Cell migration and axon growth cone guidance in Caenorhabditis elegans.

In the past year, several new components involved in cell migration and axon guidance have been identified by genetic analysis in Caenorhabditis elegans, taking us a step closer to being able to trace the pathways which mediate these processes. The completion of the C. elegans genome sequencing project has provided us with the knowledge of the full spectrum of genes that might be involved in cell migration and axon guidance, and can facilitate the analysis of components that have been shown to be important for these processes in other systems.     

Three C. elegans Rac proteins and several alternative Rac regulators control axon guidance, cell migration and apoptotic cell phagocytosis.

The Caenorhabditis elegans genome contains three rac-like genes, ced-10, mig-2, and rac-2. We report that ced-10, mig-2 and rac-2 act redundantly in axon pathfinding: inactivating one gene had little effect, but inactivating two or more genes perturbed both axon outgrowth and guidance. mig-2 and ced-10 also have redundant functions in some cell migrations. By contrast, ced-10 is uniquely required for cell-corpse phagocytosis, and mig-2 and rac-2 have only subtle roles in this process. Rac activators are also used differentially. The UNC-73 Trio Rac GTP exchange factor affected all Rac pathways in axon pathfinding and cell migration but did not affect cell-corpse phagocytosis. CED-5 DOCK180, which acts with CED-10 Rac in cell-corpse phagocytosis, acted with MIG-2 but not CED-10 in axon pathfinding. Thus, distinct regulatory proteins modulate Rac activation and function in different developmental processes.     

UNC-71, a disintegrin and metalloprotease (ADAM) protein,regulates motor axon guidance and sex myoblast migration in C. elegans

The migration of cells and growth cones is a process that is guided by extracellular cues and requires the controlled remodeling of the extracellular matrix along the migratory path. The ADAM proteins are important regulators of cellular adhesion and recognition because they can combine regulated proteolysis with modulation of cell adhesion. We report that the C. elegans gene unc-71 encodes a unique ADAM with an inactive metalloprotease domain. Loss-of-function mutations in unc-71 cause distinct defects in motor axon guidance and sex myoblast migration. Many unc-71 mutations affect the disintegrin and the cysteine-rich domains, supporting a major function of unc-71 in cell adhesion. UNC-71 appears to be expressed in a selected set of cells. Genetic mosaic analysis and tissue-specific expression studies indicate that unc-71 acts in a cell non-autonomous manner for both motor axon guidance and sex myoblast migration. Finally, double mutant analysis of unc-71 with other axon guidance signaling molecules suggests that UNC-71 probably functions in a combinatorial manner with integrins and UNC-6/netrin to provide distinct axon guidance cues at specific choice points for motoneurons.     

A genetic analysis of axon guidance in the C elegans pharynx

We wish to understand how the trajectories of the twenty pharyngeal neurons of C. elegans are established. In this study we focused on the two bilateral M2 pharyngeal motorneurons, which each have their cell body located in the posterior bulb and send one axon through the isthmus and into the metacorpus. We used a GFP reporter to visualize these neurons in cell-autonomous and cell-non-autonomous axon guidance mutant backgrounds, as well as other mutant classes. Our main findings are: 1). Mutants with impaired growth cone functions, such as unc-6, unc-51, unc-73 and sax-3, often exhibit abnormal terminations and inappropriate trajectories at the distal ends of the M2 axons, i.e. within the metacorpus; and 2). Growth cone function mutants never exhibit abnormalities in the proximal part of the M2 neuron trajectories, i.e. between the cell body and the metacorpus. Our results suggest that the proximal and distal trajectories are established using distinct mechanisms, including a growth cone-independent process to establish the proximal trajectory. We isolated five novel mutants in a screen for worms exhibiting abnormal morphology of the M2 neurons. These mutants define a new gene class designated mnm (M neuron morphology abnormal).     

ENU-3 is a novel motor axon outgrowth and guidance protein in C. elegans

During the development of the nervous system, the migration of many cells and axons is guided by extracellular molecules. These molecules bind to receptors at the tips of the growth cones of migrating axons and trigger intracellular signaling to steer the axons along the correct trajectories. We have identified a novel mutant, enu-3 (enhancer of Unc), that enhances the motor neuron axon outgrowth defects observed in strains of Caenorhabditis elegans that lack either the UNC-5 receptor or its ligand UNC-6/Netrin. Specifically, the double-mutant strains have enhanced axonal outgrowth defects mainly in DB4, DB5 and DB6 motor neurons. enu-3 single mutants have weak motor neuron axon migration defects. Both outgrowth defects of double mutants and axon migration defects of enu-3 mutants were rescued by expression of the H04D03.1 gene product. ENU-3/H04D03.1 encodes a novel predicted putative trans-membrane protein of 204 amino acids. It is a member of a family of highly homologous proteins of previously unknown function in the C. elegans genome. ENU-3 is expressed in the PVT interneuron and is weakly expressed in many cell bodies along the ventral cord, including those of the DA and DB motor neurons. We conclude that ENU-3 is a novel C. elegans protein that affects both motor axon outgrowth and guidance.     

Characterization of loss-of-function and gain-of-function Eph receptor tyrosine kinase signaling in C. elegans axon targeting and cell migration

To understand how our brains function, it is necessary to know how neurons position themselves and target their axons and dendrites to their correct locations. Several evolutionarily conserved axon guidance molecules have been shown to help navigate axons to their correct target site. The Caenorhabditis elegans Eph receptor tyrosine kinase (RTK), VAB-1, has roles in early neuroblast and epidermal cell movements, but its roles in axon guidance are not well understood. Here, we report that mutations that disrupt the VAB-1 Eph receptor tyrosine kinase cause incompletely penetrant defects in axonal targeting and neuronal cell body positioning. The predominant axonal defect in vab-1 mutant animals was an overextension axon phenotype. Interestingly, constitutively active VAB-1 tyrosine kinase signaling caused a lack of axon outgrowth or an early termination phenotype, opposite to the loss-of-function phenotype. The combination of loss-of-function and gain-of-function analyses suggests that the VAB-1 Eph RTK is required for targeting or limiting axons and neuronal cells to specific regions, perhaps by transducing a repellent or stop cue.     

The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.     

Distinct rac activation pathways control Caenorhabditis elegans cell migration and axon outgrowth

Rac GTPases act as molecular switch in various morphogenic events. However, the regulation of their activities during the development of multicellular organisms is not well understood. Caenorhabditis elegans rac genes ced-10 and mig-2 have been shown to act redundantly to control P cell migration and the axon outgrowth of D type motoneurons. We showed that ced-10 and mig-2 also control amphid axon outgrowth and amphid dendrite fasciculation in a redundant fashion. Our biochemical and genetic data indicate that unc-73, which encodes a protein related to Trio-like guanine nucleotide exchange factor, acts as a direct activator of ced-10 and mig-2 during P cell migration and axon outgrowth of D type motoneurons and amphid sensory neurons. Furthermore, rac regulators ced-2/crkII and ced-5/dock180 function genetically upstream of ced-10 and mig-2 during axon outgrowth of D type motoneurons and act upstream of mig-2 but not ced-10 during P cell migration. However, neither ced-2/crkII nor ced-5/dock180 is involved in amphid axon outgrowth. Therefore, distinct rac regulators control ced-10 and mig-2 differentially in various cellular processes.     

Signaling in glial development: differentiation migration and axon guidance.

Glial cells have diverse functions that are necessary for the proper development and function of complex nervous systems. During development, a variety of reciprocal signaling interactions between glia and neurons dictate all parts of nervous system development. Glia may provide attractive, repulsive, or contact-mediated cues to steer neuronal growth cones and ensure that neurons find their appropriate synaptic targets. In fact, both neurons and glia may act as migrational substrates for one another at different times during development. Also, the exchange of trophic signals between glia and neurons is essential for the proper bundling, fasciculation, and ensheathement of axons as well as the differentiation and survival of both cell types. The growing number of links between glial malfunction and human disease has generated great interest in glial biology. Because of its relative simplicity and the many molecular genetic tools available, Drosophila is an excellent model organism for studying glial development. This review will outline the roles of glia and their interactions with neurons in the embryonic nervous system of the fly.     

Molecular signatures of cell migration in C. elegans Q neuroblasts

Metazoan cell movement has been studied extensively in vitro, but cell migration in living animals is much less well understood. In this report, we have studied the Caenorhabditis elegans Q neuroblast lineage during larval development, developing live animal imaging methods for following neuroblast migration with single cell resolution. We find that each of the Q descendants migrates at different speeds and for distinct distances. By quantitative green fluorescent protein imaging, we find that Q descendants that migrate faster and longer than their sisters up-regulate protein levels of MIG-2, a Rho family guanosine triphosphatase, and/or down-regulate INA-1, an integrin α subunit, during migration. We also show that Q neuroblasts bearing mutations in either MIG-2 or INA-1 migrate at reduced speeds. The migration defect of the mig-2 mutants, but not ina-1, appears to result from a lack of persistent polarization in the direction of cell migration. Thus, MIG-2 and INA-1 function distinctly to control Q neuroblast migration in living C. elegans.     

UNC-119 suppresses axon branching in C. elegans

The architecture of the differentiated nervous system is stable but the molecular mechanisms that are required for stabilization are unknown. We characterized the gene unc-119 in the nematode Caenorhabditis elegans and demonstrate that it is required to stabilize the differentiated structure of the nervous system. In unc-119 mutants, motor neuron commissures are excessively branched in adults. However, live imaging demonstrated that growth cone behavior during extension was fairly normal with the exception that the overall rate of migration was reduced. Later, after development was complete, secondary growth cones sprouted from existing motor neuron axons and cell bodies. These new growth cones extended supernumerary branches to the dorsal nerve cord at the same time the previously formed axons retracted. These defects could be suppressed by expressing the UNC-119 protein after embryonic development; thus demonstrating that UNC-119 is required for the maintenance of the nervous system architecture. Finally, UNC-119 is located in neuron cell bodies and axons and acts cell-autonomously to inhibit axon branching.     

Autophagy regulates ageing in C. elegans

The role of autophagy in ageing regulation has been suggested based on studies in C. elegans, in which knockdown of the expression of bec-1 (ortholog of the yeast and mammalian autophagy genes ATG6/VPS30 and beclin 1, respectively) shortens lifespan of the daf-2(e1370) mutant C. elegans. However, Beclin1/ATG6 is also known to be involved in other cellular functions in addition to autophagy. In the current study, we knocked down two other autophagy genes, atg-7 and atg-12, in C. elegans using RNAi. We showed that RNAi shortened the lifespan of both wild type and daf-2 mutant C. elegans, providing strong support for a role of autophagy in ageing regulation.     

The secreted cell signal Folded Gastrulation regulates glial morphogenesis and axon guidance in Drosophila

During gastrulation in Drosophila, ventral cells change shape, undergoing synchronous apical constriction, to create the ventral furrow (VF). This process is affected in mutant embryos lacking zygotic function of the folded gastrulation (fog) gene, which encodes a putative secreted protein. Fog is an essential autocrine signal that induces cytoskeletal changes in invaginating VF cells. Here we show that Fog is also required for nervous system development. Fog is expressed by longitudinal glia in the central nervous system (CNS), and reducing its expression in glia causes defects in process extension and axon ensheathment. Glial Fog overexpression produces a disorganized glial lattice. Fog has a distinct set of functions in CNS neurons. Our data show that reduction or overexpression of Fog in these neurons produces axon guidance phenotypes. Interestingly, these phenotypes closely resemble those seen in embryos with altered expression of the receptor tyrosine phosphatase PTP52F. We conducted epistasis experiments to define the genetic relationships between Fog and PTP52F, and the results suggest that PTP52F is a downstream component of the Fog signaling pathway in CNS neurons. We also found that Ptp52F mutants have early VF phenotypes like those seen in fog mutants.     

Novel roles of the chemorepellent axon guidance molecule RGMa in cell migration and adhesion

The repulsive guidance molecule A (RGMa) is a contact-mediated axon guidance molecule that has significant roles in central nervous system (CNS) development. Here we have examined whether RGMa has novel roles in cell migration and cell adhesion outside the nervous system. RGMa was found to stimulate cell migration from Xenopus animal cap explants in a neogenin-dependent and BMP-independent manner. RGMa also stimulated the adhesion of Xenopus animal cap cells, and this adhesion was dependent on neogenin and independent of calcium. To begin to functionally characterize the role of specific domains in RGMa, we assessed the migratory and adhesive activities of deletion mutants. RGMa lacking the partial von Willebrand factor type D (vWF) domain preferentially perturbed cell adhesion, while mutants lacking the RGD motif affected cell migration. We also revealed that manipulating the levels of RGMa in vivo caused major migration defects during Xenopus gastrulation. We have revealed here novel roles of RGMa in cell migration and adhesion and demonstrated that perturbations to the homeostasis of RGMa expression can severely disrupt major morphogenetic events. These results have implications for understanding the role of RGMa in both health and disease.     

Lateral signaling mediated by axon contact and calcium entry regulates asymmetric odorant receptor expression in C. elegans

C. elegans detects several odorants with the bilaterally symmetric pair of AWC olfactory neurons. A stochastic, coordinated decision ensures that the candidate odorant receptor gene str-2 is expressed in only one AWC neuron in each animal--either the left or the right neuron, but never both. An interaction between the two AWC neurons generates asymmetric str-2 expression in a process that requires normal axon guidance and probably AWC axon contact. This interaction induces str-2 expression by reducing calcium signaling through a voltage-dependent Ca2+ channel and the CaM kinase II UNC-43. CaMKII activity acts as a switch in the initial decision to express str-2; thus, calcium signals can define distinct cell types during neuronal development. A cGMP signaling pathway that is used in olfaction maintains str-2 expression after the initial decision has been made.     

The heparan sulfate proteoglycans Dally-like and Syndecan have distinct functions in axon guidance and visual-system assembly in Drosophila

Heparan sulfate proteoglycans (HSPGs), a class of glycosaminoglycan-modified proteins, control diverse patterning events via their regulation of growth-factor signaling and morphogen distribution. In C. elegans, zebrafish, and the mouse, heparan sulfate (HS) biosynthesis is required for normal axon guidance, and mutations affecting Syndecan (Sdc), a transmembrane HSPG, disrupt axon guidance in Drosophila embryos. Glypicans, a family of glycosylphosphatidylinositol (GPI)-linked HSPGs, are expressed on axons and growth cones in vertebrates, but their role in axon guidance has not been determined. We demonstrate here that the Drosophila glypican Dally-like protein (Dlp) is required for proper axon guidance and visual-system function. Mosaic studies revealed that Dlp is necessary in both the retina and the brain for different aspects of visual-system assembly. Sdc mutants also showed axon guidance and visual-system defects, some that overlap with dlp and others that are unique. dlp+ transgenes were able to rescue some sdc visual-system phenotypes, but sdc+ transgenes were ineffective in rescuing dlp abnormalities. Together, these findings suggest that in some contexts HS chains provide the biologically critical component, whereas in others the structure of the protein core is also essential.     

mig-5/Dsh controls cell fate determination and cell migration in C. elegans

Cell fate determination and cell migration are two essential events in the development of an organism. We identify mig-5, a Dishevelled family member, as a gene that regulates several cell fate decisions and cell migrations that are important during C. elegans embryonic and larval development. In offspring from mig-5 mutants, cell migrations are defective during hypodermal morphogenesis, QL neuroblast migration, and the gonad arm migration led by the distal tip cells (DTCs). In addition to abnormal migration, DTC fate is affected, resulting in either an absent or an extra DTC. The cell fates of the anchor cell in hermaphrodites and the linker cells in the male gonad are also defective, often resulting in the cells adopting the fates of their sister lineage. Moreover, 2 degrees vulval precursor cells occasionally adopt the 3 degrees vulval cell fate, resulting in a deformed vulva, and the P12 hypodermal precursor often differentiates into a second P11 cell. These defects demonstrate that MIG-5 is essential in determining proper cell fate and cell migration throughout C. elegans development.     

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.