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.

SUMMARYThe 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-offunction 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.

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

Rac GTPases control cell shape by regulating downstream effectors that influence the actin cytoskeleton. UNC-115, a putative actin-binding protein similar to human abLIM/limatin, has previously been implicated in axon pathfinding. We have discovered the role of UNC-115 as a downstream cytoskeletal effector of Rac signaling in axon pathfinding. We show that unc-115 double mutants with ced-10 Rac, mig-2 Rac or unc-73 GEF but not with rac-2/3 Rac displayed synthetic axon pathfinding defects, and that loss of unc-115 function suppressed the formation of ectopic plasma membrane extensions induced by constitutively-active rac-2 in neurons. Furthermore, we show that UNC-115 can bind to actin filaments. Thus, UNC-115 is an actin-binding protein that acts downstream of Rac signaling in axon pathfinding.

Cell migration is a fundamental process in animal development, including development of the nervous system. In C. elegans, the bilateral QR and QL neuroblasts undergo initial anterior and posterior polarizations and migrations before they divide to produce neurons. A subsequent Wnt signal from the posterior instructs QL descendants to continue their posterior migration. Nck-interacting kinases (NIK kinases) have been implicated in cell and nuclear migration as well as lamellipodia formation. Studies here show that the C. elegans MIG-15 NIK kinase controls multiple aspects of initial Q cell polarization, including the ability of the cells to polarize, to maintain polarity, and to migrate. These data suggest that MIG-15 acts independently of the Wnt signal that controls QL descendant posterior migration. Furthermore, MIG-15 affects the later migrations of neurons generated from Q cell division. Finally, a mosaic analysis indicates that MIG-15 acts cell autonomously in Q descendant migration.