Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons

The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.     

Sonic hedgehog is indirectly required for intraretinal axon pathfinding by regulating chemokine expression in the optic stalk

Successful axon pathfinding requires both correct patterning of tissues, which will later harbor axonal tracts, and precise localization of axon guidance cues along these tracts at the time of axon outgrowth. Retinal ganglion cell (RGC) axons grow towards the optic disc in the central retina, where they turn to exit the eye through the optic nerve. Normal patterning of the optic disc and stalk and the expression of guidance cues at this choice point are necessary for the exit of RGC axons out of the eye. Sonic hedgehog (Shh) has been implicated in both patterning of ocular tissue and direct guidance of RGC axons. Here, we examine the precise spatial and temporal requirement for Hedgehog (Hh) signaling for intraretinal axon pathfinding and show that Shh acts to pattern the optic stalk in zebrafish but does not guide RGC axons inside the eye directly. We further reveal an interaction between the Hh and chemokine pathways for axon guidance and show that cxcl12a functions downstream of Shh and depends on Shh for its expression at the optic disc. Together, our results support a model in which Shh acts in RGC axon pathfinding indirectly by regulating axon guidance cues at the optic disc through patterning of the optic stalk.     

A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth.

In the developing retina, retinal ganglion cell (RGC) axons elongate toward the optic fissure, even though no obvious directional restrictions exist. Previous studies indicate that axon-matrix interactions are important for retinal ganglion cell axon elongation, but the factors that direct elongation are unknown. Chondroitin sulfate proteoglycan (CS-PG), a component of the extracellular matrix, repels elongating dorsal root ganglion (DRG) axons in vitro and is present in vivo in the roof plate of the spinal cord, a structure that acts as a barrier to DRG axons during development. In this study, we examined whether CS-PG may regulate the pattern of retinal ganglion cell outgrowth in the developing retina. Immunocytochemical analysis showed that CS-PG was present in the innermost layers of the developing rat retina. The expression of CS-PG moved peripherally with retinal development, always remaining at the outer edge of the front of the developing axons. CS-PG was no longer detectable with immunocytochemical techniques when RGC axon elongation in the retina is complete. Results of studies in vitro showed that CS-PG, isolated from bovine nasal cartilage and chick limb, was inhibitory to elongating RGC axons and that RGC growth cones were more sensitive to CS-PG than were DRG neurites tested at the same concentrations of CS-PG. The behavior of retinal growth cones as they encounter CS-PG was characterized using time-lapse video microscopy. Filopodia of the RGC growth cones extended to and sampled the CS-PG repeatedly. With time, the growth cones turned to avoid outgrowth on the CS-PG and grew only on laminin. While numerous studies have shown the presence of positive factors within the retina that may guide developing RGC axons, this is the first demonstration of an inhibitory or repelling molecule in the retina that may regulate axon elongation. Taken together, these data suggest that the direction of RGC outgrowth in the retina may be regulated by the proper ratio of growth-promoting molecules, such as laminin, to growth-inhibiting molecules, like CS-PG, present in the correct pattern and concentrations along the retinal ganglion cell pathway.     

Dynamic responses of Xenopus retinal ganglion cell axon growth cones to netrin-1 as they innervate their in vivo target

Netrin-1 influences retinal ganglion cell (RGC) axon pathfinding and also participates in the branching and synaptic differentiation of mature RGC axons at their target. To investigate whether netrin also serves as an early target recognition signal in the brain, we examined the dynamic behavior of Xenopus RGC axons soon after they innervate the optic tectum. Time-lapse confocal microscopy imaging of RGC axons expressing enhanced yellow fluorescent protein demonstrated that netrin-1 is involved in early axon branching, as recombinant netrin-1 halted further advancement of growth cones into the tectum and induced back branching. RGC growth cones exhibited differential responses to netrin-1 that depended on the degree of differentiation of the axon and the developmental stage of the tadpole. Netrin-1 decreased the total number of branches on newly arrived RGC growth cones at the target, but increased the dynamic branching of more mature arbors at the later developmental stage. To further explore the response of axonal growth cones to netrin, Xenopus RGC axons were followed in culture by time-lapse imaging. Exposure to netrin-1 rapidly increased the forward advancement of the axon and decreased the size and expanse of the growth cone, while also inducing back branching. Taken together, the differential in vivo and in vitro responses to netrin-1 suggest that netrin alone is not sufficient to induce the cessation of growth cone advancement in the absence of a target but can independently modulate axon branching. Collectively, our findings reveal a novel role for netrin on RGC axon branch initiation as growth cones innervate their target.     

Ephrin‐B2 elicits differential growth cone collapse and axon retraction in retinal ganglion cells from distinct retinal regions

The circuit for binocular vision and stereopsis is established at the optic chiasm, where retinal ganglion cell (RGC) axons diverge into the ipsilateral and contralateral optic tracts. In the mouse retina, ventrotemporal (VT) RGCs express the guidance receptor EphB1, which interacts with the repulsive guidance cue ephrin‐B2 on radial glia at the optic chiasm to direct VT RGC axons ipsilaterally. RGCs in the ventral retina also express EphB2, which interacts with ephrin‐B2, whereas dorsal RGCs express low levels of EphB receptors. To investigate how growth cones of RGCs from different retinal regions respond upon initial contact with ephrin‐B2, we utilized time‐lapse imaging to characterize the effects of ephrin‐B2 on growth cone collapse and axon retraction in real time. We demonstrate that bath application of ephrin‐B2 induces rapid and sustained growth cone collapse and axon retraction in VT RGC axons, whereas contralaterally‐projecting dorsotemporal RGCs display moderate growth cone collapse and little axon retraction. Dose response curves reveal that contralaterally‐projecting ventronasal axons are less sensitive to ephrin‐B2 treatment compared to VT axons. Additionally, we uncovered a specific role for Rho kinase signaling in the retraction of VT RGC axons but not in growth cone collapse. The detailed characterization of growth cone behavior in this study comprises an assay for the study of Eph signaling in RGCs, and provides insight into the phenomena of growth cone collapse and axon retraction in general. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 781–794, 2010     

Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina

Optic nerve formation requires precise retinal ganglion cell (RGC) axon pathfinding within the retina to the optic disc, the molecular basis of which is not well understood. At CNS targets, interactions between Eph receptor tyrosine kinases on RGC axons and ephrin ligands on target cells have been implicated in formation of topographic maps. However, studies in chick and mouse have shown that both Eph receptors and ephrins are also expressed within the retina itself, raising the possibility that this receptor-ligand family mediates aspects of retinal development. Here, we more fully document the presence of specific EphB receptors and B-ephrins in embryonic mouse retina and provide evidence that EphB receptors are involved in RGC axon pathfinding to the optic disc. We find that as RGC axons begin this pathfinding process, EphB receptors are uniformly expressed along the dorsal-ventral retinal axis. This is in contrast to the previously reported high ventral-low dorsal gradient of EphB receptors later in development when RGC axons map to CNS targets. We show that mice lacking both EphB2 and EphB3 receptor tyrosine kinases, but not each alone, exhibit increased frequency of RGC axon guidance errors to the optic disc. In these animals, major aspects of retinal development and cellular organization appear normal, as do the expression of other RGC guidance cues netrin, DCC, and L1. Unexpectedly, errors occur in dorsal but not ventral retina despite early uniform or later high ventral expression of EphB2 and EphB3. Furthermore, embryos lacking EphB3 and the kinase domain of EphB2 do not show increased errors, consistent with a guidance role for the EphB2 extracellular domain. Thus, while Eph kinase function is involved in RGC axon mapping in the brain, RGC axon pathfinding within the retina is partially mediated by EphB receptors acting in a kinase-independent manner.     

Retinal axon growth cones respond to EphB extracellular domains as inhibitory axon guidance cues

Axon pathfinding relies on cellular signaling mediated by growth cone receptor proteins responding to ligands, or guidance cues, in the environment. Eph proteins are a family of receptor tyrosine kinases that govern axon pathway development, including retinal axon projections to CNS targets. Recent examination of EphB mutant mice, however, has shown that axon pathfinding within the retina to the optic disc is dependent on EphB receptors, but independent of their kinase activity. Here we show a function for EphB1, B2 and B3 receptor extracellular domains (ECDs) in inhibiting mouse retinal axons when presented either as substratum-bound proteins or as soluble proteins directly applied to growth cones via micropipettes. In substratum choice assays, retinal axons tended to avoid EphB-ECDs, while time-lapse microscopy showed that exposure to soluble EphB-ECD led to growth cone collapse or other inhibitory responses. These results demonstrate that, in addition to the conventional role of Eph proteins signaling as receptors, EphB receptor ECDs can also function in the opposite role as guidance cues to alter axon behavior. Furthermore, the data support a model in which dorsal retinal ganglion cell axons heading to the optic disc encounter a gradient of inhibitory EphB proteins which helps maintain tight axon fasciculation and prevents aberrant axon growth into ventral retina. In conclusion, development of neuronal connectivity may involve the combined activity of Eph proteins serving as guidance receptors and as axon guidance cues.     

Ganglion cell axon pathfinding in the retina and optic nerve

The eye is a highly specialized structure that gathers and converts light information into neuronal signals. These signals are relayed along axons of retinal ganglion cells (RGCs) to visual centers in the brain for processing. In this review, we discuss the pathfinding tasks RGC axons face during development and the molecular mechanisms known to be involved. The data at hand support the presence of multiple axon guidance mechanisms concentrically organized around the optic nerve head, each of which appears to involve both growth-promoting and growth-inhibitory guidance molecules. Together, these strategies ensure proper optic nerve formation and establish the anatomical pathway for faithful transmission of information between the retina and the brain.     

The role of laminin and the laminin/fibronectin receptor complex in the outgrowth of retinal ganglion cell axons

Chick embryo retinal ganglion cell (RGC) axons grow to the optic tectum along a stereotyped route, as if responding to cues distributed along the pathway. We showed previously that, in culture, RGCs from embryonic Day 6 retina are responsive to the neurite-promoting effects of the extracellular matrix glycoprotein laminin and that this response is lost by RGCs at a later stage of development. Here we report that, before axon outgrowth is initiated in vivo, laminin, is expressed along the optic pathway at nonbasal lamina sites that are accessible to the growth cones of RGC axons. The distribution of laminin within the pathway is consistent with its localization at the end-feet of neuroepithelial cells that line the route, and it continues to be expressed at these marginal sites during the first week of embryonic development. At later stages, concomitant with the loss of response by RGCs in culture, laminin becomes restricted to basal laminae at the retinal inner limiting membrane and pial surface of the optic pathway. Neurofilament-positive RGC axons bind a monoclonal antibody, JG22, which recognizes the laminin/fibronectin receptor complex, and continue to do so throughout embryonic development. We show that, in vitro, the JG22 antigen expressed by RGCs appears to function as a laminin receptor, by demonstrating that JG22 antibody blocks neurite outgrowth on a substrate of laminin. These findings are consistent with the possibility that laminin defines a transient performed pathway specifically recognized by early RGC growth cones as they navigate toward their central target.     

Robo2 is required for Slit-mediated intraretinal axon guidance

The developing optic pathway has proven one of the most informative model systems for studying mechanisms of axon guidance. The first step in this process is the directed extension of retinal ganglion cell (RGC) axons within the optic fibre layer (OFL) of the retina towards their exit point from the eye, the optic disc. Previously, we have shown that the inhibitory guidance molecules, Slit1 and Slit2, regulate two distinct aspects of intraretinal axon guidance in a region-specific manner. Using knockout mice, we have found that both of these guidance activities are mediated via Robo2. Of the four vertebrate Robos, only Robo1 and Robo2 are expressed by RGCs. In mice lacking robo1 intraretinal axon guidance occurs normally. However, in mice lacking robo2 RGC axons make qualitatively and quantitatively identical intraretinal pathfinding errors to those reported previously in Slit mutants. This demonstrates clearly that, as in other regions of the optic pathway, Robo2 is the major receptor required for intraretinal axon guidance. Furthermore, the results suggest strongly that redundancy with other guidance signals rather than different receptor utilisation is the most likely explanation for the regional specificity of Slit function during intraretinal axon pathfinding.     

Semaphorin 3d guides laterality of retinal ganglion cell projections in zebrafish

The optic chiasm is an important choice point at which retinal ganglion cell (RGC) axons either cross the midline to innervate the contralateral brain or turn back to innervate the ipsilateral brain. Guidance cues that regulate this decision, particularly those directing the midline crossing of contralateral axons, are still not well understood. Here we show that Sema3d, a secreted semaphorin expressed at the midline, guides the crossing of RGC axons in zebrafish. Both Sema3d knockdown and ubiquitous overexpression induced aberrant ipsilateral projections, suggesting that Sema3d normally guides axons into the contralateral optic tract. Live imaging in vivo showed that RGC growth cones responded to ubiquitous Sema3d overexpression by pausing for extended periods and increasing their exploratory behavior at the midline, suggesting that Sema3d overexpression causes the midline environment to become less favorable for RGC axon extension. Interestingly, Sema3d overexpression did not affect growth cone behaviors before the midline, suggesting that RGC axons normally respond to Sema3d only upon reaching the midline. After Sema3d knockdown, growth cones grew across the midline but then paused or repeatedly retracted, impairing their ability to leave the midline region. Our results indicate that a proper balance of Sema3d is needed at the midline for the progression of RGC axons from the chiasm midline into the contralateral optic tract.     

Invariant Sema5A inhibition serves an ensheathing function during optic nerve development

Retinal axon pathfinding from the retina into the optic nerve involves the growth promoting axon guidance molecules L1, laminin and netrin 1, each of which governs axon behavior at specific regions along the retinal pathway. In identifying additional molecules regulating this process during embryonic mouse development, we found that transmembrane Semaphorin5A mRNA and protein was specifically expressed in neuroepithelial cells surrounding retinal axons at the optic disc and along the optic nerve. Given that growth cone responses to a specific guidance molecule can be altered by co-exposure to a second guidance cue, we examined whether retinal axon responses to Sema5A were modulated by other guidance signals axons encountered along the retinal pathway. In growth cone collapse, substratum choice and neurite outgrowth assays, Sema5A triggered an invariant inhibitory response in the context of L1, laminin, or netrin 1 signaling, suggesting that Sema5A inhibited retinal axons throughout their course at the optic disc and nerve. Antibody-perturbation studies in living embryo preparations showed that blocking of Sema5A function led to retinal axons straying out of the optic nerve bundle, indicating that Sema5A normally helped ensheath the retinal pathway. Thus, development of some CNS nerves requires inhibitory sheaths to maintain integrity. Furthermore, this function is accomplished using molecules such as Sema5A that exhibit conserved inhibitory responses in the presence of co-impinging signals from multiple families of guidance molecules.     

Perturbations of MicroRNA Function in Mouse Dicer Mutants Produce Retinal Defects and Lead to Aberrant Axon Pathfinding at the Optic Chiasm

Background

During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear.

Methodology/Principal Findings

We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype.

Conclusions

Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.     

Independent roles of Rho-GTPases in growth cone and axonal behavior

Many external signals influence growth cone motility, pathfinding, and the formation of synapses that lead to the final map formation of the retinotectal system. Chick temporal retinal ganglion cell axons (RGCs) collapse and retract after encountering posterior tectal cells in vitro. During this process lateral extensions appear along the RGC axonal shaft. Lateral extensions appear as nascent interstitial axonal branches and also as defasciculating growth cones that are trailing along the pioneer axon. RGC branching controlled by repellent tectal cues has recently been shown to be the critical event in retinotectal map development. The intracellular mechanism underlying this phenomenon, however, is not understood. Inhibiting RhoA with either C3 toxin or inhibiting p160Rock kinase, an effector of RhoA, with Y27632 inhibited collapse, retraction, and the number of axons that showed lateral extensions. Lateral extension length increased significantly. Inhibiting Rac1A and cdc42 with cell permeable peptide inhibitors did not inhibit collapse of growth cones, but did inhibit axon retraction. In addition, the number of axons that showed lateral extensions and lateral extension length were significantly reduced. A dynamic cytoskeleton is necessary to react to incoming guidance information. This study addresses the problems of how growth cone motility and branching or defasciculation are affected by Rho-GTPases as extracellular signals are transmitted to the cytoskeleton.     

NF-Protocadherin Regulates Retinal Ganglion Cell Axon Behaviour in the Developing Visual System

Cell adhesion molecules play a central role in mediating axonal tract development within the nascent nervous system. NF-protocadherin (NFPC), a member of the non-clustered protocadherin family, has been shown to regulate retinal ganglion cell (RGC) axon and dendrite initiation, as well as influencing axonal navigation within the mid-optic tract. However, whether NFPC mediates RGC axonal behaviour at other positions within the optic pathway remains unclear. Here we report that NFPC plays an important role in RGC axonogenesis, but not in intraretinal guidance. Moreover, axons with reduced NFPC levels exhibit insensitivity to Netrin-1, an attractive guidance cue expressed at the optic nerve head. Netrin-1 induces rapid turnover of NFPC localized to RGC growth cones, suggesting that the regulation of NFPC protein levels may underlie Netrin-1-mediated entry of RGC axons into the optic nerve head. At the tectum, we further reveal a function for NFPC in controlling RGC axonal entry into the final target area. Collectively, our results expand our understanding of the role of NFPC in RGC guidance and illustrate that this adhesion molecule contributes to axon behaviour at multiple points in the optic pathway.     

Fibroblast growth factors redirect retinal axons in vitro and in vivo

Growth factors have been shown previously to participate in the process of axon target recognition. We showed that fibroblast growth factor receptor (FGFR) signaling is required for Xenopus laevis retinal ganglion cell (RGC) axons to recognize their major midbrain target, the optic tectum [neuron 17 (1996), 245]. Therefore, we have hypothesized that a change in expression of a fibroblast growth factor (FGF) at the entrance of the optic tectum, the border between the diencephalon and mesencephalon, may serve as a signal to RGC axons that they have reached their target. To determine whether RGC axons can sense changes in FGF levels, we asked whether they altered their behavior upon encountering an ectopic source of FGF. We found that in vivo RGC growth cones avoided FGF-misexpressing cells along their path, and that FGF-2 directly repelled RGC growth cones in an in vitro growth cone turning assay. These data support the idea that RGC axons can sense changes in FGF levels, and as such provide a mechanism by which FGFR signaling is involved in RGC axon target recognition.     

GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.     

Independent roles of Rho‐GTPases in growth cone and axonal behavior

Many external signals influence growth cone motility, pathfinding, and the formation of synapses that lead to the final map formation of the retinotectal system. Chick temporal retinal ganglion cell axons (RGCs) collapse and retract after encountering posterior tectal cells in vitro. During this process lateral extensions appear along the RGC axonal shaft. Lateral extensions appear as nascent interstitial axonal branches and also as defasciculating growth cones that are trailing along the pioneer axon. RGC branching controlled by repellent tectal cues has recently been shown to be the critical event in retinotectal map development. The intracellular mechanism underlying this phenomenon, however, is not understood. Inhibiting RhoA with either C3 toxin or inhibiting p160Rock kinase, an effector of RhoA, with Y27632 inhibited collapse, retraction, and the number of axons that showed lateral extensions. Lateral extension length increased significantly. Inhibiting Rac1A and cdc42 with cell permeable peptide inhibitors did not inhibit collapse of growth cones, but did inhibit axon retraction. In addition, the number of axons that showed lateral extensions and lateral extension length were significantly reduced. A dynamic cytoskeleton is necessary to react to incoming guidance information. This study addresses the problems of how growth cone motility and branching or defasciculation are affected by Rho‐GTPases as extracellular signals are transmitted to the cytoskeleton. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 358–369, 2003     

Control of retinal ganglion cell axon growth: a new role for Sonic hedgehog

Retinal ganglion cell (RGC) axons grow towards the diencephalic ventral midline during embryogenesis guided by cues whose nature is largely unknown. We provide in vitro and in vivo evidence for a novel role of Sonic hedgehog (SHH) as a negative regulator of growth cone movement. SHH suppresses both the number and the length of neurites emerging from the chick retina but not from neural tube or dorsal root ganglia explants, without interfering with their rate of proliferation and differentiation. Similarly, retroviral-mediated ectopic expression of Shh along the chick visual pathway greatly interferes the growth of RGC axons. Upon SHH addition to grown neurites, the intracellular level of cAMP decreases, suggesting that the dampening of growth cone extension mediated by SHH may involve interaction with its receptor Patched which is expressed by RGC. Based on these findings, we propose that Shh expression at the chiasm border defines a constrained pathway within the ventral midline which serves to guide the progression of RGC axons.     

RACK-1 acts with Rac GTPase signaling and UNC-115/abLIM in Caenorhabditis elegans axon pathfinding and cell migration

Migrating cells and growth cones extend lamellipodial and filopodial protrusions that are required for outgrowth and guidance. The mechanisms of cytoskeletal regulation that underlie cell and growth cone migration are of much interest to developmental biologists. Previous studies have shown that the Arp2/3 complex and UNC-115/abLIM act redundantly to mediate growth cone lamellipodia and filopodia formation and axon pathfinding. While much is known about the regulation of Arp2/3, less is known about regulators of UNC-115/abLIM. Here we show that the Caenorhabditis elegans counterpart of the Receptor for Activated C Kinase (RACK-1) interacts physically with the actin-binding protein UNC-115/abLIM and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts cell-autonomously in the UNC-115/abLIM pathway in axon pathfinding and lamellipodia and filopodia formation, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a novel signaling pathway involving Rac GTPases and UNC-115/abLIM and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones.