Responses of temporal retinal growth cones to ephrinA5-coated beads

The topographic positioning of retinal axons in the optic tectum is regulated, at least in part, by ephrinA/EphA repulsive interactions. Temporal axons, expressing high levels of EphA receptors, project to the ephrinA5-poor anterior tectum and avoid the ephrinA5-rich posterior tectum. To examine the dynamic behavior of temporal growth cones when they first encounter ephrinA, we manipulated ephrinA-coated beads with a laser tweezer into desired positions around the growth cones of chick retinal axons in culture. At high concentrations of ephrinA5 on the beads, growth cones typically collapsed on contacting the bead. At low concentrations, however, growth cones showed heterogeneous responses with some growth cones showing repulsive turning and others showing attractive turning after contacting the bead. Experiments with two beads indicate that retinal axons integrate guidance information that is provided simultaneously at two discrete locations. When a time-delay was introduced between exposure to the first and the second bead, individual axons exhibited a stereotyped response to the repeated stimuli, either responding with attraction followed by attraction, or showing repulsion followed by repulsion or collapse. Our results suggest the existence of at least two retinal subpopulations from the temporal retina, one being attracted, another being repelled by low levels of ephrinA5. These findings demonstrate that temporal retinal axons are not universally repelled by ephrinA5 and suggest that their ability to respond differentially to low concentrations may help them to map in a continuous manner over the surface of the anterior tectum.     

Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals

Growing axons encounter multiple guidance cues, but it is unclear how separate signals are resolved and integrated into coherent instructions for growth cone navigation. We report that glycosylphosphatidylinositol (GPI)-anchored ephrin-As function as "reverse" signaling receptors for motor axons when contacted by transmembrane EphAs present in the dorsal limb. Ephrin-A receptors are thought to depend on transmembrane coreceptors for transmitting signals intracellularly. We show that the receptor tyrosine kinase Ret is required for motor axon attraction mediated by ephrin-A reverse signaling. Ret?also mediates GPI-anchored GFRα1 signaling in response to GDNF, a diffusible chemoattractant in the limb, indicating that Ret is a multifunctional coreceptor for guidance molecules. Axons respond synergistically to coactivation by GDNF and EphA ligands, and these cooperative interactions are gated by GFRα1 levels. Our studies uncover a hierarchical GPI-receptor signaling network that is constructed from combinatorial components and integrated through Ret using ligand coincidence detection.     

Responses of temporal retinal growth cones to ephrinA5‐coated beads

The topographic positioning of retinal axons in the optic tectum is regulated, at least in part, by ephrinA/EphA repulsive interactions. Temporal axons, expressing high levels of EphA receptors, project to the ephrinA5‐poor anterior tectum and avoid the ephrinA5‐rich posterior tectum. To examine the dynamic behavior of temporal growth cones when they first encounter ephrinA, we manipulated ephrinA‐coated beads with a laser tweezer into desired positions around the growth cones of chick retinal axons in culture. At high concentrations of ephrinA5 on the beads, growth cones typically collapsed on contacting the bead. At low concentrations, however, growth cones showed heterogeneous responses with some growth cones showing repulsive turning and others showing attractive turning after contacting the bead. Experiments with two beads indicate that retinal axons integrate guidance information that is provided simultaneously at two discrete locations. When a time‐delay was introduced between exposure to the first and the second bead, individual axons exhibited a stereotyped response to the repeated stimuli, either responding with attraction followed by attraction, or showing repulsion followed by repulsion or collapse. Our results suggest the existence of at least two retinal subpopulations from the temporal retina, one being attracted, another being repelled by low levels of ephrinA5. These findings demonstrate that temporal retinal axons are not universally repelled by ephrinA5 and suggest that their ability to respond differentially to low concentrations may help them to map in a continuous manner over the surface of the anterior tectum. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Axon guidance: receptor complexes and signaling mechanisms

The generation of a functional neuronal network requires that axons navigate precisely to their appropriate targets. Molecules that specify guidance decisions have been identified, and the signaling events that occur downstream of guidance receptors are beginning to be understood. New research shows that guidance receptor signaling can be hierarchical -- one receptor silencing the other -- thereby allowing navigating growth cones to interpret opposing guidance cues. Among the known intracellular signaling molecules shared by all guidance receptor families, Rho GTPases appear to be primary regulators of actin dynamics and growth cone guidance. Novel effector molecules complete the picture and suggest additional signaling mechanisms.     

Regulation and function of axon guidance and adhesion molecules during olfactory map formation

The olfactory system presents a practical model for investigating basic mechanisms involved in patterning connections between peripheral sensory neurons and central targets. Our understanding of olfactory map formation was advanced greatly by the discovery of cAMP signaling as an important determinant of glomerular positioning in the olfactory bulb. Additionally, several cell adhesion molecules have been identified recently that are proposed to regulate homotypic interactions among projecting axons. From these studies a model has emerged to partially explain the wiring of axons from widely dispersed neuron populations in the nasal cavity to relatively stereotyped glomerular positions. These advances have revitalized interest in axon guidance molecules in establishing olfactory topography, but also open new questions regarding how these patterns of guidance cues are established and function, and what other pathways, such as glycosylation, might be involved. This review summarizes the current state of this field and the important molecules that impact on cAMP-dependent mechanism in olfactory axon guidance.     

cAMP differentially regulates axonal and dendritic development of dentate granule cells

Neurite polarity is a morphological characteristic of dentate gyrus granule cells, which extend axons to the hilar region and dendrites in the opposite direction, i.e. to the molecular layer. This remarkable polarity must require a differential system for axon and dendrite guidance. Here, we report that the axon and dendrites of a granule cell are differentially responsive to cAMP. In developing cultures of dispersed granule cells, dendritic growth cones were increased in number after pharmacological activation of cAMP signaling and decreased after blockade of cAMP signaling. Activation of cAMP signaling antagonized dendritic collapse induced by the potent repellents Sema3F and glutamate. In contrast to dendrites, axons were protected from Sema3F-induced collapse when cAMP signaling was inhibited. Axonal and dendritic growth cones both expressed type 1 adenylyl cyclase, but only axons showed a cAMP increase in response to Sema3F, and the elevated cAMP was sufficient to collapse axonal growth cones. Thus, the axons and dendrites of dentate granule cells differ in the regulation of cAMP levels as well as responsiveness to cAMP. cAMP may be crucial for shaping the information flow polarity in the dentate gyrus circuit.     

cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury

Central neurons regenerate axons if a permissive environment is provided; after spinal cord injury, however, inhibitory molecules are present that make the local environment nonpermissive. A promising new strategy for inducing neurons to overcome inhibitory signals is to activate cAMP signaling. Here we show that cAMP levels fall in the rostral spinal cord, sensorimotor cortex and brainstem after spinal cord contusion. Inhibition of cAMP hydrolysis by the phosphodiesterase IV inhibitor rolipram prevents this decrease and when combined with Schwann cell grafts promotes significant supraspinal and proprioceptive axon sparing and myelination. Furthermore, combining rolipram with an injection of db-cAMP near the graft not only prevents the drop in cAMP levels but increases them above those in uninjured controls. This further enhances axonal sparing and myelination, promotes growth of serotonergic fibers into and beyond grafts, and significantly improves locomotion. These findings show that cAMP levels are key for protection, growth and myelination of injured CNS axons in vivo and recovery of function.     

Growth cones integrate signaling from multiple guidance cues.

Nerve growth factor (NGF) and semaphorin3A (Sema3A) are guidance cues found in pathways and targets of developing dorsal root ganglia (DRG) neurons. DRG growth cone motility is regulated by cytoplasmic signaling triggered by these molecules. We investigated interactions of NGF and Sema3A in modulating growth cone behaviors of axons extended from E7 chick embryo DRGs. Axons extending in collagen matrices were repelled by Sema3A released from transfected HEK293 cells. However, if an NGF-coated bead was placed adjacent to Sema3A-producing cells, axons converged at the NGF bead. Growth cones of DRGs raised in 10(-9) M NGF were more resistant to Sema3A-induced collapse than when DRGs were raised in 10(-11) M NGF. After overnight culture in 10(-11) M NGF, 1-hr treatment with 10(-9) M NGF also increased growth cone resistance to Sema3A. Pharmacological studies indicated that the activities of ROCK and PKG participate in the cytoskeletal alterations that lead to Sema3A-induced growth cone collapse, whereas PKA activity is required for NGF-mediated reduction of Sema3A-induced growth cone collapse. These results support the idea that growth cone responses to a guidance cue can be modulated by interactions involving coincident signaling by other guidance cues.     

The Abelson tyrosine kinase, the Trio GEF and Enabled interact with the Netrin receptor Frazzled in Drosophila

The attractive Netrin receptor Frazzled (Fra), and the signaling molecules Abelson tyrosine kinase (Abl), the guanine nucleotide-exchange factor Trio, and the Abl substrate Enabled (Ena), all regulate axon pathfinding at the Drosophila embryonic CNS midline. We detect genetic and/or physical interactions between Fra and these effector molecules that suggest that they act in concert to guide axons across the midline. Mutations in Abl and trio dominantly enhance fra and Netrin mutant CNS phenotypes, and fra;Abl and fra;trio double mutants display a dramatic loss of axons in a majority of commissures. Conversely, heterozygosity for ena reduces the severity of the CNS phenotype in fra, Netrin and trio,Abl mutants. Consistent with an in vivo role for these molecules as effectors of Fra signaling, heterozygosity for Abl, trio or ena reduces the number of axons that inappropriately cross the midline in embryos expressing the chimeric Robo-Fra receptor. Fra interacts physically with Abl and Trio in GST-pulldown assays and in co-immunoprecipitation experiments. In addition, tyrosine phosphorylation of Trio and Fra is elevated in S2 cells when Abl levels are increased. Together, these data suggest that Abl, Trio, Ena and Fra are integrated into a complex signaling network that regulates axon guidance at the CNS midline.     

Modeling Development in Retinal Afferents: Retinotopy,Segregation, and EphrinA/EphA Mutants

During neural development, neurons extend axons to target areas of the brain. Through processes of growth, branching and retraction these axons establish stereotypic patterns of connectivity. In the visual system, these patterns include retinotopic organization and the segregation of individual axons onto different subsets of target neurons based on the eye of origin (ocular dominance) or receptive field type (ON or OFF). Characteristic disruptions to these patterns occur when neural activity or guidance molecule expression is perturbed. In this paper we present a model that explains how these developmental patterns might emerge as a result of the coordinated growth and retraction of individual axons and synapses responding to position-specific markers, trophic factors and spontaneous neural activity. This model derives from one presented earlier (Godfrey et al., 2009) but which is here extended to account for a wider range of phenomena than previously described. These include ocular dominance and ON-OFF segregation and the results of altered ephrinA and EphA guidance molecule expression. The model takes into account molecular guidance factors, realistic patterns of spontaneous retinal wave activity, trophic molecules, homeostatic mechanisms, axon branching and retraction rules and intra-axonal signaling mechanisms that contribute to the survival of nearby synapses on an axon. We show that, collectively, these mechanisms can account for a wider range of phenomena than previous models of retino-tectal development.     

Repulsive guidance of axons of spinal sensory neurons in Xenopus laevis embryos: roles of Contactin and notochord-derived chondroitin sulfate proteoglycans

An immunoglobulin superfamily neuronal adhesion molecule, Contactin, has been implicated in axon guidance of spinal sensory neurons in Xenopus embryos. To identify the guidance signaling molecules that Contactin recognizes in tailbud embryos, an in situ binding assay was performed using recombinant Contactin-alkaline phosphatase fusion protein (Contactin-AP) as a probe. In the assay of whole-mount or sectioned embryos, Contactin-AP specifically bound to the notochord and its proximal regions. This binding was completely blocked by either digestion of embryo sections with chondroitinase ABC or pretreatment of Contactin-AP with chondroitin sulfate A. When the spinal cord and the notochord explants were co-cultured in collagen gel, growing Contactin-positive spinal axons were repelled by notochord-derived repulsive activity. This repulsive activity was abolished by the addition of either a monoclonal anti-Contactin antibody, chondroitin sulfate A or chondroitinase ABC to the culture medium. An antibody that recognizes chondroitin sulfate A and C labeled immunohistochemically the notochord in embryo sections and the collagen gel matrix around the cultured notochord explant. Addition of chondroitinase ABC into the culture eliminated the immunoreactivity in the gel matrix. These results suggest that the notochord-derived chondroitin sulfate proteoglycan acts as a repulsive signaling molecule that is recognized by Contactin on spinal sensory axons.     

Follow your nose: axon pathfinding in olfactory map formation

Two new studies report how discrete identities of olfactory sensory neurons are converted into a spatial map of axonal connections (Imai et al., 2006; Serizawa et al., 2006). They find that levels of cAMP signals derived from olfactory receptors (ORs) can direct targeting of axons along an axis, and that ORs and neural activity regulate expression of adhesion/guidance molecules in mosaic patterns that can sort axons into discrete locations.     

Spatial targeting of type II protein kinase A to filopodia mediates the regulation of growth cone guidance by cAMP

The second messenger cyclic adenosine monophosphate (cAMP) plays a pivotal role in axonal growth and guidance, but its downstream mechanisms remain elusive. In this study, we report that type II protein kinase A (PKA) is highly enriched in growth cone filopodia, and this spatial localization enables the coupling of cAMP signaling to its specific effectors to regulate guidance responses. Disrupting the localization of PKA to filopodia impairs cAMP-mediated growth cone attraction and prevents the switching of repulsive responses to attraction by elevated cAMP. Our data further show that PKA targets protein phosphatase-1 (PP1) through the phosphorylation of a regulatory protein inhibitor-1 (I-1) to promote growth cone attraction. Finally, we find that I-1 and PP1 mediate growth cone repulsion induced by myelin-associated glycoprotein. These findings demonstrate that the spatial localization of type II PKA to growth cone filopodia plays an important role in the regulation of growth cone motility and guidance by cAMP.     

Bifunctional action of ephrin-B1 as a repellent and attractant to control bidirectional branch extension in dorsal-ventral retinotopic mapping

We report that the EphB receptor ligand, ephrin-B1, may act bifunctionally as both a branch repellent and attractant to control the unique mechanisms in mapping the dorsal-ventral (DV) retinal axis along the lateral-medial (LM) axis of the optic tectum. EphB receptors are expressed in a low to high DV gradient by retinal ganglion cells (RGCs), and ephrin-B1 is expressed in a low to high LM gradient in the tectum. RGC axons lack DV ordering along the LM tectal axis, but directionally extend interstitial branches that establish retinotopically ordered arbors. Recent studies show that ephrin-B1 acts as an attractant in DV mapping and in controlling directional branch extension. Modeling indicates that proper DV mapping requires that this attractant activity cooperates with a repellent activity in a gradient that mimics ephrin-B1. We show that ectopic domains of high, graded ephrin-B1 expression created by retroviral transfection repel interstitial branches of RGC axons and redirect their extension along the LM tectal axis, away from their proper termination zones (TZs). In contrast, the primary RGC axons are unaffected and extend through the ectopic domains of ephrin-B1 and arborize at the topographically correct site. However, when the location of a TZ is coincident with ectopic domains of ephrin-B1, the domains appear to inhibit arborization and shape the distribution of arbors. Our findings indicate that ephrin-B1 selectively controls, through either attraction or repulsion, the directional extension and arborization of interstitial branches extended by RGC axons arising from the same DV position: branches that arise from axons positioned lateral to the correct TZ are attracted up the gradient of ephrin-B1 and branches that arise from axons positioned medial to the same TZ are repelled down the ephrin-B1 gradient. Alternatively, EphB receptor signaling may act as a 'ligand-density sensor' and titrate signaling pathways that promote branch extension toward an optimal ephrin-B1 concentration found at the TZ; branches located either medial or lateral to the TZ would encounter a gradient of increasingly favored attachment in the direction of the TZ.     

Lens-derived Semaphorin3A regulates sensory innervation of the cornea

The cornea, one of the most highly innervated tissues of the body, is innervated by trigeminal sensory afferents. During development, axons are initially repelled at the corneal margin, resulting in the formation of a circumferential nerve ring. The nature and source of guidance molecules that regulate this process remain a mystery. Here, we show that the lens, which immediately underlies the cornea, repels trigeminal axons in vivo and in vitro. Lens ablation results in premature, disorganized corneal innervation and disruption of the nerve ring and ventral plexus. We show that Semaphorin3A (Sema3A) is expressed in the lens epithelium and its receptor Neuropilin-1 (Npn1) is expressed in the trigeminal ganglion during cornea development. Inhibition of Sema3A signaling abrogates axon repulsion by the lens and cornea in vitro and phenocopies lens removal in vivo. These results demonstrate that lens-derived Sema3A mediates initial repulsion of trigeminal sensory axons from the cornea and is necessary for the proper formation of the nerve ring and positioning of the ventral plexus in the choroid fissure.     

UNC-6/netrin and its receptors UNC-5 and UNC-40/DCC modulate growth cone protrusion in vivo in C. elegans

The UNC-6/netrin guidance cue functions in axon guidance in vertebrates and invertebrates, mediating attraction via UNC-40/DCC family receptors and repulsion via by UNC-5 family receptors. The growth cone reads guidance cues and extends lamellipodia and filopodia, actin-based structures that sense the extracellular environment and power the forward motion of the growth cone. We show that UNC-6/netrin, UNC-5 and UNC-40/DCC modulated the extent of growth cone protrusion that correlated with attraction versus repulsion. Loss-of-function unc-5 mutants displayed increased protrusion in repelled growth cones, whereas loss-of-function unc-6 or unc-40 mutants caused decreased protrusion. In contrast to previous studies, our work suggests that the severe guidance defects in unc-5 mutants may be due to latent UNC-40 attractive signaling that steers the growth cone back towards the ventral source of UNC-6. UNC-6/Netrin signaling also controlled polarity of growth cone protrusion and F-actin accumulation that correlated with attraction versus repulsion. However, filopodial dynamics were affected independently of polarity of protrusion, indicating that the extent versus polarity of protrusion are at least in part separate mechanisms. In summary, we show here that growth cone guidance in response to UNC-6/netrin involves a combination of polarized growth cone protrusion as well as a balance between stimulation and inhibition of growth cone (e.g. filopodial) protrusion.     

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.     

SEXUAL SELECTION AND FEMALE CHOICE IN MOLE CRICKETS (SCAPTERISCUS: GRYLLOTALPIDAE): MODELLING THE EFFECTS OF INTENSITY AND MALE SPACING

ABSTRACT

Attraction of flying mole crickets to individual males calling in an outdoor arena was influenced significantly by the relative intensity of the males calling in the arena. Louder males attracted more individuals than males whose calling songs were less intense. Making simple assumptions about the acoustic output from calling males and about the flight pattern and response of flying females, the differential attraction can be explained by a mathematical model. Computer simulation of the model was used to examine the importance of a male's intensity relative to others and the effect of distance between males on the attraction of females. The model and its relation to active female choice and passive attraction are discussed. The model makes predictions about differences in spacing behaviour of males that maximize attraction relative to other males.     

Calcium mediates bidirectional growth cone turning induced by myelin-associated glycoprotein

Cytoplasmic second messengers, Ca2+ and cAMP, regulate nerve growth cone turning responses induced by many guidance cues, but the causal relationship between these signaling pathways has been unclear. We here report that, for growth cone turning induced by a gradient of myelin-associated glycoprotein (MAG), cAMP acts by modulating MAG-induced Ca2+ signaling. Growth cone repulsion induced by MAG was accompanied by localized Ca2+ signals on the side of the growth cone facing the MAG source, due to Ca2+ release from intracellular stores. Elevating cAMP signaling activity or membrane depolarization enhanced MAG-induced Ca2+ signals and converted growth cone repulsion to attraction. Directly imposing high- or low-amplitude Ca2+ signals with an extracellular gradient of Ca2+ ionophore was sufficient to trigger either attractive or repulsive turning, respectively. Thus, distinct Ca2+ signaling, which can be modulated by cAMP, mediates the bidirectional turning responses induced by MAG.