD-2/α Liprin, suggesting a defect in the pre-synaptic differentiation of AIY. Pre-synaptic defects in AIY caused by absence of UNC-40 could only be rescued by cell-autonomous expression of the receptor.Localization of UNC-40/DCC/Fra in the AIY interneuron is controlled by UNC-6/Netrin emanating from a pair of glial cells called the ventral cephalic sheath cells (VCSCs), which are similar to astrocytes.⁴ Wadsworth et al.¹¹ have previously shown that the VCSCs at the nerve ring express UNC-6/Netrin during neurulation. Colon-Ramos et al.⁴ found the VCSCs project deeply invaginated end-feet that form membranous lamellae, thereby ensheathing the region of AIY-RIA synapses. There is thus a very tight association between the glial cell and the two interneurons in the region of the synapses in zone 2. UNC-40 localizes to the pre-synaptic zones 2 and 3. In the absence of UNC-6, UNC-40 is more diffuse and is present along the entire neuron.The anatomical relationship between the sheath cells and synapses is instructive in mediating AIY:RIA innervations. Sheath cell morphology was altered by the absence of UNC-34/Enabled such that the glial end-feet now migrated further posteriorly to include zone 1.⁴ There was a concomitant appearance of both ectopic pre-synaptic complexes and UNC-40 localization in zone 1 due to an alteration in the source of UNC-6. In UNC-34/Enabled mutants, the trajectory of the RIA interneuron was also altered, such that it had migrated towards the new site of the synapses. Therefore, in this study UNC-40 is playing two independent roles, one in axon path-finding of the RIA axon and a second in positioning the synapses in the AIY pre-synaptic cell. Both of these activities are under the control of UNC-40''s normal ligand, UNC-6/Netrin, that is expressed by glial cells that ensheath the region of the synapses. Regulation of both processes by a single molecule allows co-ordination in circuit assembly.In contrast to the work described above, UNC-6/Netrin and its receptor UNC-5 have recently been reported to exclude synaptic vesicle and active zone components from the dendrite of the DA9 motor neuron in C. elegans (Fig. 2).¹⁴ The DA9 neuron can be divided into five zones (see insert in Fig. 2). It synapses en passant with the VD/DD motor neurons and the body wall muscles along the dorsal cord. In wild-type animals, the synapses of the DA9 neuron were detected using a fluorescently labelled RAB-3, a synaptic vesicle associated protein, and they were found mainly in the region most distant from the cell body (zone 5 of DA9 in Fig. 2). Synapses were entirely excluded from the dendrite (zone 1) and the remainder of the axon (zones 2, 3 and 4) in wild-type animals. Interestingly, UNC-5 had a somewhat complementary distribution to the pre-synaptic complexes since it was expressed mainly in the dendrite of DA9 and the ventral region of the axon (zones 1 and 2 in Fig. 2), suggesting that the presence of UNC-5 can exclude synapses.Open in a separate windowFigure 2The tail region of C. elegans is shown with the DA9 motor neuron. The DA9 axon is in blue and the dendrite in orange. The pre-synaptic structures are in black. The sources of the LIN-44/Wnt, EGL-20/Wnt and UNC-6/Netrin are shown. The insert below shows the various zones of the DA9 neuron described in the text. Redrawn from Poon et al.¹⁴ and WormAtlas²⁴ (with permission).The correct location of the pre-synaptic complexes in DA9 is dependent on both the ligand UNC-6/Netrin and the receptor UNC-5. RAB-3 was found ectopically in the dendrites of either unc-6(ev400), or unc-5(e53), both considered to be null mutants.^5,9,15 Other pre-synaptic vesicle proteins tested, including SNB-1/Synaptobrevin and SNG-1/Synaptogyrin, as well as CCB-1, an L-type voltage-gated calcium channel β subunit and the active zone protein SYD-2/α-liprin, were also mis-localized in the dendrite in the absence of either UNC-6 or UNC-5.¹⁴ UNC-5 functions cell autonomously for the exclusion of pre-synaptic complexes. Interestingly, deletion of either one of the immunoglobulin domains or one of the thrombospondin domains from the extracellular regions of an UNC-5 protein was previously shown to alter the sub-cellular localization of the protein so that is was more localized to the cell body than wild-type UNC-5.¹⁵ This suggests that the extracellular region of UNC-5 is responsible for its localization in the neuron and it would be interesting to see if synapse localization is affected in the absence of the extracellular domains.In addition to its roles in axon guidance and localizing pre-synaptic complexes, an ongoing supply of UNC-5 is required in DA9 to maintain the position of the synapses. This has been demonstrated by the use of a temperature-sensitive silencing intron construct that allowed UNC-5 expression at a permissive temperature of 25°C but not at the restrictive temperature of 16°C.¹⁴ Temperature shift experiments from the permissive temperature to the restrictive temperature at the L4 stage, after the axon was already fully developed, caused synapse mis-localization similar to that observed in the absence of UNC-5. Initial synapse mislocalization was irreversible as the reverse shift from the restrictive to the permissive temperature at L4 failed to rescue the defect. The exclusion of pre-synaptic complexes from all the compartments of DA9 except for the most distal regions (zones 4 and 5) was not simply a consequence of axon misguidance, since axons that were not misguided due to the absence of UNC-5, still exhibited altered RAB-3 localization. Additionally, animals lacking another axon guidance cue, UNC-129/TGFβ exhibited misguidance of DA9 but not mis-localization of pre-synaptic components. Dendritic localization of the pre-synaptic proteins was also not just a reversal of the axons and dendrites in DA9, since four different dendritic proteins were correctly localized in the absence of both UNC-6/Netrin and UNC-5. The need for an ongoing supply of UNC-5 accounts for the observation that both UNC-5 and UNC-6 persist into adulthood, long after axon guidance or synapse formation in worms.^11,13 The finding that UNC-5 must be present on an ongoing basis to maintain localization of pre-synaptic complexes suggests a novel role for UNC-5 in maintaining the polarized localization of the pre-synaptic complexes in a manner independent of axon guidance or initial synaptic polarization. This is an intriguing finding and one that deserves investigation for other neurons and axon guidance molecules.Two Wnt cues also control synapse localization in the DA9 neuron but in different regions than UNC-6/Netrin.¹⁶ LIN-44/Wnt emanating from the tail region (light pink patch in Fig. 2) causes exclusion of synapses from the more posterior section of the DA9 axon located in the dorsal cord (zone 4 in Fig. 2). A second Wnt, EGL-20 is also produced by tail cells (deeper pink region in Fig. 2), and it excludes synapses from the region of the axon in the ventral cord (zone 2 in Fig. 2). Both Wnts cooperate to exclude synapses from zone 3. There is a strict correlation between the presence of the LIN-44/Wnt receptor, LIN-17/Fz, in zones 2, 3 and 4 and the absence of synapses in these regions. LIN-17/Fz is required cell-autonomously in DA9 to rescue synaptic localization defects. In the absence of LIN-44/Wnt, both the receptor LIN-17/Fz and the pre-synaptic complexes were mis-localized since they were now found in both zone 4 and 5 of the axon. Therefore, LIN-44/Wnt is instructive in regulating the location of the synapses in the axon of the DA9 neuron. Both LIN-44/Wnt and EGL-20/Wnt normally work cooperatively to exclude synapses, since animals lacking both had synapses in zones 3, 4 and 5 of DA9.UNC-6/Netrin cooperates with the Wnt family members to exclude synapses from particular regions of the DA9 axon and only allow them to occur in regions free of the influence of both UNC-6/Netrin and the Wnts. Ectopic expression of UNC-6/Netrin and LIN-44/Wnt in various cells and genetic backgrounds was used to show that UNC-6/Netrin and LIN-44/Wnt could function interchangeably in excluding synapses in the DA9 neuron.¹⁴ Ectopic expression of UNC-6 in a posterior to anterior gradient close to DA9 caused RAB-3 to be localized more posteriorly in zone 5, rather than in both zone 4 and 5. The mis-localization was UNC-5 dependent and was seen regardless of whether or not DA9 was misguided. Ectopic UNC-6 could also rescue mis-localization defects in the absence of either LIN-44/Wnt or its receptor LIN-17/Frizzled. These observations suggest that UNC-6/Netrin and LIN-44/Wnt both exclude synapses and can function together to control both axon guidance and pre-synaptic complex localization. Therefore, EGL-20/Wnt and LIN-44/Wnt work cooperatively with the UNC-6/Netrin ligand to inhibit the assembly of pre-synaptic complexes from inappropriate neuronal compartments. Synapses are excluded from the dendrite (zone 1) by UNC-6/Netrin, the region of the axon proximal to the cell body (zone 2) by EGL-20/Wnt, the commissures (zone 3) by EGL-44/Wnt and EGL-20/Wnt, and the distal portion of the axon (zone 4) by LIN-44/Wnt.¹⁴It remains to be seen whether UNC-6/Netrin and its receptors are usually involved in synapse localization in C. elegans itself and in other organisms, beyond the highly specific cell contexts discussed. The involvement of these molecules in both axon guidance and synaptogenesis is likely to be a general phenomenon, as the Netrins are expressed in the adult nervous systems of vertebrates including neurons and oligodendrocytes in the adult rat.¹⁷ DCC is expressed in the adult rat forebrain.¹⁸ UNC-5 is expressed in the heart and brain of adult vertebrates.¹⁹ Ephrins have also been shown to be involved in both axon guidance and synapse formation.²⁰ Wnts have been found to play roles in regulating neuronal connectivity by controlling axon pathfinding, axon remodelling, dendrite morphogenesis and synapse formation in invertebrates and mammals.²¹ Recently, it was shown that pro- and anti-synaptogenic effects of Wnt proteins are associated with the activation of canonical and non-canonical Wnt signaling pathways in Drosophila and mouse.^22,23 It is anticipated that many more instances of axon guidance molecules involved in synapse formation will be described. For instance, in the case of the synapse between the AIY and the RIA interneurons just discussed, AIY also synapses with two other interneurons, the AIZ and RIB but these synapses are not altered significantly in the absence of UNC-40/DCC/Fra. Presumably, these synapses require other molecules to guide synapse formation. Although the two receptors UNC-40 and UNC-5 are functioning with their normal ligand UNC-6/Netrin, it is not clear whether the remainder of the signaling pathways are conserved, and this question will be an interesting topic for future work on synapse formation.     

Identification and characterization of roundabout orthologs in zebrafish

The Roundabout (Robo) family of receptors and their extracellular ligands, the Slit protein family, play important roles in repulsive axon guidance. First identified in Drosophila, Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin (Ig) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain, a transmembrane domain, and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling (Cell 92 (1998) 205; Cell 101 (2000) 703). Robo family members have now been identified in C. elegans, Xenopus, rat, mouse, and human (Cell 92 (1998) 205; Cell 92 (1998) 217; Cell 96 (1999) 807; Dev. Biol. 207 (1999) 62). Furthermore, multiple robo genes have been described in Drosophila, rat, mouse and humans, raising the possibility of potential redundancy and diversity in robo gene function. As a first step in elucidating the role of Robo receptors during vertebrate development, we identified and characterized two Robo family members from zebrafish. We named these zebrafish genes robo1 and robo3, reflecting their amino acid sequence similarity to other vertebrate robo genes. Both genes are dynamically expressed in the developing nervous system in distinct patterns. robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina. robo1 nervous system expression appears later in development and is more restricted. Moreover, both genes are expressed in non-neuronal tissues consistent with additional roles for these genes during development.     

Extracellular Matrix Regulates UNC-6 (Netrin) Axon Guidance by Controlling the Direction of Intracellular UNC-40 (DCC) Outgrowth Activity

How extracellular molecules influence the direction of axon guidance is poorly understood. The HSN axon of Caenorhabditis elegans is guided towards a ventral source of secreted UNC-6 (netrin). The axon’s outgrowth response to UNC-6 is mediated by the UNC-40 (DCC) receptor. We have proposed that in response to the UNC-6 molecule the direction of UNC-40-mediated axon outgrowth is stochastically determined. The direction of guidance is controlled by asymmetric cues, including the gradient of UNC-6, that regulate the probability that UNC-40-mediated axon outgrowth is directed on average, over time, in a specific direction. Here we provide genetic evidence that a specialized extracellular matrix, which lies ventral to the HSN cell body, regulates the probability that UNC-40-mediated axon outgrowth will be directed ventrally towards the matrix. We show that mutations that disrupt the function of proteins associated with this matrix, UNC-52 (perlecan), UNC-112 (kindlin), VAB-19 (Kank), and UNC-97 (PINCH), decrease the probability of UNC-40-mediated axon outgrowth in the ventral direction, while increasing the probability of outgrowth in the anterior and posterior directions. Other results suggest that INA-1 (α integrin) and MIG-15 (NIK kinase) signaling mediate the response in HSN. Although the AVM axon also migrates through this matrix, the mutations have little effect on the direction of AVM axon outgrowth, indicating that responses to the matrix are cell-specific. Together, these results suggest that an extracellular matrix can regulate the direction of UNC-6 guidance by increasing the probability that UNC-40-mediated axon outgrowth activity will be oriented in a specific direction.     

Localization of the netrin guidance receptor, DCC, in the developing peripheral and enteric nervous systems

Over recent years the secreted guidance cue, netrin-1, and its receptor, DCC, have been shown to be an essential guidance system driving axon pathfinding within the developing vertebrate central nervous system (CNS). Mice lacking DCC exhibit severe defects in commissural axon extension towards the floor plate demonstrating that the DCC-netrin guidance system is largely responsible for directing axonal projections toward the ventral midline in the developing spinal cord (Fazeli et al., Nature 386 (1997) 796). In addition, these mutants lack several major commissures within the forebrain, including the corpus callosum and the hippocampal commissure. In contrast to the CNS, the role of the DCC guidance receptor in the development of the mammalian peripheral and enteric nervous systems (PNS and ENS) has not been investigated. Here we demonstrate using immunohistochemical analysis that the DCC receptor is present in the developing mouse PNS where it is found on spinal, segmental, and sciatic nerves, and in developing sensory ganglia and their associated axonal projections. In addition, DCC is present in the ENS throughout the early developmental phase.     

Short- and long-range repulsion by the Drosophila Unc5 netrin receptor.

Netrins are bifunctional guidance molecules, attracting some axons and repelling others. They act through receptors of the DCC and UNC5 families. DCC receptors have been implicated in both attraction and repulsion by Netrins. UNC5 receptors are required only for repulsion. In Drosophila, Netrins are expressed by midline cells of the CNS and by specific muscles in the periphery. They attract commissural and motor axons expressing the DCC family receptor Frazzled. Here we report the identification of the Drosophila Unc5 receptor, and show that it is a repulsive Netrin receptor likely to contribute to motor axon guidance. Ectopic expression of Unc5 on CNS axons can elicit either short- or long-range repulsion from the midline. Both short- and long-range repulsion require Netrin function, but only long-range repulsion requires Frazzled.