On the turning of Xenopus retinal axons induced by ephrin-A5

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, play important roles during development of the nervous system. Frequently they exert their functions through a repellent mechanism, so that, for example, an axon expressing an Eph receptor does not invade a territory in which an ephrin is expressed. Eph receptor activation requires membrane-associated ligands. This feature discriminates ephrins from other molecules sculpturing the nervous system such as netrins, slits and class 3 semaphorins, which are secreted molecules. While the ability of secreted molecules to guide axons, i.e. to change their growth direction, is well established in vitro, little is known about this for the membrane-bound ephrins. Here we set out to investigate--using Xenopus laevis retinal axons--the properties of substratum-bound and (artificially) soluble forms of ephrin-A5 (ephrin-A5-Fc) to guide axons. We find--as expected on the basis of chick experiments - that, when immobilised in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from chick. The analysis of growth cone behaviour in a gradient of soluble ephrin-A5 in the 'turning assay' revealed a substratum-dependent reaction of Xenopus retinal axons. On fibronectin, we observed a repulsive response, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations, indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, our data indicate that ephrin-As are able to guide axons in immobilised bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown.     

A subset of signal transduction pathways is required for hippocampal growth cone collapse induced by ephrin-A5

The Eph family tyrosine kinase receptors and their ligands, ephrins, play key roles in a wide variety of physiological and pathological processes including tissue patterning, angiogenesis, bone development, carcinogenesis, axon guidance, and neural plasticity. However, the signaling mechanisms underlying these diverse functions of Eph receptors have not been well understood. In this study, effects of Eph receptor activation on several important signal transduction pathways are examined. In addition, the roles of these pathways in ephrin-A5-induced growth cone collapse were assessed with a combination of biochemical analyses, pharmacological inhibition, and overexpression of dominant-negative and constitutively active mutants. These analyses showed that ephrin-A5 inhibits Erk activity but activates c-Jun N-terminal kinase. However, regulation of these two pathways is not required for ephrin-A5-induced growth cone collapse in hippocampal neurons. Artificial Erk activation by expression of constitutively active Mek1 and B-Raf failed to block ephrin-A5 effects on growth cones, and inhibitors of the Erk pathway also failed to inhibit collapse by ephrin-A5. Inhibition of JNK had no effects on ephrin-A5-induced growth cone collapse either. In addition, inhibitors to PKA and PI3-K showed no effects on ephrin-A5-induced growth cone collapse. However, pharmacological blockade of phosphotyrosine phosphatase activity, the Src family kinases, cGMP-dependent protein kinase, and myosin light chain kinase significantly inhibited ephrin-A5-induced growth cone collapse. These observations indicate that only a subset of signal transduction pathways is required for ephrin-A5-induced growth cone collapse.     

Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains

Contact-dependent signaling between membrane-linked ligands and receptors such as the ephrins and Eph receptor tyrosine kinases controls a wide range of developmental and pathological processes. Paradoxically, many cell types coexpress both ligands and receptors, raising the question of how specific signaling readouts are achieved under these conditions. Here, we studied the signaling activities exerted by coexpressed EphA receptors and GPI-linked ephrin-A ligands in spinal motor neuron growth cones. We demonstrate that coexpressed Eph and ephrin proteins segregate laterally into distinct membrane domains from which they signal opposing effects on the growth cone: EphAs direct growth cone collapse/repulsion and ephrin-As signal motor axon growth/attraction. This subcellular arrangement of Eph-ephrin proteins enables axons to discriminate between cis- versus trans-configurations of ligand/receptor proteins, thereby allowing the utilization of both Ephs and ephrins as functional guidance receptors within the same neuronal growth cone.     

Distinct membrane compartmentalization and signaling of ephrin-A5 and ephrin-B1

Eph receptor tyrosine kinases and their membrane-bound ligand ephrins form an essential cell communication system. Both ephrin classes have been shown to localize within cell surface lipid rafts, yet regulate different biological processes. In order to provide insight into this distinct behavior, we examined ephrin-A5 and B1 localization and signaling in murine fibroblasts and tissues. Results indicated that ephrin-A5 was constitutively present in detergent-resistant membrane fractions, while ephrin-B1 displayed translocation to membrane fractions upon stimulation. Ephrin-A5 and B1 were present in detergent-resistant membrane fractions with different buoyancies in vitro and in different raft fractions in vivo. Moreover, ephrin-A5 and B1 differentially influenced actin reorganization. Finally, microarray analysis revealed unique patterns of gene expression between the two ephrin classes. We thus demonstrate that distinct localization and compartmentalization provide insight into the subcellular basis for differential signaling observed in ephrin-A and B classes.     

p75(NTR) mediates ephrin-A reverse signaling required for axon repulsion and mapping

Reverse signaling by ephrin-As upon binding EphAs controls axon guidance and mapping. Ephrin-As are GPI-anchored to the membrane, requiring that they complex with transmembrane proteins that transduce their signals. We show that the p75 neurotrophin receptor (NTR) serves this role in retinal axons. p75(NTR) and ephrin-A colocalize within caveolae along retinal axons and form a complex required for Fyn phosphorylation upon binding EphAs, activating a signaling pathway leading to cytoskeletal changes. In vitro, retinal axon repulsion to EphAs by ephrin-A reverse signaling requires p75(NTR), but repulsion to ephrin-As by EphA forward signaling does not. Constitutive and retina-specific p75(NTR) knockout mice have aberrant anterior shifts in retinal axon terminations in superior colliculus, consistent with diminished repellent activity mediated by graded ephrin-A reverse signaling induced by graded collicular EphAs. We conclude that p75(NTR) is a signaling partner for ephrin-As and the ephrin-A- p75(NTR) complex reverse signals to mediate axon repulsion required for guidance and mapping.     

Axon guidance processes in the retinotectal and vomeronasal projection are controlled by Eph receptor tyrosine kinases and ephrins

The Eph family of receptor tyrosine kinases and their ‘ligands’, the ephrins, have been shown to play key roles in a number of different developmental processes such as cell migration, boundary formation, axon guidance, synapse formation and vasculogenesis. Here, we summarize recent findings derived from investigating the role of the EphA family during development of the retinotectal and vomeronasal projection uncovering a role of ephrin-A molecules as axon guidance receptors.     

Ephrin-A5 inhibits growth of embryonic sensory neurons

EphA-ephrin signaling has recently been implicated in the establishment of motor innervation patterns, in particular in determining whether motor axons project into dorsal versus ventral nerve trunks in the limb. We investigated whether sensory axons, which grow out together with and can be guided by motor axons, are also influenced by Eph-ephrin signaling. We show that multiple EphA receptors are expressed in DRGs when limb innervation is being established, and EphA receptors are present on growth cones of both NGF-dependent (predominantly cutaneous) and NT3-dependent (predominantly proprioceptive) afferents. Both soluble and membrane-attached ephrin-A5 inhibited growth of approximately half of each population of sensory axons in vitro. On average, growth cones that collapsed in response to soluble ephrin-A5 extended more slowly than those that did not, and ephrin-A5 significantly slowed the extension of NGF-dependent growth cones that did not collapse. Finally, we show that ectopic expression of ephrin-A5 in ovo reduced arborization of cutaneous axons in skin on the limb. Together these results suggest that sensory neurons respond directly to A-class ephrins in the limb. Thus, ephrins appear to pattern sensory axon growth in two ways-both directly, and indirectly via their inhibitory effects on neighboring motor axons.     

Ephs and ephrins in cancer: ephrin-A1 signalling

Ephrin-A1 and its primary receptor, EphA2, are involved in numerous physiological processes and have been intensely studied for their roles in malignancy. Ephrin-Eph signalling is complex on its own and is also cell-type dependent, making elucidation of the exact role of ephrin-A1 in neoplasia challenging. Multiple oncogenic signalling pathways, such as MAP/ERK and PI3K are affected by ephrin-A1, and in some cases evidence suggests the promotion of a specific pathway in one cell or cancer type and inhibition of the same pathway in another type of cell or cancer. Ephrin-A1 also plays an integral role in angiogenesis and tumor neovascularization. Until recently, studies investigating ephrins focused on the ligands as GPI-anchored proteins that required membrane anchoring or artificial clustering for Eph receptor activation. However, recent studies have demonstrated a functional role for soluble, monomeric ephrin-A1. This review will focus on various forms of ephrin-A1-specific signalling in human malignancy.     

Crystal structure of the human ephrin-A5 ectodomain

The Eph receptors, the largest subfamily of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, pathfinding, and mobility in the nervous and cardiovascular systems. Recent structural studies have revealed unique molecular features that explain many of the biochemical and signaling properties of Ephs and ephrins. Nevertheless, open questions remain, including understanding the precise molecular mechanism underlining their binding-partner preferences and subclass specificity. In this study, we have determined and present the crystal structure of the extracellular domain of ephrin-A5-the first structure of an unbound A-class ephrin. The structure, determined at 2.1 A resolution, is a variation of the Greek key beta-barrel folding topology, containing eight beta-strands, and stabilized by two disulphide bonds. Overall, ephrin-A5 is structurally very similar to ephrin-B1 and ephrin-B2 but, unlike ephrin-B2, it does not show dimerization either in solution or in the crystals. Comparing free ephrin-A5 to the previously published structure of EphB2-bound ephrin-A5 reveals that significant conformational changes occur only around the G-H ephrin loop that upon binding bends toward the receptor. Interestingly, the G-H loop undergoes a very similar conformational rearrangement in ephrin-B2 upon receptor binding. The results of this study further emphasize the importance of the G-H loop for receptor recognition and selectivity, and could serve as a starting point for the development of structure-based Eph antagonists.     

Analysis of ephrin-A2 in the chick retinotectal projection using a function-blocking monoclonal antibody

Eph receptor tyrosine kinases and their ligands have been shown to be involved in processes of cell migration and axon guidance during embryonic development. Here we describe the development of a function-blocking monoclonal antibody against chick ephrin-A2, and its effect on retinal ganglion cell axons studied both in vitro and in vivo. In the stripe assay, the blocking antibody completely abolished the repulsive effect of posterior tectal membranes. In vivo, in a loss-of-function approach, hybridoma cells secreting the antiephrin-A2 antibody were applied to chick embryos from embryonic day 3 (E3) on, and the retinotectal projection was subsequently analyzed at E16. DiI tracing analyses showed that although the projection of both temporal and nasal retinal ganglion axons in the tectum was, overall, normal, occasionally diffuse and extra termination zones were observed, in addition to axons over-shooting their termination zones. These data support the idea that ephrin-A2 contributes to the establishment of the chick retinotectal projection.     

Ephrin-A5 modulates cell adhesion and morphology in an integrin-dependent manner

The ephrins are membrane-tethered ligands for the Eph receptor tyrosine kinases, which play important roles in patterning of the nervous and vascular systems. It is now clear that ephrins are more than just ligands and can also act as signalling-competent receptors, participating in bidirectional signalling. We have recently shown that ephrin-A5 signals within caveola-like domains of the plasma membrane upon engagement with its cognate Eph receptor, leading to increased adhesion of the cells to fibronectin. Here we show that ephrin-A5 controls sequential biological events that are consistent with its role in neuronal guidance. Activation of ephrin-A5 induces an initial change in cell adhesion followed by changes in cell morphology. Both effects are dependent on the activation of beta1 integrin involving members of the Src family of protein tyrosine kinases. The prolonged activation of ERK-1 and ERK-2 is required for the change in cell morphology. Our work suggests a new role for class A ephrins in specifying the affinity of the cells towards various extracellular substrates by regulating integrin function.     

Receptor-binding domain of ephrin-A1: Production in bacterial expression system and activity

Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.     

Roles for ephrins in positionally selective synaptogenesis between motor neurons and muscle fibers

Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.     

Ephrin-mediated cis-attenuation of Eph receptor signaling is essential for spinal motor axon guidance

Axon guidance receptors guide neuronal growth cones by binding in trans to axon guidance ligands in the developing nervous system. Some ligands are coexpressed in cis with their receptors, raising the question of the relative contribution of cis and trans interactions to axon guidance. Spinal motor axons use Eph receptors to select a limb trajectory in response to trans ephrins, while expressing ephrins in cis. We show that changes in motor neuron ephrin expression result in trajectory selection defects mirrored by changes in growth cone sensitivity to ephrins in vitro, arguing for ephrin cis-attenuation of Eph function. Furthermore, the relative contribution of trans-signaling and cis-attenuation is influenced by the subcellular distribution of ephrins to membrane patches containing Eph receptors. Thus, growth cone ephrins are essential for axon guidance in vivo and the balance between cis and trans modes of axon guidance ligand-receptor interaction contributes to the diversity of axon guidance signaling responses.     

Denervation-induced spatiotemporal upregulation of ephrin-A2 in the mouse hippocampus after transections of the perforant path

Transections of the entorhinal afferent fibers to hippocampus, perforant path (PP), result in the denervation in specific hippocampal subregions, which is followed by a series of plastic events including axon sprouting and reactive synaptogenesis. Many growth-associated molecules are thought to participate in these events. In the present study, we proved the upregulation of ephrin-A2 in the denervated areas of the ipsilateral hippocampus following PP transections. Interestingly, when the elevation of ephrin-A2 reached the maximum axon sprouting in the denervated areas almost finished, implying the possible inhibitory effect of ephrin-A2 on sprouting. In addition, ephrin-A2 expression was observed in synapses during reactive synaptogenesis, suggesting that this molecule might also be implicated in the formation and maturation of synapses in the denervated areas.     

Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. For the first time, we show that the ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5-treated retinal ganglion cell cultures, Rho was activated and Rac was downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduced the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway.