Neuronal clues to vascular guidance

The development of the vertebrate vascular system into a highly ordered and stereotyped network requires precise control over the branching and growth of new vessels. Recent research has highlighted the important role of genetic programs in regulating vascular patterning and in particular has established a crucial role for families of molecules previously described in controlling neuronal guidance. Like neurons, new vessels are guided along the correct path by integrating attractive and repulsive cues from the external environment. This is achieved by specialised endothelial cells at the leading tip of vessel sprouts which express receptor proteins that couple extracellular guidance signals with the cytoskeletal changes necessary to alter cell direction. Here, we review the genetic and in vitro evidence implicating four families of ligand-receptor signalling systems common to both neuronal and vessel guidance: the Ephrins and Eph receptors; Semaphorins, Neuropilins and Plexin receptors; Netrin and Unc5 receptors; and Slits and Robo receptors.     

When a diffusible axon guidance cue stops diffusing: roles for netrins in adhesion and morphogenesis

Netrins are a small family of secreted proteins that are best known for their role as secreted long-range chemotropic guidance cues. Extracellular gradients of netrin protein, established by diffusion, are thought to direct cell and axon migration during neural development. In addition to this long-range role, recent findings provide increasing support for short-range functions, in which secreted netrin protein remains closely associated with its cellular source. Emerging evidence for short-range actions of netrins suggests that they contribute to tissue morphogenesis by regulating cell-cell and cell-matrix adhesion.     

Navigating their way to the clinic: emerging roles for axon guidance molecules in neurological disorders and injury

The mechanisms underlying formation of the basic network of the nervous system are of fundamental interest in developmental neurobiology. During the wiring of the nervous system, newborn neurons send axons that travel long distances to their targets. These axons are directed by environmental cues, known as guidance cues, to their correct destinations. Through extensive studies in vertebrates and invertebrates many of the guidance cues and their receptors have been identified. Recently, guidance molecules have been suggested to have important roles in pathological conditions of the nervous system. Mutations in guidance receptors have been associated with hereditary neurological disorders, and deregulation of guidance cues might be associated with predisposition to epilepsy. In addition, it was suggested that guidance molecules play roles in the ability of the adult nervous system to recover and repair after injury. Thus, molecules that were first discovered as "developmental cues" are now emerging as important factors in neurological disease and injury in the adult.     

Mechanisms and Molecules of Neuronal Wiring: A Primer

The complex patterns of neuronal wiring in the adult nervous system depend on a series of guidance events during neural development that establish a framework on which functional circuits can be built. In this subject collection, the cellular and molecular mechanisms that underlie neuronal guidance are considered from several perspectives, ranging from how cytoskeletal dynamics within extending neuronal growth cones steer axons, to how guidance cues influence synaptogenesis. We introduce here some basic topics to frame the more detailed reviews in following articles, including the cellular strategies that define basic themes governing neuronal wiring throughout life, an enumeration of the molecular cues and receptors known to play key guidance roles during neural development, and an overview of the signaling mechanisms that transduce guidance information into growth-cone steering.Nerve processes extend toward their immediate and final targets with remarkable precision. At the tip of an extending axon is a flattened, fan-shaped structure called a growth cone, with many long, thin spikes that radiate outward much like fingers on a glove. Classical observations of neuronal growth cones and the formation of axonal and dendritic trajectories during neural development led to the conclusion that extrinsic cues must exist that have the capacity to steer extending neuronal processes. For over 100 years, neuroscientists have searched for these cues, their cell surface receptors, and an understanding of how the cues signal spatial information to the extending neuronal processes to direct neural circuit formation.A wealth of cellular observations indicate that growth cones are actively directed along their prescribed pathways. In this collection, Raper and Mason review the extensive body of experiments that support this view (Raper and Mason 2010). These studies reveal that neural wiring occurs through a combination of initial neuronal activity-independent guidance events, and that these early formed connections are subsequently refined through electrical signaling among neurons. The cues that initially guide axons and dendrites can function at both long and short ranges, and they are capable of influencing the bundling of axons together into nerves or fascicles (termed “fasciculation”) and also of mediating interactions between nerves and the substrates on which they extend (Fig. 1). Guidance cues associated with particular intermediate or final targets can be chemoattractive or chemorepulsive, and provide the information essential for selective guidance of distinct neuronal populations. Sequential responses to guidance cues as axons extend over very long distances toward their targets allow for complex pathways to develop, but this often requires that neurons extinguish their responses to certain cues and acquire responsiveness to others at key choice points. Much work over the past several decades has been devoted to identifying these guidance cues and their receptors, and to understanding how cellular responses to these cues change to allow for guidance of extending neuronal processes along discrete segments of their journey to their final targets.Open in a separate windowFigure 1.The diversity of neuronal guidance mechanisms. Neuronal processes are guided by cues that can function at long and short distances to mediate either attractive or repulsive guidance.     

<Emphasis Type="Italic">Drosophila</Emphasis> Dunc-115 mediates axon projection through actin binding

A central step in organizing the central nervous system development is the growth cone of an axon navigating through guidance cues to reach its specific target. While a great deal of this process has been understood especially in identifying the extracellular guidance cues and their membrane receptors, much less is known about how guidance signals are further relayed to the actin filaments that are central to the mobility of the growth cone. The previous results from our laboratory have shown that Drosophila gene dunc-115 regulates axon projection in the eye and the central nervous system. Furthermore, Dunc-115 has a villin-headpiece (VHD) domain, implying the possibility of binding to actin. To further characterize Dunc-115’s functions, we have identified the isoform Dunc-115L as a possible downstream target in relaying guidance cues further down to the cytoskeleton. Specifically, we have shown that Dunc-115 regulates neural connections in both the eye and the central nervous system in Drosophila and that Dunc-115 contains an actin-binding domain potentially capable of binding to actin filaments. In this report, we show that Dunc-115 binds to actin via its VHD domain directly, suggesting a possible mechanism for how Dunc-115 relays guidance signals.     

Regeneration and maintenance of the planarian midline is regulated by a slit orthologue

Several families of evolutionarily conserved axon guidance cues orchestrate the precise wiring of the nervous system during embryonic development. The remarkable plasticity of freshwater planarians provides the opportunity to study these molecules in the context of neural regeneration and maintenance. Here we characterize a homologue of the Slit family of guidance cues from the planarian Schmidtea mediterranea. Smed-slit is expressed along the planarian midline, in both dorsal and ventral domains. RNA interference (RNAi) targeting Smed-slit results in the collapse of many newly regenerated tissues at the midline; these include the cephalic ganglia, ventral nerve cords, photoreceptors, and the posterior digestive system. Surprisingly, Smed-slit RNAi knockdown animals also develop morphologically distinguishable, ectopic neural structures near the midline in uninjured regions of intact and regenerating planarians. These results suggest that Smed-slit acts not only as a repulsive cue required for proper midline formation during regeneration but that it may also act to regulate the behavior of neural precursors at the midline in intact planarians.     

Axon guidance: morphogens show the way

Hedgehog and Wnt family proteins can act as classic developmental morphogens to pattern a field of nai;ve cells. Surprising new studies show that members of these same protein families also act as guidance cues for growing axons in the developing nervous system.     

Signalling mechanisms mediating neuronal responses to guidance cues

Several families of extracellular guidance cues have been implicated in guiding neurons and axons to their appropriate destinations in the nervous system. Their receptors include single- and seven-transmembrane receptors, and their signal transduction pathways converge onto the Rho family of small GTPases, which control the cytoskeleton. A single guidance protein can use different mechanisms to regulate different kinds of motility or the motilities of different cell types. There is crosstalk between the signalling pathways initiated by distinct guidance cues. Studies of neuronal guidance mechanisms have shed light not only on neural development, but also on other processes that involve the extracellular regulation of the cytoskeleton.     

Emerging roles for neogenin and its ligands in CNS development

It is now well established that the netrin guidance cues and their receptors comprise a major molecular guidance system driving axon pathfinding during nervous system development. One netrin receptor, neogenin, is now emerging as a key regulator of many developmental processes throughout the embryo. Unexpectedly, a new family of neogenin ligands, the repulsive guidance molecule (RGM) family, has recently been identified. The functional outcome of neogenin activation is dictated by both the nature of the ligand as well as the developmental context. Netrin-1–neogenin interactions mediate chemoattractive axon guidance, while RGMa–neogenin interactions repel axons. Neogenin is required for the establishment of the pseudostratified epithelium of the neural tube, probably by promoting cell adhesion. In addition, a role for RGMa and neogenin in neuronal differentiation has been demonstrated. While neogenin signaling cascades are poorly understood, the opposing responses of neogenin to RGMa and netrin-1 in the context of axon guidance indicates that neogenin signaling is complex and subject to tight spatiotemporal regulation. In summary, neogenin is a multifunctional receptor regulating diverse developmental processes. Thus, its contribution to neural development is proving to be considerably more extensive than originally predicted.     

Secreted cell signaling molecules in axon guidance

The growth cones of developing neurons respond to specific guidance cues in their extracellular environment. Recent studies have shown that secreted signaling molecules from protein families that are best known for their roles as morphogens in specifying cell fate can function as axon guidance molecules. These signaling molecules seem to act directly on the growth cone and thus are likely to activate non-canonical signaling pathways that are coupled to the cytoskeleton.     

Semaphorins in the development,homeostasis and disease of hormone systems

Semaphorin proteins are among the best-studied families of guidance cues. Initially characterized as repulsive neuronal guidance cues, during the last decade, significant progress has been made in defining their involvement in the regulation of dynamic changes in the cellular cytoskeleton during embryonic and postnatal neuronal development, under both physiological and pathological conditions. However, semaphorins are not restricted to the nervous system but widely expressed in other tissues, where they play key roles in angiogenesis and organogenesis.In recent years, there has been an increasing emphasis on the potential influence of semaphorins on the development and homeostasis of hormone systems, and conversely, how circulating reproductive hormones regulate semaphorin expression. In this review, we summarize recent studies analyzing the contribution of semaphorin signaling to the morphogenesis, differentiation and plasticity of fundamental neuroendocrine and endocrine systems that regulate key physiological processes, such as reproduction, bone formation and the control of energy homeostasis.     

Axon guidance: asymmetric signaling orients polarized outgrowth

A network of connections is established as neural circuits form between neurons. To make these connections, neurons initiate asymmetric axon outgrowth in response to extracellular guidance cues. Within the specialized growth cones of migrating axons, F-actin and microtubules asymmetrically accumulate where an axon projects forward. Although many guidance cues, receptors and intracellular signaling components that are required for axon guidance have been identified, the means by which the asymmetry is established and maintained is unclear. Here, we discuss recent studies in invertebrate and vertebrate organisms that define a signaling module comprising UNC-6 (the Caenorhabditis elegans ortholog of netrin), UNC-40 (the C. elegans ortholog of DCC), PI3K, Rac and MIG-10 (the C. elegans ortholog of lamellipodin) and we consider how this module could establish polarized outgrowth in response to guidance cues.     

Semaphorins: a new class of immunoregulatory molecules

The immune and nervous systems play distinct roles in maintaining physiological homeostasis. Recent data indicates that these systems influence one another and share many proteins and pathways that are essential for their normal function and development. Molecules originally shown to be critical for the development of proper immune responses have recently been found to function in the nervous system. Conversely, neuronal guidance cues can modulate immune functions. Although semaphorins were originally identified as axon guidance factors active during neuronal development, several recent studies have identified indispensable functions for these molecules in the immune system. This review provides an overview of the rapidly emerging functions of semaphorins and their receptors in the immune system.     

A Slit/miR-218/Robo regulatory loop is required during heart tube formation in zebrafish

Members of the Slit family of secreted ligands interact with Roundabout (Robo) receptors to provide guidance cues for many cell types. For example, Slit/Robo signaling elicits repulsion of axons during neural development, whereas in endothelial cells this pathway inhibits or promotes angiogenesis depending on the cellular context. Here, we show that miR-218 is intronically encoded in slit2 and slit3 and that it suppresses Robo1 and Robo2 expression. Our data indicate that miR-218 and multiple Slit/Robo signaling components are required for heart tube formation in zebrafish and that this network modulates the previously unappreciated function of Vegf signaling in this process. These findings suggest a new paradigm for microRNA-based control of ligand-receptor interactions and provide evidence for a novel signaling pathway regulating vertebrate heart tube assembly.     

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.     

Emerging role of Paxillin-PKL in regulation of cell adhesion,polarity and migration

Cell adhesion and motility is of fundamental importance during development, normal physiology and pathologic conditions such as tumor metastasis. Focal adhesion (FA) proteins and their dynamic interactions play a critical role in the regulation of directed cell migration upon extracellular guidance cues. Using a combination of pharmacological inhibitors, knockout and knockdown cells, dominant negative and constitutively active mutants, we recently reported that the dynamic interaction and balancing phosphorylation of paxillin-PKL(GIT2) complex were critical for the cell polarity, thus directional migration upon cell adhesion and growth factor signaling. Similarly, restricted regulation of Arf6 and Rho families GTPase activities in polarized migrating cells is implicated in recent studies using cell culture and in vivo models.     

Regulation of growth cone actin dynamics by ADF/cofilin.

Nervous system development is reliant on neuronal pathfinding, the process in which axons are guided to their target cells by specific extracellular cues. The ability of neurons to extend over long distances in response to environmental guidance signals is made possible by the growth cone, a highly motile structure found at the end of neuronal processes. Growth cones detect directional cues and respond with either attractive or repulsive movements. The motility of growth cones is dependent on rapid reorganization of the actin cytoskeleton, presumably mediated by actin-associated proteins under the control of incoming guidance signals. This article reviews how one such family of proteins, the ADF/cofilins, are emerging as key regulators of growth cone actin dynamics. These proteins are essential for rapid actin turnover in a variety of different cell types. ADF/cofilins are heavily co-localized with actin in growth cones and are necessary for neurite outgrowth. ADF/cofilin activities are regulated through reversible phosphorylation by LIM kinases and slingshot phosphatases. LIM kinases are downstream effectors of the Rho GTPases Rho, Rac, and Cdc42. Growing evidence suggests that extracellular guidance cues may locally alter actin dynamics by regulating the activity of LIM kinase and ADF/cofilin phosphatases via the Rho GTPases. In this way, ADF/cofilins and their upstream effectors may be pivotal to our understanding of how guidance information is translated into physical alterations of the growth cone actin cytoskeleton.     

Mechanisms of astrocyte-directed neurite guidance

Astrocytes have recently become better recognized as playing vital roles in regulating the patterning of central nervous system neurites during development and following injury. In general, astrocytes have been shown to be supportive of neurite extension, but alterations in the biochemical properties of astrocytes in particular areas during development and in gliotic tissue may act to confine neurite outgrowth and thus provide guidance cues. In vivo studies indicate that restrictive astrocytes function through their altered expression of specific extracellular matrix molecules, including tenascin, chondroitin, and keratan sulfate proteoglycans. In addition, several in vitro models suggest that other cell surface molecules are utilized by restrictive astrocytes to direct neurite trajectories. Received: 5 May 1997 / Accepted: 6 June 1997     

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.