Genes required for axon pathfinding and extension in the C. elegans nerve ring.

Over half of the neurons in Caenorhabditis elegans send axons to the nerve ring, a large neuropil in the head of the animal. Genetic screens in animals that express the green fluorescent protein in a subset of sensory neurons identified eight new sax genes that affect the morphology of nerve ring axons. sax-3/robo mutations disrupt axon guidance in the nerve ring, while sax-5, sax-9 and unc-44 disrupt both axon guidance and axon extension. Axon extension and guidance proceed normally in sax-1, sax-2, sax-6, sax-7 and sax-8 mutants, but these animals exhibit later defects in the maintenance of nerve ring structure. The functions of existing guidance genes in nerve ring development were also examined, revealing that SAX-3/Robo acts in parallel to the VAB-1/Eph receptor and the UNC-6/netrin, UNC-40/DCC guidance systems for ventral guidance of axons in the amphid commissure, a major route of axon entry into the nerve ring. In addition, SAX-3/Robo and the VAB-1/Eph receptor both function to prevent aberrant axon crossing at the ventral midline. Together, these genes define pathways required for axon growth, guidance and maintenance during nervous system development.     

SYD-1C,UNC-40 (DCC) and SAX-3 (Robo) Function Interdependently to Promote Axon Guidance by Regulating the MIG-2 GTPase

During development, axons must integrate directional information encoded by multiple guidance cues and their receptors. Axon guidance receptors, such as UNC-40 (DCC) and SAX-3 (Robo), can function individually or combinatorially with other guidance receptors to regulate downstream effectors. However, little is known about the molecular mechanisms that mediate combinatorial guidance receptor signaling. Here, we show that UNC-40, SAX-3 and the SYD-1 RhoGAP-like protein function interdependently to regulate the MIG-2 (Rac) GTPase in the HSN axon of C. elegans. We find that SYD-1 mediates an UNC-6 (netrin) independent UNC-40 activity to promote ventral axon guidance. Genetic analysis suggests that SYD-1 function in axon guidance requires both UNC-40 and SAX-3 activity. Moreover, the cytoplasmic domains of UNC-40 and SAX-3 bind to SYD-1 and SYD-1 binds to and negatively regulates the MIG-2 (Rac) GTPase. We also find that the function of SYD-1 in axon guidance is mediated by its phylogenetically conserved C isoform, indicating that the role of SYD-1 in guidance is distinct from its previously described roles in synaptogenesis and axonal specification. Our observations reveal a molecular mechanism that can allow two guidance receptors to function interdependently to regulate a common downstream effector, providing a potential means for the integration of guidance signals.     

The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.     

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.     

The VAB-1 Eph receptor tyrosine kinase and SAX-3/Robo neuronal receptors function together during C. elegans embryonic morphogenesis

Mutations that affect the single C. elegans Eph receptor tyrosine kinase VAB-1 cause defects in cell movements during embryogenesis. Here, we provide genetic and molecular evidence that the VAB-1 Eph receptor functions with another neuronal receptor, SAX-3/Robo, for proper embryogenesis. Our analysis of sax-3 mutants shows that SAX-3/Robo functions with the VAB-1 Eph receptor for gastrulation cleft closure and ventral epidermal enclosure. In addition, SAX-3 functions autonomously for epidermal morphogenesis independently of VAB-1. A double-mutant combination between vab-1 and slt-1 unmasks a role for the SLT-1 ligand in embryogenesis. We provide evidence for a physical interaction between the VAB-1 tyrosine kinase domain and the juxtamembrane and CC1 region of the SAX-3/Robo receptor. Gene dosage, non-allelic non-complementation experiments and co-localization of the two receptors are consistent with a model in which these two receptors form a complex and function together during embryogenesis.     

The Arp2/3 activators WAVE and WASP have distinct genetic interactions with Rac GTPases in Caenorhabditis elegans axon guidance

In the developing nervous system, axons are guided to their targets by the growth cone. Lamellipodial and filopodial protrusions from the growth cone underlie motility and guidance. Many molecules that control lamellipodia and filopodia formation, actin organization, and axon guidance have been identified, but it remains unclear how these molecules act together to control these events. Experiments are described here that indicate that, in Caenorhabditis elegans, two WH2-domain-containing activators of the Arp2/3 complex, WVE-1/WAVE and WSP-1/WASP, act redundantly in axon guidance and that GEX-2/Sra-1 and GEX-3/Kette, molecules that control WAVE activity, might act in both pathways. WAVE activity is controlled by Rac GTPases, and data are presented here that suggest WVE-1/WAVE and CED-10/Rac act in parallel to a pathway containing WSP-1/WASP and MIG-2/RhoG. Furthermore, results here show that the CED-10/WVE-1 and MIG-2/WSP-1 pathways act in parallel to two other molecules known to control lamellipodia and filopodia and actin organization, UNC-115/abLIM and UNC-34/Enabled. These results indicate that at least three actin-modulating pathways act in parallel to control actin dynamics and lamellipodia and filopodia formation during axon guidance (WASP-WAVE, UNC-115/abLIM, and UNC-34/Enabled).     

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.     

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.     

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.     

CED-10/Rac1 mediates axon guidance by regulating the asymmetric distribution of MIG-10/lamellipodin

Axon migrations are guided by extracellular cues that induce asymmetric outgrowth activity in the growth cone. Several intracellular signaling proteins have been implicated in the guidance response. However, how these proteins interact to generate asymmetric outgrowth activity is unknown. Here, we present evidence that in C. elegans, the CED-10/Rac1 GTPase binds to and causes asymmetric localization of MIG-10/lamellipodin, a protein that regulates actin polymerization and has outgrowth-promoting activity in neurons. Genetic analysis indicates that mig-10 and ced-10 function together to orient axon outgrowth. The RAPH domain of MIG-10 binds to activated CED-10/Rac1, and ced-10 function is required for the asymmetric MIG-10 localization that occurs in response to the UNC-6/netrin guidance cue. We also show that asymmetric localization of MIG-10 in growth cones is associated with asymmetric concentrations of f-actin and microtubules. These results suggest that CED-10/Rac1 is asymmetrically activated in response to the UNC-6/netrin signal and thereby causes asymmetric recruitment of MIG-10/lamellipodin. We propose that the interaction between activated CED-10/Rac1 and MIG-10/lamellipodin triggers local cytoskeletal assembly and polarizes outgrowth activity in response to UNC-6/netrin.     

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.     

Distinct cell guidance pathways controlled by the Rac and Rho GEF domains of UNC-73/TRIO in Caenorhabditis elegans

The cytoskeleton regulator UNC-53/NAV2 is required for both the anterior and posterior outgrowth of several neurons as well as that of the excretory cell while the kinesin-like motor VAB-8 is essential for most posteriorly directed migrations in Caenorhabditis elegans. Null mutations in either unc-53 or vab-8 result in reduced posterior excretory canal outgrowth, while double null mutants display an enhanced canal extension defect, suggesting the genes act in separate pathways to control this posteriorly directed outgrowth. Genetic analysis of putative interactors of UNC-53 or VAB-8, and cell-specific rescue experiments suggest that VAB-8, SAX-3/ROBO, SLT-1/Slit, and EVA-1 are functioning together in the outgrowth of the excretory canals, while UNC-53 appears to function in a parallel pathway with UNC-71/ADAM. The known VAB-8 interactor, the Rac/Rho GEF UNC-73/TRIO operates in both pathways, as isoform specific alleles exhibit enhancement of the phenotype in double-mutant combination with either unc-53 or vab-8. On the basis of these results, we propose a bipartite model for UNC-73/TRIO activity in excretory canal extension: a cell autonomous function that is mediated by the Rho-specific GEF domain of the UNC-73E isoform in conjunction with UNC-53 and UNC-71 and a cell nonautonomous function that is mediated by the Rac-specific GEF domain of the UNC-73B isoform, through partnering with VAB-8 and the receptors SAX-3 and EVA-1.     

SAX-3 (Robo) and UNC-40 (DCC) Regulate a Directional Bias for Axon Guidance in Response to Multiple Extracellular Cues

Axons in Caenorhabditis elegans are guided by multiple extracellular cues, including UNC-6 (netrin), EGL-20 (wnt), UNC-52 (perlecan), and SLT-1 (slit). How multiple extracellular cues determine the direction of axon guidance is not well understood. We have proposed that an axon''s response to guidance cues can be modeled as a random walk, i.e., a succession of randomly directed movement. Guidance cues dictate the probability of axon outgrowth activity occurring in each direction, which over time creates a directional bias. Here we provide further evidence for this model. We describe the effects that the UNC-40 (DCC) and SAX-3 (Robo) receptors and the UNC-6, EGL-20, UNC-52, and SLT-1 extracellular cues have on the directional bias of the axon outgrowth activity for the HSN and AVM neurons. We find that the directional bias created by the cues depend on UNC-40 or SAX-3. UNC-6 and EGL-20 affect the directional bias for both neurons, whereas UNC-52 and SLT-1 only affect the directional bias for HSN and AVM, respectively. The direction of the bias created by the loss of a cue can vary and the direction depends on the other cues. The random walk model predicts this combinatorial regulation. In a random walk a probability is assigned for each direction of outgrowth, thus creating a probability distribution. The probability distribution for each neuron is determined by the collective effect of all the cues. Since the sum of the probabilities must equal one, each cue affects the probability of outgrowth in multiple directions.     

C. elegans slit acts in midline, dorsal-ventral, and anterior-posterior guidance via the SAX-3/Robo receptor

Robo receptors interact with ligands of the Slit family. The nematode C. elegans has one Robo receptor (SAX-3) and one Slit protein (SLT-1), which direct ventral axon guidance and guidance at the midline. In larvae, slt-1 expression in dorsal muscles repels axons to promote ventral guidance. SLT-1 acts through the SAX-3 receptor, in parallel with the ventral attractant UNC-6 (Netrin). Removing both UNC-6 and SLT-1 eliminates all ventral guidance information for some axons, revealing an underlying longitudinal guidance pathway. In the embryo, slt-1 is expressed at high levels in anterior epidermis. Embryonic expression of SLT-1 provides anterior-posterior guidance information to migrating CAN neurons. Surprisingly, slt-1 mutants do not exhibit the nerve ring and epithelial defects of sax-3 mutants, suggesting that SAX-3 has both Slit-dependent and Slit-independent functions in development.     

Arabidopsis GNARLED encodes a NAP125 homolog that positively regulates ARP2/3

In migrating cells, the actin filament nucleation activity of ARP2/3 is an essential component of dynamic cell shape change and motility. In response to signals from the small GTPase Rac1, alterations in the composition and/or subcellular localization of the WAVE complex lead to ARP2/3 activation. The human WAVE complex subunit, WAVE1/SCAR1, was first identified in Dictyostelium and is a direct ARP2/3 activator. In the absence of an intact WAVE complex, SCAR/WAVE protein is destabilized. Although the composition of the five-subunit WAVE complex is well characterized, the means by which individual subunits and fully assembled WAVE complexes regulate ARP2/3 in vivo are unclear. The molecular genetics of trichome distortion in Arabidopsis is a powerful system to understand how signaling pathways and ARP2/3 control multicellular development. In this paper we prove that the GNARLED gene encodes a homolog of the WAVE subunit NAP125. Despite the moderate level of amino acid identity between Arabidopsis and human NAP125, both homologs were functionally interchangeable in vivo and interacted physically with the putative Arabidopsis WAVE subunit ATSRA1. gnarled trichomes had nearly identical cell shape and actin cytoskeleton phenotypes when compared to ARP2/3 subunit mutants, suggesting that GRL positively regulates ARP2/3.     

BRICK1/HSPC300 functions with SCAR and the ARP2/3 complex to regulate epidermal cell shape in Arabidopsis

The Arp2/3 complex, a highly conserved nucleator of F-actin polymerization, is essential for a variety of eukaryotic cellular processes, including epidermal cell morphogenesis in Arabidopsis thaliana. Efficient nucleation of actin filaments by the Arp2/3 complex requires the presence of an activator such as a member of the Scar/WAVE family. In mammalian cells, a multiprotein complex consisting of WAVE, PIR121/Sra-1, Nap1, Abi-2 and HSPC300 mediates responsiveness of WAVE to upstream regulators such as Rac. Essential roles in WAVE complex assembly or function have been demonstrated for PIR121/Sra-1, Nap1 and Abi-2, but the significance of HSPC300 in this complex is unclear. Plant homologs of all mammalian WAVE complex components have been identified, including HSPC300, the mammalian homolog of maize BRICK1 (BRK1). We show that, like mutations disrupting the Arabidopsis homologs of PIR121/Sra-1, Nap1 and Scar/WAVE, mutations in the Arabidopsis BRK1 gene result in trichome and pavement cell morphology defects (and associated alterations in the F-actin cytoskeleton of expanding cells) similar to those caused by mutations disrupting the ARP2/3 complex itself. Analysis of double mutants provides genetic evidence that BRK1 functions in a pathway with the ARP2/3 complex. BRK1 is required for accumulation of SCAR1 protein in vivo, potentially explaining the apparently essential role of BRK1 in ARP2/3 complex function.     

Axon guidance: asymmetric signaling orients polarized outgrowth

A network of connections is established as neural circuits form between neurons. To make these connections, neurons initiate asymmetric axon outgrowth in response to extracellular guidance cues. Within the specialized growth cones of migrating axons, F-actin and microtubules asymmetrically accumulate where an axon projects forward. Although many guidance cues, receptors and intracellular signaling components that are required for axon guidance have been identified, the means by which the asymmetry is established and maintained is unclear. Here, we discuss recent studies in invertebrate and vertebrate organisms that define a signaling module comprising UNC-6 (the Caenorhabditis elegans ortholog of netrin), UNC-40 (the C. elegans ortholog of DCC), PI3K, Rac and MIG-10 (the C. elegans ortholog of lamellipodin) and we consider how this module could establish polarized outgrowth in response to guidance cues.     

Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex.

WAVE/SCAR protein was identified as a protein which has similarity to WASP and N-WASP, especially in its C terminal. Recently, WAVE/SCAR protein has been shown to cooperate with the Arp2/3 complex, a nucleation core for actin polymerization in vitro. However, in spite of its general function, WAVE/SCAR expression is mainly restricted to the brain, suggesting the existence of related molecule(s). We here identified two human WAVE/SCAR homologues, which cover other organs. We named the original WAVE1 and newly identified ones WAVE2 and WAVE3. WAVE2 had a very wide distribution with strong expression in peripheral blood leukocytes and mapped on chromosome Xp11.21, next to the WASP locus. WAVE3 and WAVE1 had similar distributions. WAVE3 was strongly expressed in brain and mapped on chromosome 13q12. WAVE1 was mapped on chromosome 6q21-22. Ectopically expressed WAVE2 and WAVE3 induced actin filament clusters in a similar manner with WAVE1. These actin cluster formations were suppressed by deletion of their C-terminal VPH (verproline homology)/WH2 (WASP homology 2) domain. Further, WAVE2 and WAVE3 associate with the Arp2/3 complex as does WAVE1. Our identification of WAVE homologues suggests that WAVE family proteins have general function for regulating the actin cytoskeleton in many tissues.     

SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion

Myoblast fusion is crucial for formation and repair of skeletal muscle. Here we show that active remodeling of the actin cytoskeleton is essential for fusion in Drosophila. Using live imaging, we have identified a dynamic F-actin accumulation (actin focus) at the site of fusion. Dissolution of the actin focus directly precedes a fusion event. Whereas several known fusion components regulate these actin foci, others target additional behaviors required for fusion. Mutations in kette/Nap1, an actin polymerization regulator, lead to enlarged foci that do not dissolve, consistent with the observed block in fusion. Kette is required to positively regulate SCAR/WAVE, which in turn activates the Arp2/3 complex. Mutants in SCAR and Arp2/3 have a fusion block and foci phenotype, suggesting that Kette-SCAR-Arp2/3 participate in an actin polymerization event required for focus dissolution. Our data identify a new paradigm for understanding the mechanisms underlying fusion in myoblasts and other tissues.     

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.