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.     

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.     

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     

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.     

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.     

Semaphorins and their receptors in olfactory axon guidance.

The mammalian olfactory system is capable of discriminating among a large variety of odor molecules and is therefore essential for the identification of food, enemies and mating partners. The assembly and maintenance of olfactory connectivity have been shown to depend on the combinatorial actions of a variety of molecular signals, including extracellular matrix, cell adhesion and odorant receptor molecules. Recent studies have identified semaphorins and their receptors as putative molecular cues involved in olfactory pathfinding, plasticity and regeneration. The semaphorins comprise a large family of secreted and transmembrane axon guidance proteins, being either repulsive or attractive in nature. Neuropilins were shown to serve as receptors for secreted class 3 semaphorins, whereas members of the plexin family are receptors for class 1 and V (viral) semaphorins. The present review will discuss a role for semaphorins and their receptors in the establishment and maintenance of olfactory connectivity.     

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.     

Common mechanisms underlying growth cone guidance and axon branching

During development, growth cones direct growing axons into appropriate targets. However, in some cortical pathways target innervation occurs through the development of collateral branches that extend interstitially from the axon shaft. How do such branches form? Direct observations of living cortical brain slices revealed that growth cones of callosal axons pause for many hours beneath their cortical targets prior to the development of interstitial branches. High resolution imaging of dissociated living cortical neurons for many hours revealed that the growth cone demarcates sites of future axon branching by lengthy pausing behaviors and enlargement of the growth cone. After a new growth cone forms and resumes forward advance, filopodial and lamellipodial remnants of the large paused growth cone are left behind on the axon shaft from which interstitial branches later emerge. To investigate how the cytoskeleton reorganizes at axon branch points, we fluorescently labeled microtubules in living cortical neurons and imaged the behaviors of microtubules during new growth from the axon shaft and the growth cone. In both regions microtubules reorganize into a more plastic form by splaying apart and fragmenting. These shorter microtubules then invade newly developing branches with anterograde and retrograde movements. Although axon branching of dissociated cortical neurons occurs in the absence of targets, application of a target-derived growth factor, FGF-2, greatly enhances branching. Taken together, these results demonstrate that growth cone pausing is closely related to axon branching and suggest that common mechanisms underlie directed axon growth from the terminal growth cone and the axon shaft.     

Touch and go: guidance cues signal to the growth cone cytoskeleton

Growth cones, the highly motile tips of growing axons, guide axons to their targets by responding to molecular cues. Growth cone behaviors such as advancing, retracting, turning and branching are driven by the dynamics and reorganization of the actin and microtubule cytoskeleton through signaling pathways linked to guidance cue receptors. Actin filaments play a major part in growth cone motility, and because of their peripheral locations were thought to be the primary target of molecular cues. However, recent studies have shown that dynamic microtubules can penetrate the growth cone periphery where guidance molecules can influence them directly. Moreover, guidance cues can regulate growth cone steering by modulating dynamic actin-microtubule interactions.     

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.     

Drosophila Ten-m and filamin affect motor neuron growth cone guidance

The Drosophila Ten-m (also called Tenascin-major, or odd Oz (odz)) gene has been associated with a pair-rule phenotype. We identified and characterized new alleles of Drosophila Ten-m to establish that this gene is not responsible for segmentation defects but rather causes defects in motor neuron axon routing. In Ten-m mutants the inter-segmental nerve (ISN) often crosses segment boundaries and fasciculates with the ISN in the adjacent segment. Ten-m is expressed in the central nervous system and epidermal stripes during the stages when the growth cones of the neurons that form the ISN navigate to their targets. Over-expression of Ten-m in epidermal cells also leads to ISN misrouting. We also found that Filamin, an actin binding protein, physically interacts with the Ten-m protein. Mutations in cheerio, which encodes Filamin, cause defects in motor neuron axon routing like those of Ten-m. During embryonic development, the expression of Filamin and Ten-m partially overlap in ectodermal cells. These results suggest that Ten-m and Filamin in epidermal cells might together influence growth cone progression.     

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.     

Axon guidance receptors direct growth cone pathfinding: rivalry at the leading edge

One of the earliest steps in the development of the central and peripheral nervous systems is the initiation of axon outgrowth from newly born neurons. Nascent axons then navigate towards their specific targets to establish the intricate network of axon projections found within the mature central nervous system. In doing so, the projecting axons must continually reassess their spatial environment and accurately select the correct pathways among the maze of possible routes. A variety of molecular navigational systems governing axon pathfinding have now been identified. Understanding how these individual molecular guidance systems operate at the level of a single axon, and, how these different systems work in concert to initiate and steer axonal migration is a major goal in developmental neurobiology.     

Neogenin mediates the action of repulsive guidance molecule

Repulsive guidance molecule (RGM) is a recently identified protein implicated in both axonal guidance and neural tube closure. The avoidance of chick RGM in the posterior optic tectum by growing temporal, but not nasal, retinal ganglion cell axons is thought to contribute to visual map formation. In contrast to ephrins, semaphorins, netrins and slits, no receptor mechanism for RGM action has been defined. Here, an expression cloning strategy identified neogenin as a binding site for RGM, with a sub-nanomolar affinity. Consistent with selective axonal responsiveness to RGM, neogenin is expressed in a gradient across the chick retina. Neogenin is known to be one of several netrin-binding proteins but only neogenin interacts with RGM. The avoidance of RGM by temporal retinal axons is blocked by the anti-neogenin antibody and the soluble neogenin ectodomain. Dorsal root ganglion axons are unresponsive to RGM but are converted to a responsive state by neogenin expression. Thus, neogenin functions as an RGM receptor.     

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.     

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.     

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.     

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.     

Phosphatidylinositol 3-kinase facilitates microtubule-dependent membrane transport for neuronal growth cone guidance

The activity of PI3K is necessary for polarized cell motility. To guide extending axons, environmental cues polarize the growth cone via asymmetric generation of Ca(2+) signals and subsequent intracellular mechanical events, including membrane trafficking and cytoskeletal reorganization. However, it remains unclear how PI3K is involved in such events for axon guidance. Here, we demonstrate that PI3K plays a permissive role in growth cone turning by facilitating microtubule (MT)-dependent membrane transport. Using embryonic chick dorsal root ganglion neurons in culture, attractive axon turning was induced by Ca(2+) elevations on one side of the growth cone by photolyzing caged Ca(2+) or caged inositol 1,4,5-trisphosphate. We show that PI3K activity was required downstream of Ca(2+) signals for growth cone turning. Attractive Ca(2+) signals, generated with caged Ca(2+) or caged inositol 1,4,5-trisphosphate, triggered asymmetric transport of membrane vesicles from the center to the periphery of growth cones in a MT-dependent manner. This centrifugal vesicle transport was abolished by PI3K inhibitors, suggesting that PI3K is involved in growth cone attraction at the level of membrane trafficking. Consistent with this observation, immunocytochemistry showed that PI3K inhibitors reduced MTs in the growth cone peripheral domain. Time-lapse imaging of EB1 on the plus-end of MTs revealed that MT advance into the growth cone peripheral domain was dependent on PI3K activity: inhibition of the PI3K signaling pathway attenuated MT advance, whereas exogenous phosphatidylinositol 3,4,5-trisphosphate, the product of PI3K-catalyzed reactions, promoted MT advance. This study demonstrates the importance of PI3K-dependent membrane trafficking in chemotactic cell migration.     

Peptides derived from repulsive guidance molecule act as antagonists

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system [T. Yamashita, B.K. Mueller, K. Hata, Neogenin and RGM signaling in the central nervous system, Curr. Opin. Neurobiol. 17 (2007) 29-34]. Functional studies in Xenopus and chick embryos have revealed the roles of RGM in axon guidance and laminar patterning, while those in mouse embryos have demonstrated its function in regulating the cephalic neural tube closure. Importantly, RGM inhibition enhanced the growth of injured axons and promoted functional recovery after spinal cord injury in rats. Here, we identified two RGMa-derived peptides that functioned as antagonists against RGMa. The peptides studied in vitro dose-dependently suppressed the neurite growth inhibition and growth cone collapse induced by RGMa. Thus, these peptides are promising reagents to treat injuries of the central nervous system.