Rapid changes in the distribution of GAP-43 correlate with the expression of neuronal polarity during normal development and under experimental conditions

Hippocampal neurons growing in culture initially extend several, short minor processes that have the potential to become either axons or dendrites. The first expression of polarity occurs when one of these minor processes begins to elongate rapidly, becoming the axon. Before axonal outgrowth, the growth-associated protein GAP-43 is distributed equally among the growth cones of the minor processes; it is preferentially concentrated in the axonal growth cone once polarity has been established (Goslin, K., D. Schreyer, J. Skene, and G. Banker. 1990. J. Neurosci. 10:588-602). To determine when the selective segregation of GAP-43 begins, we followed individual cells by video microscopy, fixed them as soon as the axon could be distinguished, and localized GAP-43 by immunofluorescence microscopy. Individual minor processes acquired axonal growth characteristics within a period of 30-60 min, and GAP-43 became selectively concentrated to the growth cones of these processes with an equally rapid time course. We also examined changes in the distribution of GAP-43 after transection of the axon. After an axonal transection that is distant from the soma, neuronal polarity is maintained, and the original axon begins to regrow almost immediately. In such cases, GAP-43 became selectively concentrated in the new axonal growth cone within 12-30 min. In contrast, when the axon is transected close to the soma, polarity is lost; the original axon rarely regrows, and there is a significant delay before a new axon emerges. Under these circumstances, GAP-43 accumulated in the new growth cone much more slowly, suggesting that its ongoing selective routing to the axon had been disrupted by the transection. These results demonstrate that the selective segregation of GAP-43 to the growth cone of a single process is closely correlated with the acquisition of axonal growth characteristics and, hence, with the expression of polarity.     

Substrate-cytoskeletal coupling as a mechanism for the regulation of growth cone motility and guidance

Growth cones are highly motile structures at the end of neuronal processes, capable of receiving multiple types of guidance cues and transducing them into directed axonal growth. Thus, to guide the axon toward the appropriate target cell, the growth cone carries out different functions: it acts as a sensor, signal transducer, and motility device. An increasing number of molecular components that mediate axon guidance have been characterized over the past years. The vast majority of these molecules include proteins that act as guidance cues and their respective receptors. In addition, more and more signaling and cytoskeleton-associated proteins have been localized to the growth cone. Furthermore, it has become evident that growth cone motility and guidance depends on a dynamic cytoskeleton that is regulated by incoming guidance information. Current and future research in the growth cone field will be focussed on how different guidance cues transmit their signals to the cytoskeleton and change its dynamic properties to affect the rate and direction of growth cone movement. In this review, we discuss recent evidence that cell adhesion molecules can regulate growth cone motility and guidance by a mechanism of substrate-cytoskeletal coupling.     

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.     

DSCR1 is required for both axonal growth cone extension and steering

Local information processing in the growth cone is essential for correct wiring of the nervous system. As an axon navigates through the developing nervous system, the growth cone responds to extrinsic guidance cues by coordinating axon outgrowth with growth cone steering. It has become increasingly clear that axon extension requires proper actin polymerization dynamics, whereas growth cone steering involves local protein synthesis. However, molecular components integrating these two processes have not been identified. Here, we show that Down syndrome critical region 1 protein (DSCR1) controls axon outgrowth by modulating growth cone actin dynamics through regulation of cofilin activity (phospho/dephospho-cofilin). Additionally, DSCR1 mediates brain-derived neurotrophic factor–induced local protein synthesis and growth cone turning. Our study identifies DSCR1 as a key protein that couples axon growth and pathfinding by dually regulating actin dynamics and local protein synthesis.     

A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons

We tested the contribution of the small GTPase Rho and its downstream target p160ROCK during the early stages of axon formation in cultured cerebellar granule neurons. p160ROCK inhibition, presumably by reducing the stability of the cortical actin network, triggered immediate outgrowth of membrane ruffles and filopodia, followed by the generation of initial growth cone-ike membrane domains from which axonal processes arose. Furthermore, a potentiation in both the size and the motility of growth cones was evident, though the overall axon elongation rate remained stable. Conversely, overexpression of dominant active forms of Rho or ROCK was suggested to prevent initiation of axon outgrowth. Taken together, our data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics.     

Myosin II activity facilitates microtubule bundling in the neuronal growth cone neck

The cell biological processes underlying axon growth and guidance are still not well understood. An outstanding question is how a new segment of the axon shaft is formed in the wake of neuronal growth cone advance. For this to occur, the highly dynamic, splayed-out microtubule (MT) arrays characteristic of the growth cone must be consolidated (bundled together) to form the core of the axon shaft. MT-associated proteins stabilize bundled MTs, but how individual MTs are brought together for initial bundling is unknown. Here, we show that laterally moving actin arcs, which are myosin II-driven contractile structures, interact with growing MTs and transport them from the sides of the growth cone into the central domain. Upon Myosin II inhibition, the movement of actin filaments and MTs immediately stopped and MTs unbundled. Thus, Myosin II-dependent compressive force is necessary for normal MT bundling in the growth cone neck.     

How the growth cone recognizes and responds to its environment

Four interactive processes—adhesion, guidance, migration and growth—combine to direct the axonal growth cone to its targets. It is becoming clear that the sensors of the external environment, the axonal receptors and adhesion molecules, activate second messenger systems in the growth cone. This allows a cytoplasmic integration of guidance signals acting upon the growth cone, that feeds back upon the adhesion molecules and the cytoskeleton to select the direction of growth. Movement is primarily generated by the actin microfilaments, growth is dependent upon the microtubules. This review examines the interdependence of these processes during the initial phase of axon elongation, using examples from insects to mammals.     

Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis

Background

During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined.

Results

To investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases.

Conclusions

This study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.     

En passant synaptic varicosities form directly from growth cones by transient cessation of growth cone advance but not of actin-based motility.

Formation of terminal synapses at sites such as the neuromuscular junction involves transformation of the motile growth cone into the nonmotile synaptic terminal. However, transformation does not need to be the mechanism when a neurite forms multiple widely spaced synaptic varicosities along a target in an en passant configuration. Synaptic varicosities could form here by specialization of the neurite after the growth cone has advanced past the site. We examined this issue by using cocultures of identified sensory (SN) and motor (L7) neurons from Aplysia. Living SNs were labeled with fluorescent dye and their neurites were observed at high resolution every few minutes growing along the axon of L7, allowing a fine-grained analysis of the behavior of the growth cone at the sites of synapse formation. All varicosities whose formation was observed indeed developed from the growth cone. Sensory varicosities were shown by electron microscopy to contain features characteristic of active zones for transmitter release within a day of their formation on the motor axon. Growth cone advance slowed or stopped transiently during varicosity formation, but the motile activity of the peripheral region of the growth cone (veils and filopodia) was maintained. These results suggest that target "stop signals" involved in the formation of synapses, at least of the en passant variety, may be of a different type from the growth inhibitory molecules, such as the collapsins, which guide axons to their targets.     

RhoA-kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA-kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP-actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F-actin patches, the latter being an effect attributable to ROCK-mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F-actin polymerization underlying protrusive activity in the distal axon.     

Directional guidance of nerve growth cones

The intricate connections of the nervous system are established, in part, by elongating axonal fibers that are directed by complex guidance systems to home in on their specific targets. The growth cone, the major motile apparatus at the tip of axons, explores its surroundings and steers the axon along a defined path to its appropriate target. Significant progress has been made in identifying the guidance molecules and receptors that regulate growth cone pathfinding, the signaling cascades underlying distinct growth cone behaviors, and the cytoskeletal components that give rise to the directional motility of the growth cone. Recent studies have also shed light on the sophisticated mechanisms and new players utilized by the growth cone during pathfinding. It is clear that axon pathfinding requires a growth cone to sample and integrate various signals both in space and in time, and subsequently to coordinate the dynamics of its membrane, cytoskeleton and adhesion to generate specific responses.     

RhoA‐kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA‐kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP‐actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F‐actin patches, the latter being an effect attributable to ROCK‐mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F‐actin polymerization underlying protrusive activity in the distal axon. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Mechanical tension can specify axonal fate in hippocampal neurons

Here we asked whether applied mechanical tension would stimulate undifferentiated minor processes of cultured hippocampal neurons to become axons and whether tension could induce a second axon in an already polarized neuron. Experimental tension applied to minor processes produced extensions that demonstrated axonal character, regardless of the presence of an existing axon. Towed neurites showed a high rate of spontaneous growth cone advance and could continue to grow out for 1-3 d after towing. The developmental course of experimental neurites was found to be similar to that of unmanipulated spontaneous axons. Furthermore, the experimentally elongated neurites showed compartmentation of the axonal markers dephospho-tau and L-1 in towed outgrowth after 24 h. Extension of a second axon from an already polarized neuron does not lead to the loss of the spontaneous axon either immediately or after longer term growth. In addition, we were able to initiate neurites de novo that subsequently acquired axonal character even though spontaneous growth cone advance began while the towed neurite was still no longer than its sibling processes. This suggests that tension rather than the achievement of a critical neurite length determined axonal specification.     

Early ablation of target muscles modulates the arborisation pattern of an identified embryonic Drosophila motor axon.

The Drosophila RP3 motor axon establishes a stereotypic arborisation along the adjoining edges of muscles 6 and 7 by the end of embryogenesis. The present study has examined the role of the target muscles in determining this axonal arborisation pattern. Target muscles were surgically ablated prior to the arrival of the RP3 axon. Following further development of the embryo in culture medium, the morphology of target-deprived RP3 motor axons was assayed by intracellular injection with the dye Lucifer Yellow. Axonal arborisations were formed on a variety of non-target muscles when muscles 6 and 7 were removed and these contacts were maintained into stage 16. The pattern of axonal arborisations over non-target muscles varied between preparations in terms of the number of muscles contacted, and the distribution of arborisations on individual muscles. Following removal of muscle 6, the RP3 motor axon frequently contacted muscle 7, and axonal arborisations were present along the distal edge of the muscle. In the absence of muscle 7, the RP3 axon often did not contact muscle 6 and when muscle 6 was contacted, the arborisation of RP3 was poorly developed. Axonal processes were retained on non-target muscles when only one target muscle was present. Therefore, the establishment of a stereotypic arborisation by the RP3 motor axon is apparently dependent on growth cone contact with both target muscles.     

The role of microtubules in growth cone turning at substrate boundaries

To understand the role of microtubules in growth cone turning, we observed fluorescently labeled microtubules in neurons as they encountered a substrate boundary. Neurons growing on a laminin-rich substrate avoided growing onto collagen type IV. Turning growth cones assumed heterogeneous morphologies and behaviors that depended primarily in their extent of adhesion to the substrate. We grouped these behaviors into three categories-sidestepping, motility, and growth-mediated reorientation. In sidestepping and motility-mediated reorientation, the growth cone and parts of the axon were not well attached to the substrate so the acquisition of an adherent lamella caused the entire growth cone to move away from the border and consequently reoriented the axon. In these cases, since the motility of the growth cone dominates its reorientation, the microtubules were passive, and reorientation occurred without significant axon growth. In growth-mediated reorientation, the growth cone and axon were attached to the substrate. In this case, microtubules reoriented within the growth cone to stabilize a lamella. Bundling of the reoriented microtubules was followed by growth cone collapse to form new axon, and further, polarized lamellipodial extension. These observations indicate that when the growth cone remains adherent to the substrate during turning, the reorientation and bundling of microtubules is an important, early step in growth cone turning.     

Initial stages of neural regeneration in Helisoma trivolvis are dependent upon PLA2 activity

Neuronal regeneration after damage to an axon tract requires the rapid sealing of the injured plasma membrane and the subsequent formation of growth cones that can lead regenerating processes to their appropriate target. Membrane sealing and growth cone formation are Ca(2+)-dependent processes, but the signaling pathways activated by Ca(2+) to bring about these effects remain poorly understood. An in vitro injury model was employed in which neurites from identified snail neurons (Helisoma trivolvis) were transected with a glass microknife, and the formation of new growth cones from the distal portions of transected neurites was recorded at defined times after transection. This study presents three main results. First, phospholipase A(2) (PLA(2)), a calcium-activated enzyme, is necessary for membrane sealing in vitro. Second, PLA(2) activity is also required for the formation of a new growth cone after the membrane has sealed successfully. Thus, PLA(2) plays a dual role by affecting both growth cone formation and membrane sealing. Third, the injury-induced activation of PLA(2) by Ca(2+) controls growth cone formation through the production of leukotrienes, secondary metabolites of PLA(2) activity. Taken together, these results suggest that the injury-induced Ca(2+) influx acts via PLA(2) and leukotriene production to assure growth cone formation. These findings indicate that events that cause an inhibition of PLA(2) or lipoxygenases, enzymes that produce leukotrienes, could result in the inability of neurites to regenerate.     

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.     

The region-specific activities of lipid rafts during axon growth and guidance

The generation and control of cell polarity is a fundamental mechanism for directed migration of the cell. In developing neurons, the axonal growth cone recognizes environmental molecular cues and migrates toward its correct target, thereby forming neuronal networks. The spatial information provided by environmental cues directs axon growth and guidance through generating polarity of intracellular signals and cytoskeletal organization in the growth cone. This polarization process is dependent on lipid rafts, specialized microdomains in the cell membrane. Lipid rafts in specific regions of the growth cone are involved in axon growth and guidance. For example, forward migration of the growth cone requires raft membranes in its leading front. Recent experiments have suggested that lipid rafts function as a platform for localized signaling downstream of adhesion molecules and guidance receptors. The rafts assemble into an active membrane domain that captures and reorganizes the cytoskeletal machinery. In this way, the spatial control of signaling through raft membranes plays a critical role in translating extracellular information into polarized motility of the growth cone.     

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.