Eph/ephrin interactions modulate vascular sympathetic innervation.

Ephs and ephrins are membrane-bound proteins that interact to modulate axon growth and neuronal function. We tested the hypothesis that eph/ephrin interactions affected the growth and function of vascular sympathetic innervation. Using RT-PCR analyses, we detected both classes of ephs (A and B) and both classes of ephrins (A and B) in sympathetic ganglia from neonatal and adult rats. Both classes of ephs (A and B) and both classes of ephrins (A and B) bound to the cell bodies and neurites of dissociated postganglionic sympathetic neurons. Messenger RNAs encoding for both classes of ephs (A and B) and both classes of ephrins (A and B) were also detected in sympathetically innervated arteries from neonatal and adult rats. These data suggest that ephrins/ephs on nerve fibers of postganglionic sympathetic neurons could interact with ephs/ephrins on cells in innervated arteries. We found that ephA4 reduced reinnervation of denervated femoral arteries. Reinnervation in the presence of ephA4-Fc (38.9±6.6%) was significantly less than that in the presence of IgG-Fc (62±10%; n=5; p<0.05; one-tailed unpaired t-test). These data indicate that eph/ephrin interactions modulated the growth of vascular sympathetic innervation. We also found that ephA4 increased basal release of norepinephrine from nerve terminals of isolated tail arteries. These data indicate that eph/ephrin interactions affect the growth and function of vascular sympathetic innervation.

Astrocyte-produced ephrins inhibit schwann cell migration via VAV2 signaling.

Schwann cells are a promising candidate for bridging spinal cord injuries and remyelinating axons. However, grafted Schwann cells show little intermingling with host astrocytes and therefore limited migration from transplant sites. This leads to the formation of a sharp border between host astrocytes and Schwann cells, which results in axons stalling at the graft-host interface and failing to exit the graft. We investigated the possibility that Eph/ephrin interactions are involved in the segregation of Schwann cells and astrocytes and in limiting Schwann cell migration. Using reverse transcription-PCR, we have characterized the ephrin and Eph profile in cultured Schwann cells and astrocytes, showing that astrocytes produce all the ephrinAs and Schwann cells produce the receptors EphA2, EphA4, and EphA7. Several ephrinAs inhibit Schwann cell migration on laminin, with ephrinA5 being the most effective. Blocking the EphA receptors with excess EphA4-Fc increases Schwann cell migration on astrocytes and improves Schwann-astrocyte intermingling. We show that the action of ephrinA5 on Schwann cells is mediated via VAV2. Both clustered ephrinA5 and astrocyte contact increases the phosphorylation of VAV2 in Schwann cells. Knockdown of VAV2 abrogates the inhibitory effect of clustered ephrinA5 on migration and increases the ability of Schwann cells to migrate on astrocytes. In addition, we found a role for ephrinA5 in inhibiting Schwann cell integrin signaling and function. Overall, we suggest that Eph/ephrin interactions inhibit Schwann cell migration and intermingling with astrocytes via VAV signaling affecting integrin function.

Ephrin-B reverse signaling promotes structural and functional synaptic maturation in vivo.

Ephrin-Eph signaling is involved in axon guidance during development, but it may also regulate synapse development after the axon has contacted the target cell. Here we report that the activation of ephrin-B reverse signaling in the developing Xenopus laevis optic tectum promotes morphological and functional maturation of retinotectal synapses. Elevation of ephrin-B signaling increased the number of retinotectal synapses and stabilized the axon arbors of retinal ganglion cells. It also enhanced basal synaptic transmission and activity-induced long-term potentiation (LTP) of retinotectal synapses. The functional effects were caused by a rapid enhancement of presynaptic glutamate release and a delayed increase in the postsynaptic glutamate responsiveness. The facilitated LTP induction occurred during the early phase of enhanced transmitter release and appeared to be causally related to the late-phase postsynaptic maturation via an NMDA receptor-dependent mechanism. This ephrin-B-dependent synapse maturation supports the notion that the ephrin/Eph protein families have multiple functions in neural development.

Eph receptors and their ephrin ligands are expressed in developing mouse pancreas.

Pancreas development involves branching morphogenesis concomitantly to differentiation of endocrine, exocrine and ductal cell types from a single population of pancreatic precursors. These processes depend on many signals and factors that also control development of the central nervous system. In the latter, Eph receptors and their class-A (GPI-anchored) and class-B (transmembrane) ephrin ligands control cell migration and axon-pathfinding, help establish regional patterns and act as labels for cell positioning. This raised the question as to whether and where Ephs and ephrins are expressed during pancreas development. Here we have identified the Eph and ephrin genes that are expressed in mouse embryonic pancreas, as detected by RT-PCR analysis. In situ hybridization experiments showed that Ephs and ephrins are mainly expressed in the burgeoning structures of the epithelium which differentiate into exocrine acini. Binding experiments on whole pancreas demonstrated the presence of functional Eph receptors. They showed that EphBs are expressed by the pancreatic epithelium at embryonic day (e) 12.5 and that, from e14.5 on, Ephs of both classes are expressed by the pancreatic epithelium and then become restricted to developing acini. We conclude that specific members of the Eph/ephrin family are expressed in embryonic pancreas according to a dynamic temporal and regional pattern.

Functions of ephrin/Eph interactions in the development of the nervous system: emphasis on the hippocampal system.

Ephrins and their Eph receptors are membrane-anchored proteins that have key roles in the development of the Central Nervous System. The main characteristics of ephrin/Eph interactions are that their effect is mediated by cell-to-cell contacts and that they can propagate bidirectional signals downstream of the ligand-receptor complex. These characteristics make ephrins and Eph receptors critical cues in the regulation of migrating cells or axons, and in the establishment of tissue patterns and topographic maps in distinct regions of the developing brain. In addition, ephrins and Eph receptors regulate synapse formation and plasticity. These roles would be promoted by complementary gradual expression of receptors and ligands in the neurons involved. Although, historically, ephrins and Eph receptors have been considered as repulsion signals through barriers or gradients, new evidence indicates that they may be both inhibitory and permissive/active cues depending on expression levels. The expression of distinct ligands and receptors in the developing and mature hippocampus suggests that these proteins are involved in distinct processes during the development and maturation of the hippocampal region. In fact, recent studies have shown that ephrin/Eph signaling participates in the formation of the layer-specific patterns of hippocampal afferents, in synaptogenesis and in plasticity. Therefore, ephrin/Eph interactions should be considered a crucial system in the development and maturation of the brain regions, including the hippocampus.

Eph/ephrin A- and B-family expression patterns in the leopard frog (Rana utricularia).

Eph/ephrin expression was studied in Rana utricularia larvae and adults with in situ receptor and ligand affinity probes. From stages TK-II (early limb bud) to VI (early foot paddle larva), tectal EphB expression is highest in a band extending transversely across the posterior optic tectum and grades off anteriorly and posteriorly. The ephrin-A expression gradient is parallel to the EphB gradient rather than being orthogonal to it. However, its high point occupies the posterior pole, and it runs from high-posteriorly to low-anteriorly. Tectal EphA expression is high anteriorly and low posteriorly, while ephrin-Bs are expressed only in a thin line at the dorsal midline. At later stages and in adults, tectal EphB expression becomes uniform.

Disruption of ephrin signaling associates with disordered axophilic migration of the gonadotropin-releasing hormone neurons.

Ephrin signaling is involved in repulsive and attractive interactions mediating axon guidance and cell-boundary formation in the developing nervous system. As a result of a fortuitous transgene integration event, we have identified here a potential role for EphA5 in the axophilic migration of gonadotropin-releasing hormone (GnRH) neurons from the nasal placode into the brain along ephrin-expressing vomeronasal axons. Transgene integration in the GNR23 mouse line resulted in a 26 kb deletion in chromosome 5, approximately 67 kb 3' to Epha5. This induced a profound, region-specific upregulation of EphA5 mRNA and protein expression in the developing mouse brain. The GnRH neurons in GNR23 mice overexpressed EphA5 from embryonic day 11, whereas ephrin A3 and A5 mRNA levels in olfactory neurons were unchanged. The GnRH neurons were found to be slow in commencing their migration from the olfactory placode and also to form abnormal clusters of cells on the olfactory axons, prohibiting their migration out of the nose. As a result, adult hemizygous mice had only 40% of the normal complement of GnRH neurons in the brain, whereas homozygous mice had <15%. This resulted in infertility in adult female homozygous GNR23 mice, suggesting that some cases of human hypogonadotropic hypogonadism may result from ephrin-related mutations. These data provide evidence for a role of EphA-ephrin signaling in the axophilic migration of the GnRH neurons during embryogenesis.

Ephrins and their receptors: binding versus biology.

Ephrins and Eph receptors play important roles in the development of the central nervous system and peripheral tissues by orchestrating cellular movements, resulting in events such as axonal growth cone guidance, tissue segmentation, and angiogenic remodeling. To understand the role of specific ephrin and Eph receptor interactions, it is important to identify the binding specificity between individual ligand-receptor complexes. To date, a dogma in the field suggests that there may be promiscuous binding within the subclasses of the ephrin family. However, this overlooks and contradicts several binding studies that suggest specificity within each subclass. Although binding studies only provide evidence on the dynamics and strength of protein interactions, they do not indicate whether particular interactions are physiologically relevant. Thus, distribution and gene targeted mutations of ephrins and their receptors can provide critical insights into the relevance of specific ligand-receptors interactions. This review mainly focuses on the B-class family and will evaluate the differences between binding affinities and biological functions, importance of oligomeric interactions, and structural differences and similarities between classes.

Retinal axon response to ephrin-as shows a graded, concentration-dependent transition from growth promotion to inhibition.

Ephrin-As act as retinal topographic mapping labels, but the molecular basis for two key aspects of mapping remains unclear. First, although mapping is believed to require balanced opposing forces, ephrin-As have been reported to be retinal axon repellents, and the counterbalanced force has not been molecularly identified. Second, although graded responsiveness across the retina is required for smooth mapping, a sharp discontinuity has instead been reported. Here, an axon growth assay was developed to systematically vary both retinal position and ephrin concentration and test responses quantitatively. Responses varied continuously with retinal position, fulfilling the requirement for smooth mapping. Ephrin-A2 inhibited growth at high concentrations but promoted growth at lower concentrations. Moreover, the concentration producing a transition from promotion to inhibition varied topographically with retinal position. These results lead directly to a mapping model where position within a concentration gradient may be specified at the neutral point between growth promotion and inhibition.

[Ephrins, neuronal development and plasticity]. / Efrinas, desarrollo neuronal y plasticidad.

AIMS: In this work we review the main characteristics of ephrins and their Eph receptors (ER), as well as descriptions that have been published to date of the different functions the ephrin/Eph system (EES) performs in neuronal development. DEVELOPMENT: ER constitute the largest group of tyrosine kinase receptors and are found in many different types of cells during development and in mature tissues. Their ligands, the ephrins, are membrane anchored proteins that are divided into class A ephrins, with a glycosylphosphatidylinositol bond, and class B ephrins, with a hydrophobic transmembrane region and a cytoplasmic domain. The EES is the only one that involves bidirectional signalling. Thus, the ephrin Eph interaction both activates the tyrosine kinase domain of the ER, with the resulting signal transduction in the cell that expresses Eph, and produces a reverse signal in the cells that contain the ligands. Over the last decade a number of studies have been conducted that establish the involvement of the EES in neuronal development. Although the classic function of this system is that of establishing patterns of both cellular and axonal organisation, recent reports describe how the ER and their ephrin ligands regulate synaptogenesis and the maturation of terminals during development, as well as the plasticity of the adult brain. CONCLUSIONS: Recent findings open up new expectations regarding the possible functions carried out by the interaction of ephrin and Eph. They also confirm the crucial role played by this system in all the processes involved in allowing neuronal development to take place in a correct fashion.

Analysis of EphB receptors and their ligands in the developing retinocollicular system of the wallaby reveals dynamic patterns of expression in the retina.

The expression of EphB1 and B2 receptors and ephrins-B1, -B2 and -B3 in the retina and superior colliculus of the wallaby (Macropus eugenii) was examined during the development of the retinocollicular projection, using reverse transcription-polymerase chain reaction and immunohistochemistry. There was an early transient differential expression of EphB2 that was higher in ventral retina and restricted to the outer neuroblast layer, whereas a high ventral to low dorsal gradient of ephrin-B2 expression occurred there throughout the study period. However, there was no dorsoventral gradient of receptors or ligands in retinal ganglion cells or a mediolateral gradient of ephrins in the colliculus. These findings suggest a limited role for these molecules in topographic mapping across the mediolateral colliculus in the wallaby. Early in retinal development there is a complementary pattern of expression of ephrin-B1 and -B2 in the outer neuroblast layer that overlaps with expression of EphB2. Ganglion and amacrine cells also express EphB2. As development proceeds subpopulations of putative horizontal and bipolar cells, also expressing EphB2, come to reside in the inner nuclear layer and ephrin-B1 is expressed throughout the outer nuclear layer. At the same time cells expressing ephrin-B2, and subpopulations of horizontal and bipolar cells come to reside in the inner nuclear layer and there is a corresponding decrease in ephrin-B2 expression in the outer nuclear layer. This pattern of coexpression of receptors and ligands suggests a role for them in cell migration and maintenance of laminar boundaries.

Ephs and ephrins during early stages of chick embryogenesis.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are membrane-bound proteins that mediate bidirectional signals between adjacent cells. By modulating cytoskeleton dynamics affecting cell motility and adhesion, Ephs and ephrins orchestrate cell movements during multiple morphogenetic processes, including gastrulation, segmentation, angiogenesis, axonal pathfinding, and neural crest cell migration. The full repertoire of developmental Eph/ephrin functions remains uncertain, however, because coexpression of multiple receptor and ligand family members, and promiscuous interactions between them, can result in functional redundancy. A complete understanding of expression patterns, therefore, is a necessary prerequisite to understanding function. Here, we present a comprehensive expression overview for 10 Eph and ephrin genes during the first 48 hr of chick embryo development. First, dynamic expression domains are described for each gene between Hamburger and Hamilton stages 4 and 12; second, comparative analyses are presented of Eph/ephrin expression patterns in the primitive streak, the somites, the vasculature, and the brain. Complex spatially and temporally dynamic expression patterns are revealed that suggest novel functions for Eph and ephrin family members in both known and previously unrecognized processes. This study will provide a valuable resource for further experimental investigations of Eph and ephrin functions during early embryonic development.

Chemometrical classification of ephrin ligands and Eph kinases using GRID/CPCA approach.

Eph receptor tyrosine kinases are divided on two subfamilies based on their affinity for ephrin ligands and play a crucial role in the intercellular processes such as angiogenesis, neurogenesis, and carcinogenesis. As such, Eph kinases represent potential targets for drug design, which requires the knowledge of structural features responsible for their specific interactions. To overcome the existing gap between available sequence and structure information we have built 3D models of eight ephrins and 13 Eph kinase ligand-binding domains using homology modeling techniques. The interaction energies for several molecular probes with binding sites of these models were calculated using GRID and subjected to chemometrical classification based on consensus principal component analysis (CPCA). Despite inherent limitations of the homology models, CPCA was able to successfully distinguish between ephrins and Eph kinases, between Eph kinase subfamilies, and between ephrin subfamilies. As a result we have identified several amino acids that may account for selectivity in ephrin-Eph kinase interactions. In general, although the difference in charge between ephrin and Eph kinase binding domains creates an attractive long-range electrostatic force, the hydrophobic and steric interactions are highly important for the short-range interactions between two proteins. The chemometrical analysis also provides the pharmacophore model, which could be used for virtual screening and de novo ligand design.

mRNA expression of ephrins and Eph receptor tyrosine kinases in the neonatal and adult mouse central nervous system.

Ephrins and Eph receptors are a family of molecules that have been implicated in axonal pathfinding. A unique feature of B-class ephrins and Eph receptors is their ability to transmit bidirectional signals in both ephrin- and Eph receptor-expressing cells upon cell-cell contact. These signals can lead to cytoskeletal alterations that have been attributed to regulating neuronal growth responses. Examination of gene-target knockout mice has supported this hypothesis, revealing numerous developmental defects in the nervous systems of mice mutant for both B-class ephrins and Eph receptors. To examine the potential scope of action for these genes in the nervous system, we have used in situ hybridization to study the mRNA expression of ephrins (B1, B2, and B3) and Eph receptors (B1, B2, B3, A4) in neonatal and adult mice. We found ephrins and Eph receptors to be expressed throughout the CNS. Expression was observed in the epithelium and migratory regions of the neonate and adult tissues as well as in discrete regions of high plasticity, including the adult olfactory bulb, hippocampus, and cerebellum. These studies suggest additional potential roles for these molecules in the postnatal and adult CNS and will serve as a guide in the detailed evaluation of mutant mice.