Axon Guidance at the Midline: From Mutants to Mechanisms

How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Understanding the mechanisms that mediate axon guidance will give important insight into how the nervous system is correctly wired during development and may have implications for therapeutic approaches to developmental brain disorders and nerve regeneration. Achieving this understanding will require unraveling the molecular logic that ensures the proper expression and localization of axon guidance cues and receptors, and elucidating the signaling events that regulate the growth cone cytoskeleton in response to guidance receptor activation. Studies of axon guidance at the midline of many experimental systems, from the ventral midline of Drosophila to the vertebrate spinal cord, have led to important mechanistic insights into the complex problem of wiring the nervous system. Here we review recent advances in understanding the regulation of midline axon guidance, with a particular emphasis on the contributions made from molecular genetic studies of invertebrate model systems.     

Axon guidance at the midline: from mutants to mechanisms

How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Understanding the mechanisms that mediate axon guidance will give important insight into how the nervous system is correctly wired during development and may have implications for therapeutic approaches to developmental brain disorders and nerve regeneration. Achieving this understanding will require unraveling the molecular logic that ensures the proper expression and localization of axon guidance cues and receptors, and elucidating the signaling events that regulate the growth cone cytoskeleton in response to guidance receptor activation. Studies of axon guidance at the midline of many experimental systems, from the ventral midline of Drosophila to the vertebrate spinal cord, have led to important mechanistic insights into the complex problem of wiring the nervous system. Here we review recent advances in understanding the regulation of midline axon guidance, with a particular emphasis on the contributions made from molecular genetic studies of invertebrate model systems.     

The Yin and Yang of Wnt/Ryk axon guidance in development and regeneration

In the developing embryo,nascent axons navigate towards their specific targets to establish the intricate network of axonal connections linking neurons within the mature nervous system.Molecular navigational systems comprising repulsive and attractive guidance cues form chemotactic gradients along the pathway of the exploring growth cone.Axon-bound receptors detect these gradients and determine the trajectory of the migrating growth cone.In contrast to their benevolent role in the developing nervous system,repulsive guidance receptors are detrimental to the axon’s ability to regenerate after injury in the adult.In this review we explore the essential and beneficial role played by the chemorepulsive Wnt receptor,Ryk/Derailed in axon navigation in the embryonic nervous system(the Yin function).Specifically,we focus on the role of Wnt5a/Rykmediated guidance in the establishment of two major axon tracts in the mammalian central nervous system,the corticospinal tract and the corpus callosum.Recent studies have also identified Ryk as a major suppressor of axonal regeneration after spinal cord injury.Thus,we also discuss this opposing aspect of Ryk function in axonal regeneration where its activity is a major impediment to axon regrowth(the Yang function).     

Neogenin and repulsive guidance molecule signaling in the central nervous system

The repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system. Functional studies have revealed that it has roles in axon guidance and laminar patterning in Xenopus and chick embryos, and in controlling cephalic neural tube closure in mouse embryos. The recent identification of neogenin as a receptor for RGM has provided evidence of the diverse functions of this ligand-receptor pair. Re-expression of RGM is observed after injury in the adult human and rat central nervous systems. Inhibition of RGM enhances growth of injured axons and promotes functional recovery after spinal cord injury in rats. Thus, re-expression of embryonic repulsive cues in adult tissues contributes to failure of axon regeneration in the central nervous system.     

EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.     

Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans

The molecular and cellular mechanisms that allow adult-stage neurons to regenerate following damage are poorly understood. Recently, axons of motoneurons and mechanosensory neurons in adult C. elegans were found to regrow after being snipped by femtosecond laser ablation. Here, we explore the molecular determinants of adult-stage axon regeneration using the AVM mechanosensory neurons. The first step in AVM axon development is a pioneer axonal projection from the cell body to the ventral nerve cord. We show that regeneration of the AVM axon to the ventral nerve cord lacks the deterministic precision of initial axon development, requiring competition and pruning of unwanted axon branches. Nevertheless, axons of injured AVM neurons regrow to the ventral nerve cord with over 60% reliability in adult animals. In addition, in contrast to initial development, axon guidance during regeneration becomes heavily dependent on cytoplasmic protein MIG-10/Lamellipodin but independent of UNC-129/TGF-beta repellent and UNC-40/DCC receptor, and axon growth during regeneration becomes heavily dependent on UNC-34/Ena and CED-10/Rac actin regulators. Thus, C. elegans may be used as a genetic system to characterize novel cellular and molecular mechanisms underlying adult-stage nervous system regeneration.     

Extracellular matrix of the central nervous system: from neglect to challenge

The basic concept, that specialized extracellular matrices rich in hyaluronan, chondroitin sulfate proteoglycans (aggrecan, versican, neurocan, brevican, phosphacan), link proteins and tenascins (Tn-R, Tn-C) can regulate cellular migration and axonal growth and thus, actively participate in the development and maturation of the nervous system, has in recent years gained rapidly expanding experimental support. The swift assembly and remodeling of these matrices have been associated with axonal guidance functions in the periphery and with the structural stabilization of myelinated fiber tracts and synaptic contacts in the maturating central nervous system. Particular interest has been focused on the putative role of chondroitin sulfate proteoglycans in suppressing central nervous system regeneration after lesions. The axon growth inhibitory properties of several of these chondroitin sulfate proteoglycans in vitro, and the partial recovery of structural plasticity in lesioned animals treated with chondroitin sulfate degrading enzymes in vivo have significantly contributed to the increased awareness of this long time neglected structure.     

Eph and ephrin signaling: lessons learned from spinal motor neurons

In nervous system assembly, Eph/ephrin signaling mediates many axon guidance events that shape the formation of precise neuronal connections. However, due to the complexity of interactions between Ephs and ephrins, the molecular logic of their action is still being unraveled. Considerable advances have been made by studying the innervation of the limb by spinal motor neurons, a series of events governed by Eph/ephrin signaling. Here, we discuss the contributions of different Eph/ephrin modes of interaction, downstream signaling and electrical activity, and how these systems may interact both with each other and with other guidance molecules in limb muscle innervation. This simple model system has emerged as a very powerful tool to study this set of molecules, and will continue to be so by virtue of its simplicity, accessibility and the wealth of pioneering cellular studies.     

Semaphorin-neuropilin-1 interactions in plasticity and regeneration of adult neurons

During development, axonal growth cones are guided to their appropriate targets by many attractive and repulsive cues. It has become increasingly clear over the last few years that how the growth cone responds to these cues depends both on the molecular nature of the cue and on the internal state of the neuron. The unexpected result is that the same molecule can act as an attractor or as a repellent. A number of guidance cues used by neurons during development are retained in the adult nervous system, where their function is often still unclear. Most of these molecules are implicated in plasticity in the adult nervous system and can play a role (sometimes maladaptive) in neuronal regeneration after injury. A group of axonal guidance cues that has been well studied in development is the semaphorin family of secreted and membrane-anchored proteins, which has been implicated in axon steering, fasciculation, branching and synapse formation. This review focuses on semaphorin-3A (probably the best-characterized semaphorin) and its receptors (in particular neuropilin-1) in the adult nervous system and argues that semaphorin-3A plays a role in the maintenance and regeneration of adult sensory neurons.     

Pioneering studies on the mechanisms of neuronal morphogenesis

Axon outgrowth and pathfinding occurs through a complex series of interacting biochemical signaling pathways that regulate the motility of neuronal growth cones. Over the past 30 years, Paul Letourneau and his students have explored the molecular basis of growth cone motility and have contributed immensely to this field. In celebration of his 65th birthday, this essay is written in gratitude for Paul's many contributions and training.     

Mechanisms of neuronal growth cone guidance: an historical perspective

At the distal most aspect of motile extending axons and dendrites lies the growth cone, a hand like macroorganelle of membrane bound cytoskeleton, packed with receptors, adhesion molecules, molecular motors, and an army of regulatory and signaling proteins. Splayed out along the substratum in vitro, the growth cone resembles an open hand with bundles of filamentous actin, barbed ends outstretched, as if fingers extending from a central domain of dynamic microtubule plus ends. The growth cone acts first as a sensory platform, analyzing the environment ahead for the presence of guidance cues, secondly as a mechanical dynamo establishing focal contact with the extracellular matrix to drive processive forward outgrowth, and thirdly as a forward biochemical command center where signals are interrogated to inform turning, extension, retraction, or branching. During his career, Paul Letourneau has made major contributions to our understanding of how growth cones respond to their environment. Here, we will summarize some of these major advances in their historical context. Letourneau's contributions have provided insights into cytoskeletal organization, growth cone dynamics, and signaling pathways. His recent work has described some important molecules and molecular mechanisms involved in growth cone turning. Although much remains to be understood about this important and intriguing structure, Letourneau's contributions have provided us with "growth cone guidance."     

EphA4-dependent axon guidance is mediated by the RacGAP alpha2-chimaerin

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. We show that the Rac-specific GTPase-activating protein alpha2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. alpha-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. Our results reveal an alpha-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo.     

Growth and guidance cues for regenerating axons: where have they gone?

Both attractive and repellent cues are required to guide developing axons to their targets in the central nervous system. Critical guidance molecules in the developing brain include the semaphorins, netrins, slits, and ephrins. Current research indicates that many of these molecules and their receptors are expressed in the adult central nervous system (CNS), and that injury can alter the levels of these ligands/receptors. Recent studies have begun the process of elucidating the functions of these receptors in adult mammals, and the effects that they have on the regeneration of adult neurons. This review addresses our current knowledge with respect to the response of adult CNS neurons to axonal injury, interventions for enhancing the survival and regeneration of injured neurons, and the expression of developmental axon guidance cues in the injured mature CNS, with specific focus on the retino-tectal projection.     

Calcium/calmodulin-dependent protein kinase II regulates mammalian axon growth by affecting F-actin length in growth cone

While axon regeneration is a key determinant of functional recovery of the nervous system after injury, it is often poor in the mature nervous system. Influx of extracellular calcium (Ca²⁺) is one of the first phenomena that occur following axonal injury, and calcium/calmodulin-dependent protein kinase II (CaMKII), a target substrate for calcium ions, regulates the status of cytoskeletal proteins such as F-actin. Herein, we found that peripheral axotomy activates CaMKII in dorsal root ganglion (DRG) sensory neurons, and inhibition of CaMKII impairs axon outgrowth in both the peripheral and central nervous systems (PNS and CNS, respectively). Most importantly, we also found that the activation of CaMKII promotes PNS and CNS axon growth, and regulatory effects of CaMKII on axon growth occur via affecting the length of the F-actin. Thus, we believe our findings provide clear evidence that CaMKII is a critical modulator of mammalian axon regeneration.     

Imaging voltage in neurons

The formation, maintenance, and plasticity of neural circuits rely upon a complex interplay between progressive and regressive events. Increasingly, new functions are being identified for axon guidance molecules in the dynamic processes that occur within the embryonic and adult nervous system. The magnitude, duration, and spatial activity of axon guidance molecule signaling are precisely regulated by a variety of molecular mechanisms. Here we focus on recent progress in understanding the role of protease-mediated cleavage of guidance factors required for directional axon growth, with a particular emphasis on the role of metalloprotease and γ-secretase. Since axon guidance molecules have also been linked to neural degeneration and regeneration in adults, studies of guidance receptor proteolysis are beginning to define new relationships between neurodevelopment and neurodegeneration. These findings raise the possibility that the signaling checkpoints controlled by proteases could be useful targets to enhance regeneration.     

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA-Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.     

Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons

The identification of surface proteins restricted to subsets of embryonic axons and growth cones may provide information on the mechanisms underlying axon fasciculation and pathway selection in the vertebrate nervous system. We describe here the characterization of a 135 kd cell surface glycoprotein, TAG-1, that is expressed transiently on subsets of embryonic spinal cord axons and growth cones. TAG-1 is immunochemically distinct from the cell adhesion molecules N-CAM and L1 (NILE) and is expressed on commissural and motor neurons over the period of initial axon extension. Moreover, TAG-1 and L1 appear to be segregated on different segments of the same embryonic spinal axons. These observations provide evidence that axonal guidance and pathway selection in vertebrates may be regulated in part by the transient and selective expression of distinct surface glycoproteins on subsets of developing neurons.     

Nitric oxide as a regulator of neuronal motility and regeneration in the locust embryo

Nitric oxide (NO) is known as a gaseous messenger in the nervous system. It plays a role in synaptic plasticity, but also in development and regeneration of nervous systems. We have studied the function of NO and its signaling cascade via cyclic GMP in the locust embryo. Its developing nervous system is well suited for pharmacological manipulations in tissue culture. The components of this signaling pathway are localized by histochemical and immunofluorescence techniques. We have analyzed cellular mechanisms of NO action in three examples: 1. in the peripheral nervous system during antennal pioneer axon outgrowth, 2. in the enteric nervous system during migration of neurons forming the midgut nerve plexus, and 3. in the central nervous system during axonal regeneration of serotonergic neurons after axotomy. In each case, internally released NO or NO-induced cGMP synthesis act as permissive signals for the developmental process. Carbon monoxide (CO), as a second gaseous messenger, modulates enteric neuron migration antagonistic to NO.     

Autophagy in axonal and presynaptic development

The study of autophagy in the nervous system has predominantly centered on degeneration. Evidence is now cementing crucial roles for autophagy in neuronal development and growth, especially in axonal and presynaptic compartments. A picture is emerging that autophagy typically promotes the growth of axons and reduces presynaptic stability. Nonetheless, these are not rigid principles, and it remains unclear why autophagy does not always display these relationships during axonal and presynaptic development. Recent progress has identified mechanisms underlying spatiotemporal control of autophagy in neurons and begun to unravel how autophagy is integrated with other cellular processes, such as proteasomal degradation and axon guidance. Ultimately, understanding how autophagy is regulated and its role in the developing nervous system is key to comprehending how the nervous system assembles its stereotyped yet plastic configuration. It is also likely to inform how we think about neurodevelopmental disorders and neurodegenerative diseases.     

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.