Signal transduction underlying growth cone guidance by diffusible factors.

Many diffusible axon guidance cues and their receptors have been identified recently. These cues are often found to be bifunctional, acting as attractants or repellents under different circumstances. Studies of cytoplasmic signaling mechanisms have led to the notion that the response of a growth cone to a particular guidance cue depends on the internal state of the neuron, which, in turn, is under the influence of other coincident signals received by the neuron. Furthermore, many diffusible guidance cues appear to share common cytoplasmic signaling pathways.     

Rho GTPases in growth cone guidance

It is now well established that the small GTPases of the Rho family--Rac, Cdc42 and Rho--regulate growth cone morphology. Less clear is their role in guiding the growth cone. Do they act permissively, providing the dynamic actin structures needed for guidance? Or do they act instructively, transducing specific guidance signals? Recent studies have provided the first strong evidence for an instructive role: extracellular guidance cues can modulate Rho GTPase activities in vitro, and Rho GTPase activators function in growth cone guidance in vivo. The pathways linking Rho GTPases and the actin cytoskeleton are also rapidly coming into view, revealing further points of regulation by extracellular guidance cues. The growth cone is therefore guided by signals transduced both via and independently of Rho GTPases.     

Regulation of growth cone extension by SNARE proteins.

Recent studies have suggested that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion with the surface plasma membrane, which leads to neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. We studied this mechanism by overexpressing GFP-fusion SNAREs including VAMP-2, SNAP-25A, and syntaxin1A in PC12 cells to investigate the role of SNAREs in neurite outgrowth. When overexpressed in PC12 cells, VAMP-2 promoted neurite elongation, whereas SNAP-25A stimulated neurite sprouting. On the other hand, overexpression of syntaxin1A neither promoted nor inhibited neurite outgrowth. Thus, VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.     

Regulation of growth cone actin filaments by guidance cues

The motile behaviors of growth cones at the ends of elongating axons determine pathways of axonal connections in developing nervous systems. Growth cones express receptors for molecular guidance cues in the local environment, and receptor-guidance cue binding initiates cytoplasmic signaling that regulates the cytoskeleton to control growth cone advance, turning, and branching behaviors. The dynamic actin filaments of growth cones are frequently targets of this regulatory signaling. Rho GTPases are key mediators of signaling by guidance cues, although much remains to be learned about how growth cone responses are orchestrated by Rho GTPase signaling to change the dynamics of polymerization, transport, and disassembly of actin filaments. Binding of neurotrophins to Trk and p75 receptors on growth cones triggers changes in actin filament dynamics to regulate several aspects of growth cone behaviors. Activation of Trk receptors mediates local accumulation of actin filaments, while neurotrophin binding to p75 triggers local decrease in RhoA signaling that promotes lengthening of filopodia. Semaphorin IIIA and ephrin-A2 are guidance cues that trigger avoidance or repulsion of certain growth cones, and in vitro responses to these proteins include growth cone collapse. Dynamic changes in the activities of Rho GTPases appear to mediate responses to these cues, although it remains unclear what the changes are in actin filament distribution and dynamic reorganization that result in growth cone collapse. Growth cones in vivo simultaneously encounter positive and negative guidance cues, and thus, growth cone behaviors during axonal pathfinding reflect the complex integration of multiple signaling activities.     

Gradients and growth cone guidance of grasshopper neurons.

The generation of a functional nervous system is dependent on precise pathfinding of axons during development. This pathfinding is directed by the distribution of local and long-range guidance cues, the latter of which are believed to be distributed in gradients. Gradients of guidance cues have been associated with growth cone function for over a hundred years. However, little is known about the mechanisms used by growth cones to respond to these gradients, in part owing to the lack of identifiable gradients in vivo. In the developing grasshopper limb, two gradients of the semaphorin Sema-2a are necessary for correct neuronal pathfinding in vivo. The gradients are found in regions where growth cones make critical steering decisions. Observations of different growth cone behaviors associated with these gradients have provided some insights into how growth cones respond to them. Growth cones appear to respond more faithfully to changes in concentration, rather than absolute levels, of Sema-2a expression, whereas the absolute levels may regulate growth cone size.     

Discrete roles for secreted and transmembrane semaphorins in neuronal growth cone guidance in vivo

From the initial stages of axon outgrowth to the formation of a functioning synapse, neuronal growth cones continuously integrate and respond to multiple guidance cues. To investigate the role of semaphorins in the establishment of appropriate axon trajectories, we have characterized a novel secreted semaphorin in grasshopper, gSema 2a. Sema 2a is expressed in a gradient in the developing limb bud epithelium during Ti pioneer axon outgrowth. We demonstrate that Sema 2a acts as chemorepulsive guidance molecule critical for axon fasciculation and for determining both the initial direction and subsequent pathfinding events of the Ti axon projection. Interestingly, simultaneous perturbation of both secreted Sema 2a and transmembrane Sema I results in a broader range and increased incidence of abnormal Ti pioneer axon phenotypes, indicating that different semaphorin family members can provide functionally distinct guidance information to the same growth cone in vivo.     

Electrical activity modulates growth cone guidance by diffusible factors

Brief periods of electrical stimulation of cultured Xenopus spinal neurons resulted in a marked alteration in the turning responses of the growth cone induced by gradients of attractive or repulsive guidance cues. Netrin-1-induced attraction was enhanced, and the repulsion induced by myelin-associated glycoprotein (MAG) or myelin membrane fragments was converted to attraction. The effect required the presence of extracellular Ca(2+) during electrical stimulation and appeared to be mediated by an elevation of both cytoplasmic Ca(2+) and cAMP. Thus, electrical activity may influence the axonal path finding of developing neurons, and intermittent electrical stimulation may be effective in promoting nerve regeneration after injury.     

Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ

Directed outgrowth of neural processes must involve transmission of signals from the tips of filopodia to the central region of the growth cone. Here, we report on the distribution and dynamics of one possible element in this process, actin, in live growth cones which are reorienting in response to in situ guidance cues. In grasshopper embryonic limbs, pioneer growth cones respond to at least three types of guidance cues: a limb axis cue, intermediate target cells, and a circumferential band of epithelial cells. With time-lapse imaging of intracellularly injected rhodamine-phalloidin and rhodamine-actin, we monitored the distribution of actin during growth cone responses to these cues. In distal limb regions, accumulation of actin in filopodia and growth cone branches accompanies continued growth, while reduction of actin accompanies withdrawal. Where growth cones are reorienting to intermediate target cells, or along the circumferential epithelial band, actin selectively accumulates in the proximal regions of those filopodia that have contacted target cells or are extending along the band. Actin accumulations can be retrogradely transported along filopodia, and can extend into the central region of the growth cone. These results suggest that regulation and translocation of actin may be a significant element in growth cone steering.     

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.     

Semaphorins: mediators of repulsive growth cone guidance

During development, neuronal growth cones encounter a variety of guidance cues while mediating axon path finding, target recognition and synapse formation. It is clear that repulsive guidance mechanisms play an essential role in these processes. The semaphorin gene family, which is conserved from invertebrates to mammals, includes members that mediate repulsive guidance. Molecular and cellular analysis of this gene family is providing insight into how inhibitory cues function during neurodevelopment.     

Axon guidance: opposing EPHects in the growth cone

Cells communicate with other cells via (trans) interaction between membrane-linked ephrins and Eph receptors. In this issue of Cell, Pfaff and colleagues (Marquardt et al., 2005) demonstrate that coexpressed ephrin-As and Ephs do not interact in cis but rather segregate into separate membrane domains, from which they signal opposing effects during motor axon guidance.     

Myosin functions in Xenopus retinal ganglion cell growth cone motility in vivo

The role of myosins in Xenopus retinal ganglion cell growth cone motility in the optic tract was studied using two pharmacologic inhibitors with different specificities. 2,3-Butanedione monoxime (BDM) disrupts myosin—actin interactions of all myosins, and ML-7 specifically inhibits activation of myosin II. Both inhibitors caused growth cones to assume a collapsed morphology and decreased growth cone speed. Similar effects were observed in vitro. Interestingly, the effects of the two inhibitors, while similar, were clearly distinguishable, raising the possibility that different myosins may have different functional roles in growth cone motility. BDM caused growth cones to withdraw lamellipodia and some filopodia and eventually to freeze, whereas ML-7 caused total collapse and retraction. Concentrations of BDM and ML-7 that had no effect when applied independently stopped growth cones when applied simultaneously, suggesting that these inhibitors act synergistically on myosin function, thus providing evidence of specificity. These results imply that normal growth cone motility in the molecularly and spatially complex environment of the living brain requires myosin function. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 567–578, 1997     

Phosphorylation of DCC by Fyn mediates Netrin-1 signaling in growth cone guidance

Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1-DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1-dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1-dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.     

A stochastic model of neuronal growth cone guidance regulated by multiple sensors

Neuronal growth cones migrate directionally under the control of axon guidance molecules, thereby forming synapses in the developing brain. The signal transduction system by which a growth cone detects surrounding guidance molecules, analyzes the detected signals, and then determines the overall behavior remains undetermined. In this study, we describe a novel stochastic model of this behavior that utilizes multiple sensors on filopodia to respond to guidance molecules. Overall growth cone behavior is determined by using only the concentration gradients of guidance molecules in the immediate vicinity of each sensor. The detected signal at each sensor, which is treated as a vector quantity, is sent to the growth cone center and then integrated to determine axonal growth in the next step by means of a simple vector operation. We compared the results of computer simulations of axonal growth with observations of actual axonal growth from co-culture experiments using olfactory bulb and septum. The probabilistic distributions of axonal growth generated by the computer simulation were consistent with those obtained from the culture experiments, indicating that our model accurately simulates growth cone behavior. We believe that this model will be useful for elucidating the as yet unknown mechanisms responsible for axonal growth in vivo.     

Role of laminin and integrin interactions in growth cone guidance

Laminin is well known to promote neuronal adhesion and axonal growth, but recent experiments suggest laminin has a wider role in guiding axons, both in development and regeneration. In vitro experiments demonstrate that laminin can alter the rate and direction of axonal growth, even when growth cone contact with laminin is transient. Investigations focused on a single neuronal type, such as retinal ganglion cells (RGCs), strongly implicate laminin as an important guidance molecule in development and suggest the involvement of integrins. Integrins are receptors for laminin, and neurons express multiple types of integrins that bind laminin. Morphologically, integrins cluster in point contacts, specialized regions of the growth cone that may coordinately regulate adhesion and motility. Recent evidence suggests that the structure and regulation of point contacts may differ from that of their nonneuronal counterparts, focal contacts. In part, this may be because the interaction of the cytoplasmic domain of integrin with the cytoskeleton is different in point contacts and focal contacts. Mutational studies where the cytoplasmic domain is truncated or altered are leading to a better understanding of the role of the α and β subunit in regulating integrin clustering and binding to the cytoskeleton. In addition, whereas integrins may regulate motility through direct physical linkages to the growth cone cytoskeleton, an equally important role is their ability to elicit signaling, both through protein tyrosine phosphorylation and modulating calcium levels. Through such mechanisms integrins likely regulate the dynamic attachment and detachment of the growth cone as it moves on laminin substrates.     

Cytoskeletal dynamics and transport in growth cone motility and axon guidance

Recent studies indicate the actin and microtubule cytoskeletons are a final common target of many signaling cascades that influence the developing neuron. Regulation of polymer dynamics and transport are crucial for the proper growth cone motility. This review addresses how actin filaments, microtubules, and their associated proteins play crucial roles in growth cone motility, axon outgrowth, and guidance. We present a working model for cytoskeletal regulation of directed axon outgrowth. An important goal for the future will be to understand the coordinated response of the cytoskeleton to signaling cascades induced by guidance receptor activation.     

Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth cone guidance

Although a growing body of evidence supports that Wnt-Frizzled signaling controls axon guidance from vertebrates to worms, whether and how this is mediated by planar cell polarity (PCP) signaling remain elusive. We show here that the core PCP components are required for Wnt5a-stimulated outgrowth and anterior-posterior guidance of commissural axons. Dishevelled1 can inhibit PCP signaling by increasing hyperphosphorylation of Frizzled3 and preventing its internalization. Vangl2 antagonizes that by reducing Frizzled3 phosphorylation and promotes its internalization. In commissural axon growth cones, Vangl2 is predominantly localized on the plasma membrane and is highly enriched on the tips of the filopodia as well as in patches of membrane where new filopodia emerge. Taken together, we propose that the antagonistic functions of Vangl2 and Dvl1 (over Frizzled3 hyperphosphorylation and endocytosis) allow sharpening of PCP signaling locally on the tips of the filopodia to sense directional cues, Wnts, eventually causing turning of growth cones.     

Mechanisms of growth cone guidance and motility in the developing grasshopper embryo

During neuronal pathfinding in vivo, growth cones must reorient their direction of migration in response to extracellular guidance cues. The developing grasshopper limb bud has proved to be a model system in which to examine mechanisms of growth cone guidance and motility in vivo. In this review we examine the contributions of adhesion and multiple guidance cues (semaphorins 1 and 2) in directing a growth cone steering event. Recent observations have suggested that the tibial pioneer growth cones are not directed via mechanisms of differential adhesivity. We present a model of growth cone steering that suggests a combination of adhesive and guidance receptors are important for a correct steering event and that guidance molecules may be important regulators of adhesive interactions with the actin cytoskeleton.     

FAK: Dynamic integration of guidance signals at the growth cone

During the formation of neural circuitry, axons are known to be guided to their specific targets by a relatively small arsenal of guidance signals. However, the molecular integration of this guidance information inside the axonal growth cone (GC) is still baffling. Focal adhesion kinase (FAK) is a cytosolic kinase which interacts with a complex molecular network via multiple phosphorylation sites. Paradoxically, FAK activation is required by both attractive and repulsive cues to control respectively axon outgrowth and disassembly of adhesive structures together with cytoskeletal dynamics. It was suggested that FAK might work as a versatile molecular integrator switching to different functions depending on its activation state. Two studies published recently by our group and Woo et al. shed light on this issue: for the first time, these works report a detailed molecular analysis of FAK activation and phosphorylation pattern in primary neuronal cultures in response to the repulsive cues Semaphorin3A and ephrinA1 respectively. Here we comment on the major novelties provided by these papers in the context of previous literature and we speculate on the future avenues of investigation opened by these works.Key words: FAK, growth cone, semaphorin, ephrin, netrin     

Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance.

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-gamma (PLC-gamma) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform exposure of TrkA-expressing neurons to NGF resulted in a cross-desensitization of turning responses induced by a gradient of netrin-1, brain-derived neurotrophic factor (BDNF), or myelin-associated glycoprotein (MAG) but not by a gradient of collapsin-1/semaphorin III/D or neurotrophin-3 (NT-3). These results, together with the effects of pharmacological inhibitors, support the notion that there are common cytosolic signaling pathways for two separate groups of guidance cues, one of which requires coactivation of PLC-gamma and PI3-kinase pathways.