InteractiveFly: GeneBrief

Eph receptor tyrosine kinase: Biological Overview | Regulation | Developmental Biology | Effects of Mutation and RNAi | Evolutionary Homologs |References

Gene name - Eph receptor tyrosine kinase

Synonyms -

Cytological map position - 102C6

Function - receptor

Keywords - axon pathfinding

Symbol - Eph

FlyBase ID: FBgn0023093

Genetic map position -

Classification - receptor tyrosine kinase

Cellular location - surface transmembrane

NCBI link: Entrez Gene

Eph orthologs: Biolitmine
Recent literature
Buhl, E., Kim, Y. A., Parsons, T., Zhu, B., Santa-Maria, I., Lefort, R. and Hodge, J. J. L. (2022). Effects of Eph/ephrin signalling and human Alzheimer's disease-associated EphA1 on Drosophila behaviour and neurophysiology. Neurobiol Dis 170: 105752. PubMed ID: 35569721
Alzheimer's disease (AD) is the most prevalent neurodegenerative disease placing a great burden on people living with it, careers and society. Yet, the underlying patho-mechanisms remain unknown and treatments limited. To better understand the molecular changes associated with AD, genome-wide association studies (GWAS) have identified hundreds of candidate genes linked to the disease, like the receptor tyrosine kinase EphA1. However, demonstration of whether and how these genes cause pathology is largely lacking. Utilising fly genetics, this study generated the first Drosophila model of human wild-type and P460L mutant EphA1 and tested the effects of Eph/ephrin signalling on AD-relevant behaviour and neurophysiology. EphA1 mis-expression did not cause neurodegeneration, shorten lifespan or affect memory but flies mis-expressing the wild-type or mutant receptor were hyper-aroused, had reduced sleep, a stronger circadian rhythm and increased clock neuron activity and excitability. Over-expression of endogenous fly Eph and RNAi-mediated knock-down of Eph and its ligand ephrin affected sleep architecture and neurophysiology. Eph over-expression led to stronger circadian morning anticipation while ephrin knock-down impaired memory. A dominant negative form of the GTPase Rho1, a potential intracellular effector of Eph, led to hyper-aroused flies, memory impairment, less anticipatory behaviour and neurophysiological changes. These results demonstrate a role of Eph/ephrin signalling in a range of behaviours affected in AD. This presents a starting point for studies into the underlying mechanisms of AD including interactions with other AD-associated genes, like Rho1, Ankyrin, Tau and APP with the potential to identify new targets for treatment.

The Eph receptors constitute the largest subfamily of receptor tyrosine kinases; they have been implicated in pattern formation, cell migration, and axon pathfinding (for reviews see Bruckner, 1998; Hsueh, 1998; Lemke, 1998; Ernst, 1998; Flanagan, 1998; O'Leary, 1999; McLaughlin, 1999, and Holder, 1999). To date, over 15 distinct Eph receptors and 8 of their ligands, the ephrins (see Ephrin), have been identified. Members of the Eph subfamily are characterized by their extracellular structure, which consists of a globular domain, a cysteine-rich region unrelated to those of other receptor protein tyrosine kinases (RTKs), and two fibronectin (FN) type III repeats. The cytoplasmic structure of Eph receptors contains conserved catalytic domains and a unique sterile alpha motif (SAM) domain, which is thought to be involved in binding SH2 proteins and is not found in other RTKs. The Eph subfamily is divided into two classes, EphA and EphB, based on the amino acid sequence homology of their extracellular domains. Ligands of the Ephrin-A subclass are attached to the membrane via a glycosylphosphatidylinositol linkage, while the Ephrin-B ligands span the membrane via a transmembrane domain. A Drosophila Eph receptor has now been characterized. Drosophila Eph possesses all the domains characteristic of the Eph subfamily of RTKs and is equally similar in sequence to both the EphA and the EphB subclasses. Drosophila Eph is expressed on the growth cones and axons of embryonic interneurons and larval photoreceptor cells (Scully, 1999).

Eph was isolated in a screen designed to isolate novel Drosophila RTKs. An adult cDNA library was screened by polymerase chain reaction (PCR) using degenerate primers corresponding to amino acid sequences HRDLAARN and DVWSYGV within the conserved catalytic domain of receptor tyrosine kinases. An amino acid comparison of the open reading frame (ORF) of one of the isolated clones revealed homology to the vertebrate Eph subfamily (Scully, 1999).

In order to begin to address its potential role in axon pathfinding, Eph was misexpressed using the UAS-GAL4 system. To reliably follow the complete translation of Eph, five c-myc epitopes were added in frame to the C-terminal end of the Eph coding sequence. Transformants carrying the full-length myc-tagged Eph construct, UAS-Eph-myc, were initially screened for high expression levels of the c-myc epitope by crossing them to a panneural GAL4 line. Individuals carrying four copies of the highest-expressing UAS-Eph-myc insertions were crossed to a number of GAL4 lines that drive expression in various tissues at different developmental times. The progeny were assayed for phenotypes at embryonic, larval, and adult stages. Immunostaining with anti-Eph and anti-myc antibodies reveals that Eph-myc is expressed at high levels when crossed to these GAL4 lines; the protein is targeted to axons and growth cones in a manner indistinguishable from endogenous Eph protein. The axonal targeting of Eph-myc allows the examination of subsets of neurons and their processes without an additional tau-based axon-targeted reporter. Despite the observation that Eph-myc protein is being expressed at robust levels, misexpression in motor neurons or overexpression in interneurons of Eph-myc protein does not appear to generate a phenotype in any of the GAL4 assays (Scully, 1999).

The SAM domain, as well as the terminal PDZ binding motif, may have biological functions in Eph (Hock, 1998; Stein, 1996; Stein, 1998b) and may be disrupted by the addition of the c-myc epitopes. Therefore, a full-length version of Eph without an epitope tag was tested. As with Eph-myc, UAS-Eph individuals over- and mis-expressing Eph with the panel of GAL4 drivers are viable and appear normal. The non-myc-tagged Eph does not grossly affect axon pathfinding in the VNC, although axon fascicles appear somewhat defasciculated when UAS-Eph is driven by scabrous (sca)-GAL4 together with elav-GAL4. This sca-GAL4; elav-GAL4 combination initiates GAL4 expression at the neuroblast stage and continues its expression in postmitotic neurons throughout the CNS. Overexpression of Eph, using pGMR-GAL4 (which drives expression in the photoreceptor cells of the larval eye disc) does not affect axon pathfinding in the developing visual system. These results indicate that altering the levels or patterns of Eph in the CNS by ectopic expression has little effect on axon pathfinding (Scully, 1999).

An attempt was made to alter the normal function of endogenous Eph by expressing a kinase-inactive form of the protein containing the critical lysine (K759) changed to methionine. To assay expression levels, the mutant derivative was myc-tagged at the C-terminus. Flies were transformed with UAS-Eph-K759M-myc and screened as described above for high expression levels. Individuals carrying four copies of the highest-expressing UAS-Eph-K759M- myc insertions were crossed to a panel of GAL4 lines and assayed for phenotypes at embryonic, larval, and adult stages. Although Eph-K759M-myc protein, like Eph-myc, is expressed at robust levels, its expression does not appear to generate a phenotype in any of the assays (Scully, 1999).

In spite of the negative results obtained from misexpression of Eph, the highly localized expression of Eph on axons in the developing nervous system suggests that the kinase will have a role in axon pathfinding, especially in light of the evidence from vertebrate Eph studies that has demonstrated a role for Eph RTKs in axon guidance. It has been suggested that the tagged proteins may not have given an overexpression phenotype due to the interference of the epitopes with the proper functioning of the Eph protein. There could be several reasons for the lack of phenotypes with full-length Eph. (1) The level of Eph expression may not be sufficiently high to affect axon pathfinding or other putative biological functions in which Eph may be involved; (2) for misexpression in motor neurons, the Eph ligand may not be present on those cells that interact with motor neurons; therefore, the signaling cascade of Eph might not be initiated upon misexpression of Eph in those cells;(3) for overexpression in interneurons, the levels of Eph may not be critical, in contrast to systems in which disturbing gradients of Eph receptors or ephrins gives rise to pathfinding phenotypes, and (4) though less likely, considering the evidence from vertebrate Eph receptor studies, the endogenous Eph receptor may not play a role in axon guidance; in this case, misexpression of Eph would not be expected to perturb axon pathfinding. In vertebrates, redundancy has been an issue in uncovering the function of the Eph RTK subfamily and its signaling pathway(s). Analysis of Eph receptors and their ligands may be more amenable in a simpler genetic system such as C. elegans or Drosophila. Currently 14 distinct vertebrate Eph receptors have been identified, but Eph represents the only Drosophila Eph receptor isolated to date. A single Eph receptor, VAB-1, has been identified in the recently completly sequenced C. elegans genome (George, 1998). Therefore, although additional Drosophila Eph receptors may exist, it is unlikely that this organism will have the large number of Eph receptors present in vertebrates. Further studies of Eph function in Drosophila employing loss-of-function mutants may produce insight into the roles of Eph receptors in the developing nervous system (Scully, 1999).

Dendritic Eph organizes dendrodendritic segregation in discrete olfactory map formation in Drosophila

Proper function of the neural network results from the precise connections between axons and dendrites of presynaptic and postsynaptic neurons, respectively. In the Drosophila olfactory system, the dendrites of projection neurons (PNs) stereotypically target one of approximately 50 glomeruli in the antennal lobe (AL), the primary olfactory center in the brain, and form synapses with the axons of olfactory receptor neurons (ORNs). This study shows that Eph and Ephrin, the well-known axon guidance molecules, instruct the dendrodendritic segregation during the discrete olfactory map formation. The Eph receptor tyrosine kinase is highly expressed and localized in the glomeruli related to reproductive behavior in the developing AL. In one of the pheromone-sensing glomeruli (DA1), the Eph cell-autonomously regulates its dendrites to reside in a single glomerulus by interacting with Ephrins expressed in adjacent PN dendrites. These data demonstrate that the trans interaction between dendritic Eph and Ephrin is essential for the PN dendritic boundary formation in the DA1 olfactory circuit, potentially enabling strict segregation of odor detection between pheromones and the other odors (Anzo, 2017).

The proper assembly of neural circuits during development is necessary for the formation of functional neural networks. One of the key steps for establishing a functional neural circuit is to construct a precise connection between the axons and dendrites of presynaptic and postsynaptic neurons, respectively. In the visual and auditory systems, neighboring neurons in the input field target the neighboring regions in the output field. In the olfactory systems of mammals and insects, the axons of the primary olfactory receptor neurons (ORNs) that express the same olfactory or ionotropic receptors converge to one specific glomerulus in the primary olfactory center. The ORN axons form synaptic connections with dendrites of second-order neurons that also typically target one particular glomerulus among those discretely distributed. Unlike in other sensory systems, there is less spatial correlation between axon and dendrite targeting in the olfactory system. Thus, the neuronal wiring in the olfactory system can be the most striking example of specific targeting achieved by both axons and dendrites among the neural targeting events during development. The previous studies have shown that the topographic mapping in the visual system and the neuronal wiring in the olfactory system are mostly governed by axon guidance. In comparison, dendrite targeting is far less understood not only due to the complex morphology and diversity of dendrites but also because its historical background has received little attention (Anzo, 2017).

The Drosophila olfactory system is a suitable model to study the mechanisms underlying dendrite targeting. The primary olfactory center, the antennal lobe (AL), consists of ~50 discrete structures called glomeruli that are identifiable from their shape, relative size, and position. Most of the projection neuron (PN) dendrites invade one particular glomerular space and form synapses with axons of a single ORN class. In addition, genetic tools such as mosaic analysis with a repressible cell marker (MARCM) allow labelling of specific subsets of PNs at a single-cell resolution in vivo and simultaneously manipulate genes in the labeled neurons (Anzo, 2017).

By taking advantage of the Drosophila olfactory system, the cell surface molecules that regulate dendrite targeting have been gradually revealed. Cell surface molecules such as Semaphorin-1a (Sema-1a) and Toll-6 cell-autonomously regulate dendrite targeting along the dorsolateral-ventromedial axis and mediolateral axis, respectively. The PN dendrites determine their coarse position in the AL along the axes depending on the expression level of Sema-1a or Toll-6 therein. The leucine-rich repeat transmembrane protein Capricious (Caps) is differentially expressed in a subset of PNs and represents a mosaic pattern in the developing AL. The differential Caps expression cell-autonomously instructs glomerular-specific PN targeting, especially the segregation of Caps-positive and Caps-negative PN classes. These findings indicate that besides axial information, discrete determinants also provide positional information to the PN dendrites. Moreover, the cell adhesion molecule N-cadherin (Ncad) and immunoglobulin superfamily protein Dscam act as attractive or repulsive signals in most of the PN classes that restrict the dendritic field to the appropriate glomerular space. In addition to these findings, it was found in this study that the dendritic boundary formation between specific subtypes of PNs are instructed by cell surface molecules, Eph, and Ephrin (Anzo, 2017).

The Eph receptor and its ligand, Ephrin, are the largest family of receptor tyrosine kinases (RTKs) and are widely conserved from invertebrates to mammals. Eph and Ephrin have been well studied as axon guidance molecules in retinotectal topographic mapping. In the vertebrate tectum/superior colliculus (SC), the EphA/EphrinA and EphB/EphrinB countergradients are formed along the anterior-posterior and nasal-temporal axes, respectively. The axons of retinal ganglion cells (RGCs) determine their target field by recognizing the relative position based on the expression levels of their ligands at the tectum/SC. For example, the temporal RGC axon expressing EphA receives a repulsive signal from EphrinA expressed in the tectum/SC, which causes the temporal axon to avoid the posterior tectum/SC. In vertebrates, Ephrins are divided into two groups based on the type of membrane linkage: GPI-anchored EphrinAs (EphrinA1-6) and transmembrane EphrinBs (EphrinB1-3). Ephs are also divided into two subtypes depending on the affinity to Ephrins: EphAs (EphA1-8 and Eph10) bind to multiple EphrinAs, and, similarly, EphBs (EphB1-4 and Eph6) bind to multiple EphrinBs, with the exception of EphA4 binding to both EphrinAs and EphrinBs. Since both Ephs and Ephrins are membrane-bound proteins, the signal is essentially activated via contact-dependent cell-cell interaction. The bidirectional Eph/Ephrin signal works repulsively in a majority of the cases, although an adhesive response has also been described. Since Drosophila has only a single pair of Eph-Ephrin, the overlapping function of their family members as is considered in vertebrate studies can be excluded. Drosophila Eph shows ~71% identity with both vertebrate EphA3 and EphB2, and Drosophila Ephrin has a vertebrate EphrinB-like cytoplasmic domain. A previous study of topographic mapping in the Drosophila visual system strongly suggested that Drosophila Eph/Ephrin signal functions in an evolutionarily conserved fashion (Anzo, 2017).

This study found that Eph/Ephrin signal instructs dendrodendritic segregation during discrete olfactory map formation. Unlike Ncad or Dscam, which affects most of the PN classes, Eph/Ephrin signal selectively functions in only specific PN classes. High Eph RTK expression was observed specifically in the glomeruli associated with reproductive behavior in the developing AL. In addition, genetic data indicate that Eph/Ephrin trans interaction between neighboring glomeruli plays a central role in local dendrodendritic segregation through bidirectional repulsive responses (Anzo, 2017).

This study demonstrates that the trans interaction between the DA1 dendritic Eph and Ephrin on the adjacent dendrites is required for proper dendritic boundary formation. How can this be possible considering the patterned expression of Eph and the ubiquitous expression of Ephrin in the developing AL? It is proposed that the restricted expression of Eph in the DA1 dendrites could effect the activation of differential signal transduction between the dendrites in the DA1 and the adjacent glomeruli even though Ephrin is expressed ubiquitously throughout the developing AL. As the result of the trans interaction between the DA1 dendritic Eph and the adjacent dendritic Ephrin, the Eph forward signal seems to be transmitted to the Eph-expressing DA1 l-PNs, and the Ephrin reverse signal seems to be transmitted to the adjacent PNs. This transinteraction model involving Eph forward and Ephrin reverse signals also fits well with the result that the Eph-null mutant (EphX652), but not Eph-shRNA expression in the VA1d ad-PNs, exhibited dendritic spillover from the VA1d to the DA1 glomerulus. The loss of Eph in the EphX652 mutant could weaken the Ephrin reverse signal in addition to the Eph forward signal, resulting in dendritic spillover from both the DA1 and VA1d dendrites. In contrast, the cell-autonomous reduction of Eph in the VA1d dendrites with Eph-shRNA has no way to reduce the Ephrin reverse signal. In vertebrates, the trans Eph-Ephrin interaction leads to the formation of higher signaling clusters. Oligomers of Ephs and Ephrins are terminated by bidirectional transendocytosis or its cleavage by ADAM-type proteases, leading to the activation of Rho family GTPases. This activation of Rho family GTPases by Eph/Ephrin oligomerization modulates actin cytoskeletal dynamics, which induce cell-cell repulsion in the most cases. Hence, the segregation model is proposed as follows. The transinteraction between the DA1 dendritic Eph and the adjacent dendritic Ephrin results in a bidirectional repulsive Eph forward and Ephrin reverse signal running in both DA1 and VA1d PNs, respectively; thus, they segregate from each other, thereby forming a proper dendritic glomerular boundary. Additional studies may be required to describe the molecular features of the Drosophila Ephrin reverse signal in the future (Anzo, 2017).

When Eph was knocked down, the dendritic spillover phenotype was observed specifically near the DA1 and DL3 glomeruli. Why is the Eph/Ephrin system used to form the dendritic boundary of such specific glomeruli? Among 50 glomeruli, the DA1, DL3, VA1lm, and VL2a glomeruli exhibited high Eph expression during development, holding the line against the other glomeruli by their unique function. Unlike other glomeruli, the DA1, DL3, VA1lm, and VL2a glomeruli receive inputs from the axons of ORNs (Or67d, Or65a, Or47b, and Ir84a, respectively) dedicated to sensing odors related to reproductive behavior, such as pheromones and food-derived odors promoting male courtship behaviors. Among them, Or67d, the primary neuron of the DA1 olfactory circuit, detects Drosophila male-specific pheromone 11 cis-vaccenyl acetate (cVA) and triggers sex-specific courtship behavior in both male and female flies. In addition, the DA1 PNs show sexually dimorphic neural circuitry. The primary neuron of DL3 (Or65a) also responds to cVA when flies are exposed to it for a long period. DL3 olfactory neurons suppress pheromonal activation of DA1 olfactory neurons. Furthermore, as in Drosophila, the pheromone-sensing organs of Manduca sexta (macroglomerular complex [MGC]) and mice (vomeronasal organ [VNO]) express Ephrin, and their adjacent regions express Eph (Anzo, 2017).

Although Drosophila shows an opposite pattern of Eph/Ephrin (Eph in the pheromone-sensing circuit and Ephrin in the adjacent region), the same signaling machinery seems to have a conserved role in glomerular boundary formation across species. Interestingly, the mouse accessory olfactory bulb receiving input from the VNO, the moth MGC, and the Drosophila pheromone sensory glomeruli are all clustered and located dorsally to the other ordinary glomeruli in the mouse main olfactory bulb and the moth/Drosophila ALs, respectively. This conserved anatomical feature also suggests a notion that a unique signaling pathway is playing a role to secure the strict segregation between the pheromone-sensing circuits and the other olfactory circuits. Taken together, it is hypothesized that the reproductive behavior circuit is highly specific and segregated from the others using the Eph/Ephrin signal. Since Eph and Ephrin are both membrane-bound proteins, the signal is activated in a contact-dependent manner. In addition, the bidirectional signal transduction characteristic of the Eph/Ephrin signal system is reasonable for the local dendrodendritic segregation. It is possible that proper segregation in a dendrite level is necessary for building a well-organized neural network, thus allowing the optimal transfer of pheromone-related information to a higher brain center while controlling the courtship behavior (Anzo, 2017).


Promoter Structure

To establish the identity of the Eph-expressing neurons, transgenic lines were generated carrying the Eph neural enhancer fused to an axon-targeted reporter gene. Eight genomic fragments covering 20 kb were tested for enhancer activity by examining transgenic individuals, each carrying a fragment fused to either tau-myc or tau-lacZ. As judged by reporter gene expression, genomic fragments dekD (for Drosophila Eph kinaseD) through dekH do not demonstrate any enhancer activity. Fragments dekA and dekB drive reporter gene expression in patterns completely unrelated to that of Eph and thus appear to contain enhancers for a different gene located upstream of the Eph locus. In contrast, dekC, a 5.5-kb fragment located 2.2 kb upstream of the putative transcriptional start site of Eph, contains the Eph neural enhancer. In dekC-tau-lacZ embryos, tau-beta-gal expression closely resembles the pattern of Eph expression and is confined to a large subset of interneurons that project axons in the commissures and connectives of the VNC. Similar to the antibody result, no reporter expression could be detected in motor neurons. dekC also drives expression of tau-beta-gal in third-instar larval photoreceptor cells and their axon projections into the optic brain lobes (Scully, 1999).

Protein Interactions

To test if Drosophila Ephrin is able to bind to axons, the secreted EphrinDeltaC-term truncation, which has an intact receptor binding domain, was expressed. Expression of EphrinDeltaC-term in muscles overlying the CNS (GAL4 line 24B) results in a specific accumulation of the truncated protein on axons. In vertebrates, injection of secreted forms of ephrins give a dominant negative phenotype. However, expression of EphrinDeltaC-term in CNS or muscles (elav-GAL4, sim-GAL4 and GAL424B) failed to cause any obvious defects. This lack of phenotypes is most likely due to insufficient levels of expression (Bossing, 2002).

The accumulation of EphrinDeltaC-term around axons suggests that Ephrin may bind to Eph receptor tyrosine kinase (Eph). The binding between Ephrin and Eph was confirmed in cell culture. Drosophila S2 cells were transfected with a UAS construct encoding the extracellular part of Eph fused to GFP (Ephex-GFP). After incubation with Ephex-GFP-containing medium, non-permeabilized Schneider cells can be labelled with anti-GFP. Hence, Ephex-GFP can bind onto the surface of S2 cells that express endogenous Ephrin. To confirm that Ephex-GFP binds to Ephrin the level of Ephrin expression was lowered by incubation of S2 cells with dsEphrin mRNA. Indeed, Ephrin RNAi treatment of S2 cells diminishes the binding of Ephex-GFP. Control incubation of Schneider cells with dsGFP RNA (GFP RNAi) or dsEph RNA (Eph RNAi) does not interfere with the binding (Bossing, 2002).

The Amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors

VAP proteins (human VAPB/ALS8, Drosophila VAP33, and C. elegans VPR-1) are homologous proteins with an amino-terminal major sperm protein (MSP) domain and a transmembrane domain. The MSP domain is named for its similarity to the C. elegans MSP protein, a sperm-derived hormone that binds to the Eph receptor and induces oocyte maturation. A point mutation (P56S) in the MSP domain of human VAPB is associated with Amyotrophic lateral sclerosis (ALS), but the mechanisms underlying the pathogenesis are poorly understood. This study shows that the MSP domains of VAP proteins are cleaved and secreted ligands for Eph receptors. The P58S mutation in VAP33 leads to a failure to secrete the MSP domain as well as ubiquitination, accumulation of inclusions in the endoplasmic reticulum, and an unfolded protein response. It is proposed that VAP MSP domains are secreted and act as diffusible hormones for Eph receptors. This work provides insight into mechanisms that may impact the pathogenesis of ALS (Tsuda, 2008).

The mechanisms that underlie ALS are poorly understood. ALS is associated with the dysfunction or death of motor neurons in the motor cortex, brain stem, and spinal cord. About 10%-15% of all ALS cases are familial, whereas 85%-90% are sporadic. The most common form of familial ALS is caused by mutations in superoxide dismutase 1 (SOD1). Another gene, ALS8, that has been identified that causes familial ALS. This gene encodes the VAMP (synaptobrevin)-associated protein B (VAPB). Lesions in SOD1 and ALS8 have been shown to cause a wide variety of symptoms that typically include motor neuron death, but vary widely in the age of onset, the speed of progression, and the motor neuron populations that are affected. For example, a single amino acid change in ALS8 (P56S) causes typical ALS, atypical slowly progressive ALS, and spinal muscular atrophy (SMA) with an age of onset between 25 and 52 years and a speed of progression between 2 and 30 years. The cause of this variation may be due to genetic modifiers, partial redundancy, or environment (Tsuda, 2008).

VAPB is closely related to VAPA, which has been shown to associate with the cytoplasmic face of the endoplasmic reticulum (ER) and the Golgi apparatus. Human VAPB (hereafter named hVAP) protein is about 30 kDa and has homologs in C. elegans (VPR-1), Drosophila (VAP33-A, hereafter named dVAP), and numerous other species, including yeast (Scs2p). VAPs consist of an amino (N)-terminal domain of about 125 residues called the major sperm protein (MSP) domain, which is conserved among all VAP family members. The central region is predicted to form a coiled-coil motif. The hydrophobic carboxy (C)-terminus acts as a membrane anchor. The MSP domain is named for its similarity to nematode MSPs, the most abundant proteins in nematode sperm. MSP and VAP MSP domains fold into evolutionarily conserved immunoglobulin-type seven-stranded β sandwiches, suggesting a common function (Tsuda, 2008).

The main difference between VAPs and MSP is their proposed functions. C. elegans MSPs do not contain a coiled-coil motif or a transmembrane domain. MSPs have an intracellular cytoskeletal function, which depends on their ability to polymerize in the absence of actin or myosin and an extracellular signaling function during fertilization. MSP is secreted from the sperm cytosol into the reproductive tract by an unconventional process. Extracellular MSP directly binds to the VAB-1 Eph receptor and other yet-to-be-identified receptors on oocyte and ovarian sheath cell surfaces. MSP induces oocyte maturation, which prepares oocytes for fertilization and embryogenesis, and sheath contraction (Tsuda, 2008 and references therein).

The Eph receptors are an evolutionarily conserved class of receptor tyrosine kinases that bind to membrane-attached ligands called Ephrins. Ephrins act in parallel to gap junctions to inhibit oocyte maturation, and MSP antagonizes this inhibitory circuit. MSP induces activation of the MAP kinase and Ca2+/calmodulin-dependent protein kinase II cascades as well as reorganization of the oocyte microtubule cytoskeleton (Tsuda, 2008 and references therein).

The biological function of VAPs is not well understood. Yeast Scs2p is involved in phosphatidylinositol-4-phosphate synthesis and ceramide transport. VAPs have been reported to associate with the ER. Overexpression of hVAP in human cells affects the structural integrity of the ER through interaction with Nir (N-terminal domain-interacting receptor) proteins. VAPs also interact with oxysterol-binding protein (OSBP) and ceramide transfer protein. These interactions are each mediated through FFAT (two phenylalanines in an acidic tract) domains. Taken together, the results suggest that VAPs might play a role in fatty acid metabolism (Tsuda, 2008 and references therein).

To further define the role of VAPs, Drosophila dVAP has been characterized. dVAP modulates the number and size of neuromuscular junction (NMJ) boutons. Loss of dVAP disrupts the presynaptic microtubule architecture and causes an increase in miniature excitatory junctional potential (mEJP) size as well as an increase in postsynaptic glutamate receptor clustering (Tsuda, 2008).

This study presents evidence that VAP MSP domains are secreted ligands for Eph receptors. It is proposed that secreted MSP domains function as trophic factors by binding to Eph receptors and other cell-surface receptors. The P56S mutation that causes ALS8 (P58S in dVAP) induces insoluble aggregates that are ubiquitinated in flies. The mutation also leads to an accumulation of mutant and wild-type protein in the ER, an unfolded protein response (UPR), and a failure to secrete the MSP domain. Collectively, these results suggest that P56S affects a cell-autonomous pathway involving the ER and UPR as well as a cell nonautonomous pathway involving Eph receptor signaling (Tsuda, 2008).

ALS is a disease caused by death of anterior horn motor neurons in the spinal cord and neurons in motor cortex, after decades of apparently normal development and function. Familial and sporadic ALS cases as well as mouse models induced by overexpressing mutant SOD1 indicate that all forms lead to intracellular cytoplasmic protein inclusions containing ubiquitinated proteins. In flies expressing P58S dVAP, cytoplasmic inclusions and other key characteristics of ALS were found. (1) P58S dVAP protein induces ubiqutinated inclusions. (2) The protein inclusions are associated with the ER and appear to be electron-dense ER expansions. (3) Several key ER proteins colocalize with these inclusions. Finally, mutant dVAP induces a unfolded protein response (UPR). These data show at least three important parallels with ALS and SOD1 mouse models: cytoplasmic inclusions, ubiquitination, and the UPR. The UPR-induced stress caused by P58S dVAP could eventually result in cellular damage or neuronal death (Tsuda, 2008).

Another feature associated with ALS is that the disease may have a cell-non-autonomous component. VAP MSP domains can be secreted, although not all cell types appear capable of secretion in flies. The VAP proteins, including the yeast homolog SCS2, have been proposed to be type II-membrane proteins (Kagiwada, 1998). Since the proteins lack an N-terminal signal sequence, similar to MSP, secretion is likely to occur by an unconventional mechanism as observed for the C.elegans MSP proteins. In addition, the hVAP MSP domain is present in blood serum). The MSP in serum may be able to bind to Eph receptors present on endothelial cells, which regulate angiogenesis. Indeed, SOD1 mutants display defects in the tight junctions between endothelial cells, and endothelial damage occurs prior to motor neuron degeneration. Interestingly, it has recently been reported that VAPB is significantly decreased in the spinal cord of SOD1 mutants and human patients with sporadic ALS. It is therefore possible that reduced signaling by the hVAP MSP domain is a mechanism responsible for some nonautonomous features associated with ALS pathogenesis (Tsuda, 2008).

This study shows that secreted MSP domains bind to Eph receptors on the surfaces of cells. Eph receptors also bind to ligands called Ephrins. MSP domains function in vivo to antagonize Ephrin signaling during oocyte maturation and, possibly, amphid neuron migration. Competition assays are consistent with MSP domains competing with Ephrin for Eph receptor binding. In other processes, including worm-DTC cell migration, ovarian sheath contraction, and fly MB formation, MSP domains seem to be required for Eph receptor signaling. Hence, the relationship between MSP and Ephrin ligands to Eph receptor signaling may depend on the developmental context, as previously observed for Ephrins and Eph receptors in mammals. Multiple Ephrins and Eph receptors including EphA4 and A7 are expressed throughout the adult nervous system and in skeletal muscle of vertebrate species. Eph receptors regulate the survival of cultured spinal cord motor neurons and influence proliferation and apoptosis in the adult mammalian CNS. VAP MSP may play a role in motor neuron survival or muscle function through interactions with Eph receptors (Tsuda, 2008).

Glutamate excitotoxicity is likely to play a role in the pathogenesis of ALS. Three lines of evidence suggest that VAP MSP domains might regulate glutamate receptor signaling. (1) Eph receptors directly associate with NMDA-subtype glutamate receptors and regulate clustering in cultured neurons. (2) Loss of dVAP function or overexpression of P58S in flies is associated with increased glutamate receptor clustering and increased amplitudes of mEJPs at the NMJs. (3) MSP and the VAB-1 Eph receptor regulate NMDA receptor function during worm oocyte maturation (Tsuda, 2008 and references therein).

The following model is proposed for the pathogenesis of ALS8. The P56S hVAP protein accumulates in the ER, while the wild-type protein is functional. In time, the aggregates become more prominent, P56S hVAP becomes ubiquitinated, and functional wild-type proteins become trapped in the inclusions. These protein inclusions initiate a UPR that eventually affects cell viability and lead to a decrease in MSP domain secretion. Impaired secretion decreases signaling by Eph receptors and other receptors. The mutant protein therefore causes two different defects: a cell-autonomous defect in the ER that creates a UPR and a cell non-autonomous defect resulting from reduced secretion of VAP MSP, which may function as an autocrine or paracrine signal. Both defects may synergize to produce the key features of ALS pathology. This model provides testable hypotheses and raises questions to be addressed in the future (Tsuda, 2008).

hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction

Motor neuron diseases (MNDs) are progressive neurodegenerative disorders characterized by selective death of motor neurons leading to spasticity, muscle wasting and paralysis. Human VAMP-associated protein B (hVAPB) is the causative gene of a clinically diverse group of MNDs including amyotrophic lateral sclerosis (ALS), atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation is inherited in a dominant manner. Drosophila VAMP-associated protein of 33 kDa A (DVAP-33A) is the structural homologue of hVAPB and regulates synaptic remodeling by affecting the size and number of boutons at neuromuscular junctions. Associated with these structural alterations are compensatory changes in the physiology and ultrastructure of synapses, which maintain evoked responses within normal boundaries. DVAP-33A and hVAPB are functionally interchangeable and transgenic expression of mutant DVAP-33A in neurons recapitulates major hallmarks of the human diseases including locomotion defects, neuronal death and aggregate formation. Aggregate accumulation is accompanied by a depletion of the endogenous protein from its normal localization. These findings pinpoint to a possible role of hVAPB in synaptic homeostasis and emphasize the relevance of the fly model in elucidating the patho-physiology underlying motor neuron degeneration in humans (Chai, 2008).

hVAPB has been shown to be the causative gene of late-onset autosomal dominant forms of motor neuron disorders, including typical and atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation predicts a substitution of a Serine for a conserved Proline (P56). One of the hallmarks associated with loss-of-function and neuronal overexpression of DVAP-33A is decreased and increased bouton formation at the NMJ, respectively. Despite this structural alteration, synaptic transmission is maintained within a wt range. At the mechanistic level, muscles respond to a decreased number of boutons and quantal content by upregulating quantal size; conversely muscles compensate an increase in number of boutons and quantal content by downregulating quantal size. Compensatory changes in quantal size during synaptic homeostasis are thought to be determined, largely, by the properties of transmitter receptors. At the Drosophila NMJ, there are two classes of glutamate receptors: one set containing the subunit IIA and another one containing the subunit IIB. In DVAP-33A loss-of-function mutations, the increase in quantal size is associated with an increase in the number and average cluster volume of subunit IIA. Conversely, the decrease in quantal size in the oversprouting mutants is accompanied by a decrease in the level of post-synaptic receptor subunit IIA and a reduction in the average cluster volume for several subunits. In agreement with these data, the IIA subunit receptors have been shown to affect quantal size and receptor channel open time. Similar to the oversprouting mutants, in synapses lacking the receptor subunit IIA, a homeostatic increase in neurotransmitter release compensates for the reduction in quantal size and the evoked response is maintained within normal values. These data indicate that expression levels of VAP proteins play a crucial role in synaptic homeostasis by coordinating structural remodeling and post-synaptic sensitivity to neurotransmitter to ensure synaptic efficacy (Chai, 2008).

Interestingly, expression of hVAPB in neurons rescues lethality, morphological and electrophysiological phenotypes associated with DVAP-33A loss-of-function mutations. Moreover, neuronal expression of hVAPB in a wt background induces phenotypes similar to the overexpression of DVAP-33A. These data clearly indicate that DVAP-33A and hVAPB perform homologous functions at the synapse and as a consequence, information gained by studying DVAP-33A is expected to be relevant for hVAPB function as well. Surprisingly, neuronal expression of mutant VAP proteins also rescues all phenotypes associated with mutations in DVAP-33A. Two alternative scenarios could be proposed to explain these data: the mutation is irrelevant for the ALS8 pathogenesis or the mutant allele has a pathogenic effect while retaining certain functional properties of the wt protein. The second hypothesis is favored for the following reasons. (1) The P56S mutation in hVAPB has been reported to be causative for an inherited form of MNDs in humans. This mutation affects nine related families totaling 1500 individuals of which 200 suffer from motor neuron disorders. (2) A genetic model for MNDs was generated where the expression of the aberrant VAP recapitulates major hallmarks of the human disease, clearly indicating that the mutation has a pathogenic effect. (3) The data suggest that both the Drosophila and the human mutant proteins retain some functional wt properties such as the ability to self-oligomerize. However, neuronal expression of the pathogenic protein induces aggregate formation and depletes the wt protein from its normal localization. These effects are not observed when the wt protein is overexpressed, suggesting that the mutant protein has acquired a new, potentially toxic property (Chai, 2008).

Indeed, one of the most common features of MNDs and nearly all neurodegenerative diseases is the accumulation of aggregates that are intensively immuno-reactive to disease-related proteins. Each disease, however, differs with respect to the anatomical location and morphology of the aggregates. The major component of the aggregates is usually the protein encoded by the gene mutated in the familial forms, which is also unique to each disease. Despite this diversity, a bulk of circumstantial evidence support the hypothesis that aggregates are typical hallmarks of neurodegenerative diseases and have a toxic effect on neurons. While no autopsy material is available for familial cases with the P56S mutation, SOD1-positive inclusions have been reported in human sporadic and familial ALS cases as well as in SOD1 mouse models. This study found the presence of aggregates that are intensively immuno-reactive for DVAP-33A both in neuronal cell bodies and in nerve fibers of the MND model. Interestingly, hVAPB carrying the pathogenic mutation has also been shown to undergo intracellular aggregation when expressed in a cell culture system. However, similarities between human disease and the fly model are not limited to aggregate formation as flies expressing transgenic VAP proteins carrying the ALS8 mutation, exhibit other hallmarks of the human disease such as neuronal cell death, muscle wasting and defective locomotion behavior (Chai, 2008).

Although it remains to be established whether the VAP protein in the aggregates represents the mutant protein, the endogenous protein or a mixture of both, a regional decrease in the level of the endogenous protein is clearly observed. The DVAP-33A protein that is normally associated with the plasma membrane in neuronal cell bodies and at the neuromuscular synapses is nearly undetectable in DVAPP58S transgenic animals. As a consequence of the decrease in synaptic levels of the endogenous protein, a decrease in the number of boutons is observed. It has been previously shown that DVAP-33A regulates bouton formation at the synapse in a dosage-dependent manner. Despite these structural alterations a homeostatic mechanism is established to maintain synaptic efficacy within functional boundaries. It is speculated that the depletion of the endogenous protein from its normal localization and the formation of aggregates would affect the homeostatic mechanism linking structural remodeling and synaptic efficacy controlled by DVAP-33A. Although not directly tested in this model, experiments in cell culture show that overexpression of mutant hVAPB induces formation of aggregates in which the endogenous wt protein is recruited. This would suggest that the pathogenic allele functions as a dominant negative. However, the depletion of the endogenous protein from its normal localization cannot be the principal mechanism of the disease as mutants lacking DVAP-33A do not develop MND. It is therefore possible that the pathogenic allele has acquired an abnormal, new toxic activity. Similar to what has been proposed for other neurodegenerative diseases, the formation of aggregates may directly interfere with critical cellular processes and/or compromise the ability of the system to keep up with the degradation of aggregated proteins (Chai, 2008).

Taken together these data offer experimental support to the hypothesis that VAP proteins play a conserved role in synaptic homeostasis and emphasize the relevance of this fly model in fostering an understanding of the molecular mechanisms underlying VAP-induced motor neuron degeneration in humans (Chai, 2008).


In situ hybridization to whole-mount embryos reveals that Eph transcripts are present in precellular blastoderm stage embryos, as well as in unfertilized eggs, demonstrating that EPH mRNA is maternally supplied. A higher concentration of Eph transcripts is evident in the posterior pole of the embryo. In the embryo, zygotic transcription of Eph is confined to the nervous system. Expression commences in a large subset of neurons within the brain and ventral nerve cord (VNC) at stage 13 when neurons begin elongating axons. Eph continues to be expressed in the larval CNS and imaginal discs, as well as pupal and adult stages as assayed by Northern blot analysis. To further define which neurons express Eph, antibodies were generated to the cytoplasmic portion of the Eph protein. Immunostaining with an affinity-purified mouse antibody reveals that Eph is highly targeted to axons and growth cones of developing neurons within the VNC. Highest levels are present on axons within the connectives; lower levels are detectable in the commissures. Based upon the numbers and morphology of the staining axons, Eph is expressed by a large subset of interneurons and does not appear to be expressed by motor neurons (Scully, 1999).


Roles for Eph receptor tyrosine kinase signaling in the formation of topographic patterns of axonal connectivity have been well established in vertebrate visual systems. A role for a Drosophila Eph receptor tyrosine kinase (Eph) in the control of photoreceptor axon and cortical axon topography in the developing visual system is described. Although uniform across the developing eye, Eph is expressed in a concentration gradient appropriate for conveying positional information during cortical axon guidance in the second-order optic ganglion, the medulla. Disruption of this graded pattern of Eph activity by double-stranded RNA interference or by ectopic expression of wild-type or dominant-negative transgenes perturbs the establishment of medulla cortical axon topography. In addition, abnormal midline fasciculation of photoreceptor axons results from the eye-specific expression of the dominant-negative Eph transgene. These observations reveal a conserved role for Eph kinases as determinants of topographic map formation in vertebrates and invertebrates (Dearborn, 2002).

EPH expression coincides spatially and temporally with the differentiation and outgrowth of photoreceptor and cortical cell axons in the developing eye and optic ganglia, respectively. Eph antigen accumulates on the axons and growth cones of these neurons. Interestingly, the level of Eph immunoreactivity varies in a position-specific manner within each tissue. As photoreceptor axons grow into the lamina, Eph antigen is most strongly concentrated on the older photoreceptor growth cones that terminate at the posterior of the lamina. Eph antigen is also most strongly concentrated in the prospective posterior medulla neuropil that contains the axons of the earliest differentiating cortical neurons and R7-R8 photoreceptors. One might suppose that this distribution of antigen reflects the accumulation of Eph with time after the onset of differentiation. However, the observation that the anteroposterior gradient on these axons and growth cones persists into the early pupal stage suggests that it reflects spatially distinct expression or stability of Eph. Eph also displays a symmetrical concentration gradient on the dorsoventral axis of the medulla. Cortical neurons at the prospective midline of the medulla express the highest levels of Eph. In analogy with vertebrate Eph family members, the position-specific distribution of Drosophila Eph might reflect a role in the guidance of cortical cell axons to correct topographic positions. Consistent with this model, the single ephrin-like molecule encoded in the Drosophila genome (see Ephrin) is expressed in a gradient pattern that is complimentary to the Eph dorsoventral pattern in the medulla. The centripetal trajectories of cortical cell axons might thus rely on a repulsive interaction between Eph-bearing midline growth cones and a dorsoventral localized ephrin ligand. The apparently uniform expression of Eph on the dorsoventral axis of the eye does not preclude a role in the dorsoventral guidance of photoreceptor axons. In the chick, the response of retinal growth cones to target-derived ephrin can be modulated by nonuniform coexpression of an ephrin ligand by retinal ganglion neurons (Dearborn, 2002).

To gain insight into the role of Eph in the establishment of topographic connectivity, double-stranded RNA interference was used to reduce or eliminate Eph expression. eph dsRNA was injected into syncytial stage embryos to perturb eph expression at the larval time points relevant to axon targeting in the adult visual system. The reliability of RNAi was enhanced by using unique regions of eph as dsRNA template and by carefully determining the level of Eph antigen in the visual systems of dsRNA-injected animals. The data reveal that defects in photoreceptor and medulla cortical axon projections are associated with the loss of Eph expression. In the 20% of specimens that display a significant reduction or complete loss of Eph expression, the eye and medulla cortex formed with apparently normal size and cellular organization. The severe defects in medulla neuropil topography observed were most consistent with mistargeting of cortical axons. Given the severity of these defects in this target destination for the R7-R8 photoreceptor axons, it cannot be concluded that loss of Eph expression affected photoreceptor axons directly. The low penetrance of dsRNA-mediated effects (~20%) is consistent with previous reports on the effects of embryonic introduction of dsRNA on postembryonic and adult gene expression. Thus, RNAi-mediated reduction or elimination of Eph expression indicates that Eph is required for normal optic ganglia formation (Dearborn, 2002).

This conclusion was supported and refined by examining the consequences of expressing wild-type (UAS-eph+) and dominant-negative (UAS-ephDN) transgenes in the visual system. In the developing eye, transgene expression was driven in differentiating ommatidial cell clusters with ey-GAL4 and GMR-GAL4. Photoreceptor axon fascicles from each ommatidial unit (R1-R8) are normally bundled together as they traverse the optic stalk and then separate on the dorsoventral axis as they turn toward retinotopic destinations in the lamina field. With the expression of ephDN, the photoreceptor axon fascicles located near the midline were affected at the entrance into the lamina, in which they remained bundled together and often projected out of the lamina field. Axons of dorsally and ventrally located photoreceptors projected to topographically appropriate locations, despite their expression of ephDN. These defects were also observed when the FLP-out GAL4 driver was used to express ephDN in clones restricted to the developing eye. These observations are at odds with those of Scully (1999), who reported GMR-GAL4-driven expression of a putative dominant-negative eph construct did not cause defects in photoreceptor axon pathfinding. However, their construct was made by introducing a single amino acid substitution into the Eph kinase domain to eliminate kinase activity. It is possible that the fasciculation phenotype observed does not require kinase activity but relies on signaling from other Eph intracellular domains that are deleted in the ephDN construct. These observations are consistent with the idea that repulsion mediated by Eph activity is required to separate the axon fascicles as they emerge from the optic stalk. Endogenously truncated isoforms of vertebrate Eph RTKs have been found to promote adhesive interactions when coexpressed with full-length receptors in vitro (Dearborn, 2002).

The possibility that the dorsoventral gradient of Eph expression is necessary for the establishment of medulla cortical axon topography was examined by expressing the eph+ and ephDN transgenes in specific cortical cell populations. The omb-GAL4 driver was used to express the eph+ transgene in dorsally and ventrally located cortical cell populations that normally express little Eph, thus disrupting the Eph gradient on this axis. This resulted in the disruption of the projections of dorsal and ventral cortical cells. Similarly, when an ap-GAL4 driver was used to misexpress eph+ in a subset of cortical cells distributed along the dorsoventral axis, only those cells located in dorsal and ventral locations displayed axon projection defects. Although the omb-GAL4 driver would also yield eph+ expression at the dorsal and ventral margins of the eye and in a subset of optic lobe glia, the similar outcome resulting with ap-GAL4-driven expression (which is not expressed in either of those cell populations) indicates that cortical cell expression of eph+ underlies the axon projection defects. In contrast, ap-GAL4-driven expression of ephDN results in cortical cell axon projection defects at the midline, in which cells normally express the highest levels of Eph. These results are consistent with an interpretation that the requirement for Eph activity is highest at the midline, which coincides with the distribution of Eph along this axis. These observations are also consistent with the activity of the putative ephrin as a growth cone repellent for Eph-positive axons. This ephrin transcript is expressed in a pattern that is complimentary to the Eph pattern on the dorsoventral axis (Dai and Kunes, unpublished observations reported in Dearborn, 2002). More restricted, mosaic expression of the ephDN transgene in both eye and brain tissues using the FLP-out GAL4 driver further confirms a role for Eph in the formation of both retinotopic and cortical cell topographic projections and suggests that relative levels of Eph activity are critical to the establishment of medulla axon topography, observations consistent with studies performed in the mouse (Dearborn, 2002).

In summary, disruption of wild-type Eph expression and/or activity in both photoreceptor and medulla cortical cells results in defects in the axon projections of these cell types consistent with a position-dependent requirement for Eph signaling. These observations provide the first evidence that the underlying mechanisms directing axons to topographically appropriate sites within the brain during visual system development are conserved in vertebrates and invertebrates, relying on position-specific levels of Eph signaling (Dearborn, 2002).

Both Ephrin and Eph map to the fourth chromosome, for which it is very difficult to obtain and maintain mutants by classical genetic techniques. For this reason RNAi was used to inhibit expression. RNAi has rapidly become an accepted technique for generating mutant phenotypes. In test injections of dsEphrin RNA only two out of nine injected embryos show a nearly complete loss of Ephrin, while the remainder retains about 20%-50% of wild-type expression. Therefore, it was not expected that Ephrin RNAi would lead to a mutant phenotype in all injected embryos nor that all segments per embryo would be affected. Indeed, only 65% (13/20) of embryos injected with dsEphrin showed an aberrant phenotype and in total 39% (77/200) of all segments are affected. In four injected embryos all segments were affected. The phenotypes include fused commissures, loss of commissures and breaks in the connectives. Although Ephrin RNAi impedes commissure formation, it does not interfere with the differentiation of midline glia. Injection with dsCFP or buffer does not reduce Ephrin expression but occasionally results in phenotypes similar to dsEphrin injections. However, only 30% (5/15) of dsCFP injected embryos and 23% (4/17) of buffer injected embryos show a phenotype. The number of affected segments is reduced to 6% (9/148, dsCFP) or 8% (13/167, buffer) (Bossing, 2002).

Eph RNAi also results in fused commissures, loss of commissures and breaks in the connectives. The phenotype of Eph RNAi is more severe than for Ephrin RNAi. 80% (12/15) of all embryos had a phenotype and in total 69% (98/142) of all segments were affected. In five embryos all segments were abnormal. The difference in the strength of phenotype could either indicate that additional ligands besides Ephrin signal through Eph or the difference might be caused by the efficiency of RNAi, which varies between different genes (Bossing, 2002).

RNAi against Ephrin and Eph results in the fusion or loss of commissures and breaks in the connectives. Using a general axon marker, the origin of these phenotypes is not clear. Therefore the behaviour of single axons was followed in RNAi-treated embryos. The Gal4 line CY27 primarily drives expression of UAS-taumGFP6 in 2 interneurons per hemisegment, the vMP2 and dMP2 neuron. The MP2 neurons are among the first neurons to extend their axons along the connectives. In differentiated embryos the projections of these neurons form a tight fascicle which extends close and in parallel to the midline. Loss of Ephrin or Eph causes the axons of the MP2 neurons to project aberrantly out of the CNS. In 75% of embryos (15/20, Ephrin RNAi) and 82% of embryos (14/17, Eph RNAi), MP2 axons exiting the CNS were found. In the GAL4 line CY27, additional interneurons (i.e., UMI neurons) start to express GFP in late embryogenesis. No attempt was made to examine these weak projections in detail but it was noticed that many of these interneuronal axons also project out of the CNS. Therefore, signalling between Ephrin and Eph plays a role in confining interneuronal axons to the connectives (Bossing, 2002).

In vertebrates activation of Eph receptors in axonal growth cones is able to repel axons. It was speculated that despite the structural differences between vertebrate ephrins and Ephrin, the repulsive ability of Ephrin/Eph signalling might be conserved. Ephrin expression along the outer edge of the connectives and between the commissures could create barriers preventing axon extension. Absence of these barriers would be expected to result in fusion of commissures and the exit of interneuronal axons from the CNS, as was observed in RNAi experiments. To test whether Ephrin can act as an axonal repellent, Ephrin was ectopically expressed (Bossing, 2002).

Only 4-6 out of about 20 midline neurons express Ephrin. Ectopic expression of Ephrin in all midline cells (sim-GAL4) causes fusion, severe thinning or loss of commissures without affecting midline glial cell differentiation. Single cell labelling of neural precursors reveals that ectopic Ephrin in midline cells is able to prevent the midline crossing of axons. In all clones with contralateral axons, the axons are stalled at the midline. Ectopic midline Ephrin does not affect the extension of ipsilateral axons immediately adjacent to the midline or the determination of midline neurons (judged by the expression of Engrailed, Futsch and Odd-skipped) (Bossing, 2002).

Axonal repulsion by Slit, secreted from midline cells, is one of the major mechanisms controlling axons crossing the midline. It is possible that Ephrin expression at the midline exerts its repulsive effect by upregulating the expression or secretion of Slit. To test if repulsion by Ephrin depends on Slit, Ephrin was expressed ectopically in the midline of embryos mutant for Slit and Robo1, one of the receptors for Slit. Expression of Ephrin in slit/robo double mutants forces axons out of the midline. Therefore, Ephrin/Eph signalling at the ventral midline can act independently of Slit and Robo1 (Bossing, 2002).

Ephrin is expressed in nearly all neurons but not in the longitudinal glia that enwrap the connectives. Ephrin-expressing longitudinal glia were generated by injecting UAS-Ephrin plasmids into the syncytial blastoderm of GAL4MZ1580 embryos. When longitudinal glial cells express Ephrin, breaks are observed in the connectives. The breaks are always located near the glial cell. No breaks are observed when neurons express Ephrin. GFP-expressing longitudinal glial cells also do not disrupt axon extension (UAS-tau-mGFP6 plasmid). In summary, ectopic expression of Ephrin blocks axon extension (Bossing, 2002).

Activation of Eph on axons may be the reason that axons stall at Ephrin-expressing midline cells. In that case lowering the level of Eph activation by reducing Eph expression might allow these axons to overcome this repulsion and restore the commissures. To test this hypothesis Eph expression was lowered by Eph RNAi. Embryos with ectopic midline expression of Ephrin show a strong phenotype. Only 2% (2/100) of all segments have wild-type commissures and embryos are never found in which all segments have normal commissures. Injection of dsEph RNA rescues the commissures. In 30% (8/27) of all injected embryos all segments were restored to wild type. In contrast, in all embryos injected with buffer or dsGFP, segments are found with fused or absent commissures, indicating that ectopic Ephrin is still able to repel axons. In dsEph-injected embryos 33% of all segments have wild-type commissures, whereas in control-injected embryos even fewer segments show normal commissures (Bossing, 2002).

Presumably, it is possible to rescue the commissures with Eph RNAi because dsEph-injected embryos do not always show a loss or fusion of commissures. 20% of injected embryos and 31% of all segments have no phenotype. In the rescued embryos, Eph expression might be lowered enough to overcome the repulsion by ectopic midline Ephrin but not low enough to result in fused or lost commissures (Bossing, 2002).

An RNA interference screen for genes required to shape the anteroposterior compartment boundary in Drosophila identifies the Eph receptor

The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. This study reports on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in Drosophila wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, a single novel candidate was identified that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. These results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development (Umetsu, 2014: PubMed).


Eph receptors: Domain structure and function

The large subfamily of receptor tyrosine kinases (RTKs) for which EPH is the prototype have likely roles in intercellular communication during normal mammalian development, but the biochemical signaling pathways utilized by this family are poorly characterized. Two in vitro autophosphorylation sites have been identified within the juxtamembrane domain of the Eph family member Sek, and a candidate binding protein for the activated Sek kinase. Specific antibodies define Sek as a 130 kDa glycoprotein with protein kinase activity expressed in keratinocytes, while a bacterially expressed gst-Sek kinase domain fusion protein autophosphorylates exclusively on tyrosine residues, confirming that Sek encodes an authentic protein tyrosine kinase. Two dimensional phosphopeptide mapping and site-directed mutagenesis define juxtamembrane residue Y602 as a major site of in vitro autophosphorylation in Sek, while Y596 is phosphorylated to a lower stoichiometry. Complimentary approaches of in vitro binding assays and BIAcore analysis reveal a high affinity association between the Y602 Sek autophosphorylation site and the cytoplasmic tyrosine kinase p59fyn, an interaction mediated through the SH2 domain of this intracellular signaling molecule. Moreover, these data identify the novel phosphotyrosyl motif pYEDP as mediating high affinity association with fyn-SH2, extending the previously defined consensus motif for this interaction. The extensive conservation of this fyn-binding motif within the juxtamembrane domain of Eph family RTKs suggests that signaling through fyn, or fyn-related, tyrosine kinases may be utilized by many members of this large subclass of transmembrane receptors (Ellis, 1996).

The Eph family of receptor protein-tyrosine kinases (RTKs) has recently been implicated in patterning and wiring events in the developing nervous system. Eph receptors are unique among other RTKs: they fall into two large subclasses that show distinct ligand specificities and they themselves might function as 'ligands', thereby activating bidirectional signaling. To gain insight into the mechanisms of ligand-receptor interaction, the ligand binding domain in Eph receptors has been mapped. By using a series of deletion and domain substitution mutants, an N-terminal globular domain of the Nuk/Cek5 receptor has been shown to be the ligand binding domain of the transmembrane ligand Lerk2. The Cek5 globular domain is sufficient to confer Lerk2-dependent transforming activity on the Cek9 orphan receptor. The same domain is used for binding of both transmembrane and glycosylphosphatidyl-anchored ligands. These studies have determined the first structural elements involved in ligand-receptor interaction and will allow more fine-tuned genetic experiments to elucidate the mechanism of action for these important guidance molecules (Labrador, 1997).

Reported here is the crystal structure at 2.9 A resolution of the amino-terminal ligand-binding domain of the EphB2 receptor (also known as Nuk). The domain folds into a compact jellyroll beta-sandwich composed of 11 antiparallel beta-strands. Using structure-based mutagenesis, an extended loop has been identified that is important for ligand binding and class specificity. This loop, which is conserved within but not between Eph RTK subclasses, packs against the concave beta-sandwich surface near positions at which missense mutations cause signaling defects, localizing the ligand-binding region on the surface of the receptor (Himanen, 1998).

The sterile alpha motif (SAM) domain is a protein interaction module that is present in diverse signal-transducing proteins. SAM domains are known to form homo- and hetero-oligomers. The crystal structure of the SAM domain from an Eph receptor tyrosine kinase, EphB2, reveals two large interfaces. In one interface, adjacent monomers exchange amino-terminal peptides that insert into a hydrophobic groove on each neighbor. A second interface is composed of the carboxyl-terminal helix and a nearby loop. A possible oligomer, constructed from a combination of these binding modes, may provide a platform for the formation of larger protein complexes (Thanos, 1999).

The EphA4 receptor tyrosine kinase regulates the formation of the corticospinal tract (CST), a pathway controlling voluntary movements, and of the anterior commissure (AC), connecting the neocortical temporal lobes. To study EphA4 kinase signaling in these processes, mice were generated expressing mutant EphA4 receptors either lacking kinase activity or with severely downregulated kinase activity. EphA4 is required for CST formation as a receptor for which it requires an active kinase domain. In contrast, the formation of the AC is rescued by kinase-dead EphA4, suggesting that in this structure EphA4 acts as a ligand for which its kinase activity is not required. Unexpectedly, the cytoplasmic sterile-alpha motif (SAM) domain is not required for EphA4 functions. These findings establish both kinase-dependent and kinase-independent functions of EphA4 in the formation of major axon tracts (Kullander, 2001b).

The Eph receptor tyrosine kinase family is regulated by autophosphorylation within the juxtamembrane region and the kinase activation segment. The X-ray crystal structure has been solved to 1.9 Å resolution of an autoinhibited, unphosphorylated form of EphB2 comprised of the juxtamembrane region and the kinase domain. The structure, supported by mutagenesis data, reveals that the juxtamembrane segment adopts a helical conformation that distorts the small lobe of the kinase domain, and blocks the activation segment from attaining an activated conformation. Phosphorylation of conserved juxtamembrane tyrosines would relieve this autoinhibition by disturbing the association of the juxtamembrane segment with the kinase domain, while liberating phosphotyrosine sites for binding SH2 domains of target proteins. It is proposed that the autoinhibitory mechanism employed by EphB2 is a more general device through which receptor tyrosine kinases are controlled (Wybenga-Groot, 2001).

Eph receptors: Interaction with ligands

The Eph-related family of receptor tyrosine kinases consists of at least 13 members, several of which display distinctive expression patterns in the developing and adult nervous system. In order to study functional interactions between B61-related ligands and Eph-related receptors, chimeric receptors, containing an Eph-related ectodomain and the cytoplasmic domain of the TrkB neurotrophin receptor were constructed. Expression and activation of such chimeric receptors in NIH 3T3 cells induce transformation in focus formation assays. Membrane-bound LERK2 ligand signals through three different Eph-related receptors: Cek5, Cek10 and Elk. LERK2, however, fails to interact functionally with the Cek9 receptor. Quantitative analysis including binding assays indicates that Cek10 is the preferred LERK2 receptor. Preliminary mutagenesis of the LERK2 protein suggests a negative regulatory role for its cytoplasmic domain in LERK2 signaling (Brambilla, 1995).

The Eph family of receptor tyrosine kinases and their cell surface bound ligands have been implicated in a number of developmental processes, including axon pathfinding and fasciculation, as well as patterning in the central nervous system. To better understand the complex signaling events taking place, a comparative analysis has been undertaken of ligand-receptor interactions between a subset of ligands, those that are tethered to the cell surface via a transmembrane domain, and a subset of Eph receptors, the so-called Elk subclass. Based on binding characteristics, receptor autophosphorylation, and cellular transformation assays, it has been found that the transmembrane-type ligands Lerk2 and Elf2 have common and specific receptors within the Elk subclass of receptors. The common receptors Cek10 and Elk bind and signal in response to Lerk2 and Elf2, whereas the Myk1 receptor is specific for Elf2. Elf2, however, fails to signal through Cek5 in a cellular transformation assay, suggesting that Lerk2 may be the preferred Cek5 ligand in vivo. A recently identified third transmembrane-type ligand, Elf3, binds Cek10 specifically, but weakly, and only induces focus formation when activated by C-terminal truncation. This suggests that the physiological Elf3 receptor may have yet to be identified. Knowledge regarding functional ligand-receptor interactions as presented in this study will be important for the design and interpretation of in vivo experiments, e.g., loss-of-function studies in transgenic mice (Brambilla, 1996).

Many Eph-related receptor tyrosine kinases, and each of their numerous membrane-bound ligands, can be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan (Gale, 1996).

The Eph receptors are the largest known family of receptor protein tyrosine kinases; they play important roles, along with their ligands (ephrins), in the neural development, angiogenesis, and vascular network assembly. Ephrin-A2, -A3 and -A5 bind to, and activate the EphA8 receptor tyrosine kinase. An examination was performed to see if there are other additional ephrin ligands interacting with the EphA8 receptor tyrosine kinase expressed in NIH3T3 fibroblasts. For this purpose, chimeric ephrin-A1, -A4, -B1, -B2 or -B3 ligands were constructed consisting of the Fc portion of human IgG fused to ephrin ligand carboxyl-terminus. Both ephrin-A1 and ephrin-A4 chimeric ligands efficiently bind to the EphA8 receptor expressed in NIH3T3 fibroblasts, whereas the transmembrane ligands including ephrin-B1, -B2 and -B3 do not. Both the EphA8-TrkB chimeric receptor and the EphA8 receptor expressed in NIH3T3 fibroblasts are efficiently tyrosine-phosphorylated upon stimulating with epthin-A1 or -A4 but none of transmembrane ephrin-B proteins. These results strongly indicate that the EphA8 receptor functions exclusively as an glycosyl phosphatidylinositol (GPI)-linked ephrin ligand-dependent receptor protein tyrosine kinase (Choi, 1999b).

The Eph family is thought to exert its function through the complementary expression of receptors and ligands. EphA receptors colocalize on retinal ganglion cell (RGC) axons with EphA ligands, which are expressed in a high-nasal-to-low-temporal pattern. In the stripe assay, only temporal axons are normally sensitive for repellent axon guidance cues of the caudal tectum. However, overexpression of ephrinA ligands on temporal axons abolishes this sensitivity, whereas treatment with PI-PLC both removes ephrinA ligands from retinal axons and induces a striped outgrowth of formerly insensitive nasal axons. In vivo, retinal overexpression of ephrinA2 leads to topographic targeting errors of temporal axons. These data suggest that differential ligand expression on retinal axons is a major determinant of topographic targeting in the retinotectal projection (Hornberger, 1999).

Eph receptor tyrosine kinases and their ligands (ephrins) are highly conserved protein families implicated in patterning events during development, particularly in the nervous system. In a number of functional studies, strict conservation of structure and function across distantly related vertebrate species has been confirmed. Soluble human EphA3 (HEK) exerts a dominant negative effect on somite formation and axial organization during zebrafish embryogenesis: this observation has been used to probe receptor function. Based on exon structure, the extracellular region of the EphA3 receptor has been dissected into evolutionarily conserved subdomains; kinetic BIAcore analysis, mRNA injection into zebrafish embryos, and receptor transphosphorylation analysis were all used to study the function of these domains. Ligand binding is restricted to the N-terminal region encoded by exon III, and an independent, C-terminal receptor-dimerization domain is identified. Recombinant proteins encoding either region in isolation can function as receptor antagonists in zebrafish. A two-step mechanism for Eph receptor activation with distinct ligand binding and ligand-independent receptor-receptor oligomerization events is proposed (Lackmann, 1998).

Eph receptors and oocyte meiotic maturation in C. elegans

Fertilization in the female reproductive tract depends on intercellular signaling mechanisms that coordinate sperm presence with oocyte meiotic progression. To achieve this coordination in C. elegans, sperm release an extracellular signal, the major sperm protein (MSP), to induce oocyte meiotic maturation and ovulation. MSP binds to multiple receptors, including the VAB-1 Eph receptor protein-tyrosine kinase on oocyte and ovarian sheath cell surfaces. Canonical VAB-1 ligands called ephrins negatively regulate oocyte maturation and MPK-1 mitogen-activated protein kinase (MAPK) activation. MSP and VAB-1 regulate the signaling properties of two Ca2+ channels that are encoded by the NMR-1 N-methyl D-aspartate type glutamate receptor subunit and ITR-1 inositol 1,4,5-triphosphate receptor. Ephrin/VAB-1 signaling acts upstream of ITR-1 to inhibit meiotic resumption, while NMR-1 prevents signaling by the UNC-43 Ca2+/calmodulin-dependent protein kinase II (CaMKII). MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation, and UNC-43 acts redundantly in oocytes to promote oocyte maturation and MAPK activation. These results support a model in which VAB-1 switches from a negative regulator into a redundant positive regulator of oocyte maturation upon binding to MSP. NMR-1 mediates this switch by controlling UNC-43 CaMKII activation at the oocyte cortex (Corrigan, 2005).

In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and embryo production; sperm release the major sperm protein (MSP) signal to trigger meiotic resumption. Meiotic arrest depends on the parallel function of the oocyte VAB-1 MSP/Eph receptor and somatic G protein signaling. MSP promotes meiotic maturation by antagonizing Eph receptor signaling and counteracting inhibitory inputs from the gonadal sheath cells. This study presents evidence suggesting that in the absence of the MSP ligand, the VAB-1 Eph receptor inhibits meiotic maturation while either in or in transit to the endocytic-recycling compartment. VAB-1::GFP localization to the RAB-11-positive endocytic-recycling compartment is independent of ephrins but is antagonized by MSP signaling. Two negative regulators of oocyte meiotic maturation, DAB-1/Disabled and RAN-1, interact with the VAB-1 receptor and are required for its accumulation in the endocytic-recycling compartment in the absence of MSP or sperm (hereafter referred to as MSP/sperm). Inactivation of the endosomal recycling regulators rme-1 or rab-11.1 causes a vab-1-dependent reduction in the meiotic-maturation rate in the presence of MSP/sperm. Further, Gαs signaling in the gonadal sheath cells, which is required for meiotic maturation in the presence of MSP/sperm, affects VAB-1::GFP trafficking in oocytes. It is concluded that regulated endocytic trafficking of the VAB-1 MSP/Eph receptor contributes to the control of oocyte meiotic maturation in C. elegans. Eph receptor trafficking in other systems may be influenced by the conserved proteins DAB-1/Disabled and RAN-1 and by crosstalk with G protein signaling in neighboring cells (Cheng, 2008).

CDC-42 orients cell migration during epithelial intercalation in the Caenorhabditis elegans epidermis.

Cell intercalation is a highly directed cell rearrangement that is essential for animal morphogenesis. As such, intercalation requires orchestration of cell polarity across the plane of the tissue. CDC-42 is a Rho family GTPase with key functions in cell polarity, yet its role during epithelial intercalation has not been established because its roles early in embryogenesis have historically made it difficult to study. To circumvent these early requirements, this study used tissue-specific and conditional loss-of-function approaches to identify a role for CDC-42 during intercalation of the Caenorhabditis elegans dorsal embryonic epidermis . CDC-42 activity is enriched in the medial tips of intercalating cells, which extend as cells migrate past one another. Moreover, CDC-42 is involved in both the efficient formation and orientation of cell tips during cell rearrangement. Using conditional loss-of-function it was shown that the PAR complex (see Drosophila PAR complex) functions in tip formation and orientation. Additionally, the sole C. elegans Eph receptor, VAB-1 (see Drosophila Eph), was found to function during this process in an Ephrin-independent manner. Using epistasis analysis, it was shown that vab-1 lies in the same genetic pathway as cdc-42 and is responsible for polarizing CDC-42 activity to the medial tip. Together, these data establish a previously uncharacterized role for polarized CDC-42, in conjunction with PAR-6, PAR-3 and an Eph receptor, during epithelial intercalation.

Ephrin-Eph signaling drives the asymmetric division in Ciona embryos

Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. This study uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. The segregation of notochord and neural fates is an autonomous property of the mother cell, and the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. These cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. It has been demonstrated that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. A model is proposed whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification (Picco, 2007).

Eph receptors: Interaction with transmembrane and cytoplasmic proteins

Ret is a receptor protein tyrosine kinase that has been implicated in the development of the enteric nervous, endocrine, and renal systems. Mutations associated with multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B) have been shown to activate the intrinsic kinase and transforming ability of ret. Using the cytoplasmic domain of Ret as bait in a yeast two-hybrid screen of a mouse embryonic library, it was discovered that the src homology 2 (SH2) domain containing protein Grb10 binds Ret. Grb10 belongs to an emerging family of SH2 containing adapter proteins, the prototypical member being Grb7. The SH2 domain of Grb10 has been shown to specifically interact with Ret. Additionally, using an EGFR/Ret chimera, it has been shown that Grb10 binds Ret in an activation-dependent manner in vivo. This is the first description of a receptor protein tyrosine kinase that utilizes Grb10 as a signaling intermediate (Pandey, 1995b).

Pagliaccio (Pag) is a receptor tyrosine kinase of the Eph family that is expressed in Xenopus embryos in a diverse set of localized tissues. Pag is the Xenopus homolog of Hek-8 (human), Sek-1 (mouse), cek8 (chicken), and RTK-1 (zebrafish). The function of this protein has been investigated by injecting RNA encoding an epidermal growth factor receptor-Pag chimera into early Xenopus embryos. Activation of the chimeric receptor results in a kinase-dependent loss of cell-cell adhesion. This dissociation can be reversed by co-injection of RNA encoding C-cadherin, suggesting that one or more cadherins could be the functional targets for Pag activity (Winning, 1996).

The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated. In vivo tyrosine phosphorylation sites of the Eph receptors (EphA3, EphA4, and EphB2) in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase. Furthermore, tyrosine-phosphorylated EphB2 is detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate. Interestingly, a form of Src with reduced electrophoretic mobility and increased tyrosine phosphorylation was detected in Cos cells expressing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src. Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrates that phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src. In contrast, binding of the carboxy-terminal SH2 domain of phospholipase Cgamma is not abolished upon mutation of tyrosine 611 in EphB2. Phosphopeptide mapping of autophosphorylated full-length EphB2, and wild-type and tyrosine-to-phenylalanine mutants of the EphB2 cytoplasmic domain fused to LexA, show tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in EphB2. The mutational analysis also indicates that tyrosines 605 and 611 are important for EphB2 kinase activity. It is proposed that Src kinase is a downstream effector that mediates the neuron's response to Eph receptor activation (Zisch, 1998).

Eph family receptor tyrosine kinases (including EphA3, EphB4) direct neuronal pathfinding within migratory fields of cells expressing gradients of membrane-bound ligands. Other Eph family RTKs (EphB1 and EphA2) direct vascular network assembly, affecting endothelial migration, capillary morphogenesis, and angiogenesis. To explore how ephrins could provide positional labels for cell targeting, a test was performed to see whether endogenous endothelial and P19 cell receptors [EphB1 (ELK) and EphB2 (Nuk)] discriminate between different oligomeric forms of an ephrin-B1/Fc fusion ligand. Receptor tyrosine phosphorylation is stimulated by both dimeric and clustered multimeric ephrin-B1, yet only ephrin-B1 multimers (tetramers) promote endothelial capillary-like assembly, cell attachment, and the recruitment of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) to receptor complexes. Cell-cell contact among cells expressing both EphB1 and ephrin-B1 is required for EphB1 activation and recruitment of LMW-PTP to EphB1 complexes. The EphB1-binding site for LMW-PTP was mapped and shown to be required for tetrameric ephrin-B1 to recruit LMW-PTP and to promote attachment. Thus, distinct EphB1-signaling complexes are assembled and different cellular attachment responses are determined by a receptor switch mechanism responsive to distinct ephrin-B1 oligomers (Stein, 1998a).

The large subfamily of receptor tyrosine kinases (RTKs) for which EPH is the prototype are likely to have roles in intercellular communication during normal mammalian development, but the biochemical signaling pathways utilized by this family are poorly characterized. Two in vitro autophosphorylation sites have been identified within the juxtamembrane domain of the Eph family member Sek, and a candidate binding protein for the activated Sek kinase. Specific antibodies define Sek as a 130 kDa glycoprotein with protein kinase activity expressed in keratinocytes, while a bacterially expressed gst-Sek kinase domain fusion protein autophosphorylates exclusively on tyrosine residues, confirming that Sek encodes an authentic protein tyrosine kinase. Two dimensional phosphopeptide mapping and site-directed mutagenesis define juxtamembrane residue Y602 as a major site of in vitro autophosphorylation in Sek, whilst Y596 is phosphorylated to a lower stoichiometry. Complimentary approaches of in vitro binding assays and BIAcore analysis reveal a high affinity association between the Y602 Sek autophosphorylation site and the cytoplasmic tyrosine kinase p59fyn, an interaction mediated through the SH2 domain of this intracellular signaling molecule. Moreover, these data identify the novel phosphotyrosyl motif pYEDP as mediating high affinity association with fyn-SH2, extending the previously defined consensus motif for this interaction. The extensive conservation of this fyn-binding motif within the juxtamembrane domain of Eph family RTKs suggests that signaling through fyn, or fyn-related, tyrosine kinases may be utilized by many members of this large subclass of transmembrane receptors (Ellis, 1996).

Eph-related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, the EphB2 receptor (formerly Nuk/Cek5/Sek3) was expressed in neuronal NG108-15 cells, and the tyrosine phosphorylation of multiple cellular proteins was observed upon activation of EphB2 by its ligand, ephrin-B1 (formerly Elk-L/Lerk2). The activated EphB2 receptor induces the tyrosine phosphorylation of a 62-64 kDa protein (p62[dok]), which in turn forms a complex with the Ras GTPase-activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also binds through its SH2 domains to tyrosine-phosphorylated EphB2 in vitro, and complexes with activated EphB2 in vivo. An in vitro RasGAP-binding site has been localized to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2; it has been demonstrated that substitution of these amino acids abolishes ephrin-B1-induced signaling events in EphB2-expressing NG108-15 cells. These tyrosine residues are followed by proline at the +3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2-activated signaling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture (Holland, 1997).

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. Two-hybrid interaction cloning was used to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. The EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, were mapped to the juxtamembrane region. Within this subdomain, Tyr594 is required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruits Nck to native receptor complexes and activates c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocks Nck recruitment to EphB1, attenuated downstream JNK activation, and blocks cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK (Stein, 1998b).

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, the yeast two-hybrid system was applied. The carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis reveal that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells, endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. These observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs (Hock, 1998).

Localizing cell surface receptors to specific subcellular positions can be critical for their proper functioning, as most notably demonstrated at neuronal synapses. PDZ proteins apparently play critical roles in such protein localizations. Receptor tyrosine kinases have not been previously shown to interact with PDZ proteins in vertebrates. Eph receptors and their membrane-linked ligands all contain PDZ recognition motifs and can bind and be clustered by PDZ proteins. Eph receptors and ligands colocalize with PDZ proteins at synapses. Thus, PDZ proteins may play critical roles in localizing vertebrate receptor tyrosine kinases and/or their ligands and may be particularly important for Eph function in guidance or patterning or at the synapse (Torres, 1998).

The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized cell-cell junctions. To identify potential receptor-binding targets of AF-6, the PDZ domain of AF-6 was screened against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors. Phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain (Buchert, 1999).

Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encode polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies have demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bind ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells (Lin, 1999).

An examination was carried out of the functional roles of two major autophosphorylation sites, Tyr-615 and Tyr-838, in the EphA8 receptor. Two-dimensional phosphopeptide mapping analysis demonstrates that Tyr-615 and Tyr-838 constitute major autophosphorylation sites in EphA8. Tyr-615 is phosphorylated to the highest stoichiometry, suggesting that phosphorylation at this site may have a physiologically important role. Upon conservative mutation of Tyr-838 located in the tyrosine kinase domain, the catalytic activity of EphA8 is strikingly reduced, both in vitro and in vivo, whereas a mutation at Tyr-615 in the juxtamembrane domain does not impair the tyrosine kinase activity. In vitro binding experiments reveal that phosphorylation at Tyr-615 in EphA8 mediates the preferential binding to Fyn-SH2 domain rather than Src and Ras GTPase-activating protein (Ras GAP)-SH2 domains. Additionally, a high level of EphA8 is detected in Fyn immunoprecipitates in intact cells, indicating that EphA8 and Fyn can physically associate in vivo. In contrast, the association of full-length Fyn to EphA8 containing mutation at either Tyr-615 or Tyr-838 is greatly reduced. These data indicate that phosphorylation of Tyr-615 is critical for determining the association with Fyn, whereas the integrity of Tyr-838 phosphorylation is required for efficient phosphorylation at Tyr-615 as well as other major sites. Finally, it was observed that cell attachment responses are attenuated by overexpression of wild type EphA8 receptor but to a much lesser extent by EphA8 mutants lacking phosphorylation at either Tyr-615 or Tyr-838. Furthermore, transient expression of kinase-inactive Fyn in EphA8-overexpressing cells blocks cell attachment responses attenuated by the EphA8 signaling. It is proposed that Fyn kinase is one of the major downstream targets for the EphA8 signaling pathway leading to a modification of cell adhesion, and that autophosphorylation at Tyr-838 is critical for positively regulating the EphA8 signaling event (Choi, 1999a).

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. 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 is activated and Rac is 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 reduces 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. Currently not much is known as to how ligand-induced activation of EphA receptor tyrosine kinases regulates the Rho and the Cdc42/Rac pathways. EphA receptors might interact via autophosphorylated juxtamembrane tyrosine residues, with RasGAP (Ras GTPase-activating protein), which is constitutively associated with RhoGAP (see Drosophila RhoGAP). RhoGAP is a negative regulator of Rho, and the strong activation of Rho by fc-ephrin-A5 in these experiments would require inactivation of RhoGAP activity. It remains to be shown if additional elements (p62 dok) of the RasGAP-RhoGAP complex are responsible for such an inhibition (Wahl, 2000).

EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB. The kinase activity of EphB may be important for subsequent steps in synapse formation, because perturbation of EphB tyrosine kinase activity affects the number of synaptic specializations that form in cultured neurons. These findings indicate that EphrinB activation of EphB promotes an association of EphB with NMDA receptors that may be critical for synapse development or function (Dalva, 2000).

As more examples of molecules that regulate aspects of synapse formation or maturation are described, it is becoming clear that there may be a variety of factors that regulate the process of synapse development. EphB receptors are localized at the postsynaptic membrane and associate with molecules such as PICK1 and AF6 that are established structural components of the synapse. In addition, EphB receptors interact with a host of intracellular effector molecules including Src, Nck, and Grb2, although the role for these molecules in synapse development is not yet clear. The binding of ephrinB to EphB2 may lead to the direct recruitment of NMDA receptor subunit NR1 and its associated subunits (NR2A-B) to the EphB complex. The binding of ephrinB to EphB also results in the recruitment of other proteins, including CaMKII and Grb10, to the EphB/NMDA receptor complex. These results indicate that EphB receptors are linked to structural and signaling molecules at the synapse that may enable an EphB receptor-driven signal to contribute to the development or function of synapses (Dalva, 2000).

The observation that EphB and NMDA receptors interact raises the intriguing possibility that cross-talk exists between these receptors to elicit changes in the functional properties of these proteins. One possibility is that the association of EphB and NMDA receptors could lead to changes in NMDA receptor function, perhaps via the phosphorylation of NMDA receptor subunits. The EphB RTK may phosphorylate the NMDA receptor either directly or via an associated kinase. Members of the Src family of tyrosine kinases, Src and Fyn, are good candidates to mediate EphB-dependent tyrosine phosphorylation of the NMDA receptor, since both of these Src family members bind Eph receptors and have been shown to regulate NMDA receptor function. CaMKII is another good candidate, because CaMKII is recruited to the EphB/NMDA receptor complex, and CaMKII has been shown to phosphorylate the NMDA receptor. Phosphorylation of the NMDA receptor may alter its channel conductance and NMDA receptor phosphorylation may underlie aspects of LTP. Thus, the assembly of the EphB and NMDA receptor complex may lead to changes in the channel properties of the NMDA receptor that could play an important role in synapse development or plasticity. This conclusion is supported by the observations that ephrin stimulation of EphBs enhances the formation of both pre- and post-synaptic specializations, and that blocking the ability of EphBs to signal via their receptor tyrosine kinase activity suppresses the number of postsynaptic specializations (Dalva, 2000).

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. The cloning and characterization of ephexin is reported. Ephexin is a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility (Shamah, 2001).

The genomes of C. elegans and Drosophila (see CG3799) each contain a single gene with a high degree of homology to ephexin. In humans, in addition to an ephexin ortholog, there are at least three other genes that are highly homologous to ephexin: the previously characterized Dbl family member TIM and two uncharacterized cDNAs -- Neuroblastoma and KIAA0915. Using the ClustalX program, phylogenetic analysis of the DH-PH domains of the ephexin-related proteins was performed and they were compared to the DH-PH domains of Dbl and Vav1, two other Dbl family GEFs. This analysis revealed that the DH-PH domains of ephexin-related GEFs are more closely related to each other than to the DH-PH domains of Dbl and Vav1, and identified ephexin-related GEFs as a subfamily within the larger Dbl family of GEFs. Alignment of ephexin subfamily proteins revealed a high degree of conservation within the DH, PH, and SH3 domains, but little or no homology in the N-terminal regions, including the 14 amino acid hydrophobic sequence present in ephexin orthologs (Shamah, 2001).

For growth cones to respond to extracellular guidance cues in a directional manner, guidance receptors must transduce highly localized signals to the actin cytoskeleton specifically in the region of the growth cone where receptor activation occurs. The finding that ephexin interacts directly with EphA4 receptors independent of receptor kinase activity suggests a mechanism for localized actin regulation by EphA receptors. Specifically, EphA activation would result in the modulation of ephexin activity only at the site of ligand presentation, while ephexin molecules in other regions of the growth cone remain unaffected. Furthermore, because ephexin is directly associated with EphA receptors, its downstream effects on Rho GTPases may remain confined to the vicinity of the activated receptors. Thus, ephexin may be modulated by EphA receptors to elicit local changes in Rho GTPase activity and to induce spatially restricted retraction of the growth cone at points of contact with ephrin-A (Shamah, 2001).

The mechanism through which EphA receptors modulate ephexin activity is currently not clear. One intriguing possibility is that substrate specificity within the catalytic domain of ephexin can be differentially regulated to yield inhibitory effects toward some GTPases and potentiating effects toward others. Alternatively, EphA4 inhibition of ephexin signaling to Rac1 and Cdc42 might be an indirect effect of elevated RhoA activity, or vice versa. In addition, the access of ephexin to different Rho GTPases might be regulated by EphA receptors as a result of changes in Rho GTPase subcellular localization, and this could contribute to the observed changes in Rho GTPase activities (Shamah, 2001).

The EphA receptor-induced change in ephexin activity might result from a posttranslational modification of ephexin. One possibility is that EphA receptor activation leads to changes in the phosphorylation state of ephexin due to the activation of specific kinases or phosphatases. Ephexin might also be modified as a result of EphA-induced regulation of the PI3-kinase signaling pathway, since EphA4 receptors bind to multiple PI3-kinase regulatory subunits and several Eph receptors are known to activate PI3-kinase. Also, PI3-kinase lipid products have been shown to regulate the activity of Vav and alphaPIX, a Pak-interacting GEF (Shamah, 2001).

Alternatively, since the association of EphA4 receptors occurs in the catalytic DH-PH domains of ephexin, EphA modulation of ephexin might occur through reversible steric or allosteric hindrance of GEF activity. Further studies utilizing specific pharmacologic and dominant-negative reagents as well as more detailed structure-function analyses should help to elucidate the mechanism of regulation of ephexin by EphA receptors (Shamah, 2001).

In summary, the experiments described here identify ephexin as an important molecular link between EphA receptors and the Rho family of GTPases, and suggest a model for how EphA receptors may locally regulate the actin cytoskeleton during axon guidance. Additional studies with specific inhibitors of ephexin and the genetic disruption of ephexin will be required to determine the role of ephexin in axon guidance and cell migration events during development (Shamah, 2001).

Eph tyrosine kinase receptors and their membrane-bound ligands, ephrins, are presumed to regulate cell-cell interactions. The major consequence of bidirectional activation of Eph receptors and ephrin ligands is cell repulsion. In this study, Xenopus Dishevelled (Xdsh) is found to form a complex with Eph receptors and ephrin-B ligands and mediate the cell repulsion induced by Eph and ephrin. In vitro re-aggregation assays with Xenopus animal cap explants revealed that co-expression of a dominant-negative mutant of Xdsh affects the sorting of cells expressing EphB2 and those expressing ephrin-B1. Co-expression of Xdsh induces the activation of RhoA and Rho kinase in the EphB2-overexpressing cells and in the cells expressing EphB2-stimulated ephrin-B1. Therefore, Xdsh mediates both forward and reverse signaling of EphB2 and ephrin-B1, leading to the activation of RhoA and its effector protein Rho kinase. The inhibition of RhoA activity in animal caps significantly prevents the EphB2- and ephrin-B1-mediated cell sorting. It is proposed that Xdsh, which is expressed in various tissues, is involved in EphB and ephrin-B signaling related to regulation of cell repulsion via modification of RhoA activity (Tanaka, 2003).

The function of the multi-PDZ domain scaffold protein GRIP1 (glutamate receptor interacting protein 1) in neurons is unclear. To explore the function of GRIP1 in hippocampal neurons, RNA interference (RNAi) was used to knock down the expression of GRIP1. Knockdown of GRIP1 by small interfering RNA (siRNA) in cultured hippocampal neurons causes a loss of dendrites, associated with mislocalization of the GRIP-interacting proteins GIuR2 (AMPA receptor subunit), EphB2 (receptor tyrosine kinase) and KIF5 (also known as kinesin 1; microtubule motor). The loss of dendrites by GRIP1-siRNA was rescued by overexpression of the extracellular domain of EphB2, and was phenocopied by overexpression of the intracellular domain of EphB2 and extracellular application of ephrinB-Fc fusion proteins. Neurons from EphB1-EphB2-EphB3 triple knockout mice showed abnormal dendrite morphogenesis. Disruption of the KIF5-GRIP1 interaction inhibited EphB2 trafficking and strongly impaired dendritic growth. These results indicate an important role for GRIP1 in dendrite morphogenesis by serving as an adaptor protein for kinesin-dependent transport of EphB receptors to dendrites (Hoogenraad, 2005).

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. This study shows that the Rac-specific GTPase-activating protein α2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. α-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. These results reveal an α-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo (Wegmeyer, 2007).

PTEN is one of the most commonly lost tumor suppressors in human cancer and is known to inhibit insulin signaling. Eph receptor tyrosine kinases (RTKs) have also been implicated in cancer formation and progression, and they have diverse functions, including nervous and vascular system development. This study shows that in C. elegans, the VAB-1 Eph kinase domain physically interacts with and phosphorylates PTEN (DAF-18), diminishing its protein levels and function. vab-1 mutants show increased longevity and sensitivity to dauer conditions, consistent with increased DAF-18/PTEN activity and decreased insulin-like signaling. Moreover, daf-18 mutations suppress vab-1 oocyte maturation phenotypes independent of PI3K signaling. Evidence is presented that DAF-18 has protein phosphatase activity to antagonize VAB-1 action. Possible implications for human cancers are discussed, based on the idea that mutually inhibitory interactions between PTEN and Eph RTKs may be conserved (Brisbin, 2009).

Eph receptors: 'reverse' signaling through ligands

Receptor tyrosine kinases of the EPH class have been implicated in the control of axon guidance and fasciculation, in regulating cell migration, and in defining compartments in the developing embryo. Efficient activation of EPH receptors generally requires that their ligands be anchored to the cell surface, either through a transmembrane (TM) region or a glycosyl phosphatidylinositol (GPI) group. These observations have suggested that EPH receptors can transduce signals initiated by direct cell-cell interaction. Genetic analysis of Nuk, a murine EPH receptor that binds TM ligands, has raised the possibility that these ligands might themselves have a signaling function. Consistent with this, the three known TM ligands have a highly conserved cytoplasmic region, with multiple potential sites for tyrosine phosphorylation. Challenging cells that express the TM ligands Elk-L or Htk-L with the clustered ectodomain of Nuk induces phosphorylation of the ligands on tyrosine, a process that can be mimicked both in vitro and in vivo by an activated Src tyrosine kinase. Co-culture of cells expressing a TM ligand with cells expressing Nuk leads to tyrosine phosphorylation of both the ligand and Nuk. These results suggest that the TM ligands are associated with a tyrosine kinase, and are inducibly phosphorylated upon binding Nuk, in a fashion reminiscent of cytokine receptors. Furthermore, TM ligands, as well as Nuk, are phosphorylated on tyrosine in mouse embryos, indicating that this is a physiological process. EPH receptors and their TM ligands therefore mediate bidirectional cell signaling (Holland, 1996).

Axonal pathfinding in the nervous system is mediated in part by cell-to-cell signaling events involving members of the Eph receptor tyrosine kinase (RTK) family and their membrane-bound ligands. Genetic evidence suggests that transmembrane ligands may transduce signals in the developing embryo. The cytoplasmic domain of the transmembrane ligand Lerk2 becomes phosphorylated on tyrosine residues after contact with the Nuk/Cek5 receptor ectodomain; this suggests that Lerk2 has receptorlike intrinsic signaling potential. Moreover, Lerk2 is an in vivo substrate for the platelet-derived growth factor receptor, which suggests crosstalk between Lerk2 signaling and signaling cascades activated by tyrosine kinases. It is proposed that transmembrane ligands of Eph receptors act not only as conventional RTK ligands but also as receptorlike signaling molecules (Bruckner, 1997).

Transmembrane ephrinB proteins have important functions during embryonic patterning as ligands for Eph receptor tyrosine kinases and presumably as signal-transducing receptor-like molecules. Consistent with 'reverse' signaling, ephrinB1 is localized in sphingo-lipid/cholesterol-enriched raft microdomains, platforms for the localized concentration and activation of signaling molecules. Glutamate receptor-interacting protein (GRIP) and a highly related protein, termed GRIP2, are recruited into these rafts through association with the C-terminal PDZ target site of ephrinB1. Stimulation of ephrinB1 with soluble EphB2 receptor ectodomain causes the formation of large raft patches that also contain GRIP proteins. Moreover, a GRIP-associated serine/threonine kinase activity is recruited into ephrinB1-GRIP complexes. These findings suggest that GRIP proteins provide a scaffold for the assembly of a multiprotein signaling complex downstream of ephrinB ligands (Bruckner, 1999).

Eph proteins are receptors with tyrosine-kinase activity which, with their ephrin ligands, mediate contact-dependent cell interactions that are implicated in the repulsion mechanisms that guide migrating cells and neuronal growth cones to specific destinations. Ephrin-B proteins have conserved cytoplasmic tyrosine residues that are phosphorylated upon interaction with an EphB receptor, and may transduce signals that regulate a cellular response. Because Eph receptors and ephrins have complementary expression in many tissues during embryogenesis, bidirectional activation of Eph receptors and ephrin-B proteins could occur at interfaces of their expression domains, for example at segment boundaries in the vertebrate hindbrain. Previous work has implicated Eph receptors and ephrin-B proteins in the restriction of cell intermingling between hindbrain segments. An analysis was carried out to see whether complementary expression of Eph receptors and ephrins restricts cell intermingling, and whether this requires bidirectional or unidirectional signaling. Bidirectional but not unidirectional signaling restricts the intermingling of adjacent cell populations, whereas unidirectional activation is sufficient to restrict cell communication through gap junctions. These results reveal that Eph receptors and ephrins regulate two aspects of cell behaviour that can stabilize a distinct identity of adjacent cell populations (Mellitzer, 1999).

Transmembrane B ephrins and their Eph receptors signal bidirectionally. However, neither the cell biological effects nor signal transduction mechanisms of the reverse signal are well understood. A cytoplasmic protein, PDZ-RGS3, is described that binds B ephrins through a PDZ domain, and has a regulator of heterotrimeric G protein signaling (RGS) domain. PDZ-RGS3 can mediate signaling from the ephrin-B cytoplasmic tail. SDF-1, a chemokine with a G protein-coupled receptor, and BDNF, both act as a chemoattractants for cerebellar granule cells, with SDF-1 action being selectively inhibited by soluble EphB receptor. This study reveals a pathway that links reverse signaling to cellular guidance, uncovers a novel mode of control for G proteins, and demonstrates a mechanism for selective regulation of responsiveness to neuronal guidance cues (Lu, 2001).

To investigate reverse signaling at a molecular level, a screen was performed for proteins that bind the B ephrin cytoplasmic domain, leading to identification of PDZ-RGS3 in a yeast two-hybrid assay. In situ hybridization shows a close overlap of expression patterns for PDZ-RGS3 with any one of the three known B ephrins in several parts of the nervous system. Taken together, these results indicate that PDZ-RGS3 is a genuine biological interaction partner of B ephrins (Lu, 2001).

PDZ domains are known to bind to a short conserved motif at the C terminus of many membrane proteins. A sequence fitting this motif is found at the C terminus of all known B ephrins, and the PDZ domain of PDZ-RGS3 binds the ephrin-B C terminus. Tyrosine residues are found in the binding motif (YYKV-carboxy terminus) suggesting potential control of binding by phosphorylation, although no evidence of this was seen, and the interaction did not appear to be regulated by EphB receptor binding. The presence of an RGS domain suggests PDZ-RGS3 might interact with downstream effector pathways. Accordingly, a Xenopus embryo cell dissociation assay PDZ-RGS3 mediates effects of the B ephrin cytoplasmic tail, in a manner dependent on both its PDZ and RGS domains. The involvement of the RGS domain in signaling, as well as the cerebellar expression of ephrins, led the authors to test cerebellar granule cells for an effect of reverse signaling on the action of the chemokine SDF-1, which acts through a GPCR. Soluble EphB2-Fc selectively regulates the guidance response to SDF-1, and this regulation is blocked by a truncated version of PDZ-RGS3 lacking the RGS domain (Lu, 2001).

Regarding the mechanism for signal transduction across the cell membrane, as with other PDZ proteins that bind B ephrins, the association with PDZ-RGS3 is seen constitutively, and does not appear to be modulated by treating cells with soluble EphB2-Fc. This suggests regulated association between B ephrin and PDZ-RGS3 is not a likely mechanism of signal transduction. An alternative could be regulation of clustering or subcellular localization. It is known that EphB2-Fc can cluster B ephrins and associated PDZ proteins into membrane rafts. Heterotrimeric G proteins have also been localized to rafts. Therefore, one model could be that Eph receptor binding clusters B ephrins into rafts, or other subcellular structures, and this could bring associated PDZ-RGS3 into proximity with the appropriate G proteins, resulting in inhibition of their activity. It is worth noting that not only the PDZ binding motif, but at least 33 amino acids of the B ephrin cytoplasmic tail are strongly conserved, and it is likely that additional protein interactions play a role in signaling, either through independent pathways or in collaboration with PDZ-RGS3 (Lu, 2001).

At the level of cell biological effects, these results show that reverse signaling induced by Eph receptor can regulate cellular guidance. Specifically, soluble EphB2-Fc selectively inhibits SDF-1 chemoattraction of cultured cerebellar granule neurons. Although reverse signaling through B ephrins has been investigated more extensively, soluble EphA receptors can affect adhesion in cell lines, and it will be interesting to see if this may reflect similar developmental functions or signaling pathways. The observations on the regulation of cerebellar granule cell guidance by EphB2-Fc, SDF-1, and BDNF led to a model for control of cell migration in cerebellar development. In principle, these observations could also fit with other developmental functions proposed for B ephrin reverse signaling in blood vessel formation, rhombomere compartmentation, and axon pathway selection -- all involving regulation of migration or morphogenesis (Lu, 2001).

The inward migration of cerebellar granule cells from the EGL is one of the best-characterized models of neuronal migration. The genetic demonstration that SDF-1 and its receptor CXCR4 are required for normal granule cell migration provided the first evidence of chemokines as regulators of neural development. Specifically, the phenotype of premature granule cell migration, taken together with the embryonic expression of SDF-1 in the pia mater overlying the cerebellum, suggest a model where SDF-1 would prevent premature inward migration of cerebellar granule cells by chemoattracting them toward the pia. The results support this model: SDF-1 expression occurs in the pia at postnatal stages that span the onset of granule cell migration, and SDF-1 acts as a chemoattractant for cultured cerebellar granule cells. Reverse signaling induced by soluble EphB2-Fc can inhibit the effect of SDF-1 on cerebellar granule cells. This provides functional evidence for an effect of ephrin signaling on cerebellar granule cells. A developmental role for the interaction of these signaling pathways is supported by the correlated expression of ephrin-B2, SDF-1, and their receptors during cerebellar development (Lu, 2001).

The number of cells in an organ is regulated by mitogens and trophic factors that impinge on intrinsic determinants of proliferation and apoptosis. This study reports on the identification of an additional mechanism to control cell number in the brain: EphA7 induces ephrin-A2 reverse signaling, which negatively regulates neural progenitor cell proliferation. Cells in the neural stem cell niche in the adult brain proliferate more and have a shorter cell cycle in mice lacking ephrin-A2. The increased progenitor proliferation is accompanied by a higher number of cells in the olfactory bulb. Disrupting the interaction between ephrin-A2 and EphA7 in the adult brain of wild-type mice disinhibits proliferation and results in increased neurogenesis. The identification of ephrin-A2 and EphA7 as negative regulators of progenitor cell proliferation reveals a novel mechanism to control cell numbers in the brain (Holmberg, 2005).

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. The p75 neurotrophin receptor (NTR) serves this role in retinal axons. p75NTR 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 p75NTR, but repulsion to ephrin-As by EphA forward signaling does not. Constitutive and retina-specific p75NTR 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. It is concluded that p75NTR is a signaling partner for ephrin-As and the ephrin-A- p75NTR complex reverse signals to mediate axon repulsion required for guidance and mapping (Lim, 2008).

Eph receptors are affected by other receptor systems

EphA2 is regulated by E-cadherin. In nonneoplastic epithelia, EphA2 is tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties require the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 is decreased, and EphA2 is redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restores a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreases cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2 (Zantek, 1999).

EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation

In humans, fetal cytotrophoblasts leave the placenta and enter the uterine wall, where they preferentially remodel arterioles. The fundamental mechanisms that govern these processes are largely unknown. Invasive cytotrophoblasts express several chemokines, as well as the receptors with which they interact. These ligand-receptor interactions stimulate cytotrophoblast migration to approximately the same level as a growth factor cocktail that includes serum. Additionally, cytotrophoblast commitment to uterine invasion is accompanied by rapid downregulation of EPHB4, a transmembrane receptor associated with venous identity, and upregulation of ephrin B1. Within the uterine wall, the cells also upregulated expression of ephrin B2, an EPH transmembrane ligand that is associated with arterial identity. In vitro cytotrophoblasts avoid EPHB4-coated substrates; upon co-culture with 3T3 cells expressing this molecule, their migration is significantly inhibited. As to the mechanisms involved, cytotrophoblast interactions with EPHB4 downregulate chemokine-induced but not growth factor-stimulated migration. It is proposed that EPHB4/ephrin B1 interactions generate repulsive signals that direct cytotrophoblast invasion toward the uterus, where chemokines stimulate cytotrophoblast migration through the decidua. the mucous membrane lining the uterus. When cytotrophoblasts encounter EPHB4 expressed by venous endothelium, ephrin B-generates repulsive signals and a reduction in chemokine-mediated responses limit cytotrophoblast interaction with veins. When cytotrophoblasts encounter ephrin B2 ligands expressed in uterine arterioles, migration is permitted. The net effect is preferential cytotrophoblast remodeling of arterioles, a hallmark of human placentation (Red-Horse, 2005).

Balancing of ephrin/Eph forward and reverse signaling as the driving force of adaptive topographic mapping

The retinotectal projection, which topographically maps retinal axons onto the tectum of the midbrain, is an ideal model system with which to investigate the molecular genetics of embryonic brain wiring. Corroborating Sperry's seminal hypothesis, ephrin/Eph counter-gradients on both retina and tectum were found to represent matching chemospecificity markers. Intriguingly, however, it has never been possible to reconstitute topographically appropriate fiber growth in vitro with these cues. Moreover, experimentally derived molecular mechanisms have failed to provide explanations as to why the mapping adapts to grossly diverse targets in some experiments, while displaying strict point-to-point specificity in others. In vitro, ephrin-A/EphA forward, as well as reverse, signaling mediate differential repulsion to retinal fibers, instead of providing topographic guidance. It is argued that those responses are indicative of ephrin-A and EphA being members of a guidance system that requires two counteracting cues per axis. Experimentally, it was demonstrate by introducing novel double-cue stripe assays that the simultaneous presence of both cues indeed suffices to elicit topographically appropriate guidance. The peculiar mechanism, which uses forward and reverse signaling through a single receptor/ligand combination, entails fiber/fiber interactions. It is therefore proposed to extend Sperry's model to include ephrin-A/EphA-based fiber/fiber chemospecificity, eventually out-competing fiber/target interactions. By computational simulation, it was shown that this model is consistent with stripe assay results. More importantly, however, it not only accounts for classical in vivo evidence of point-to-point and adaptive topographic mapping, but also for the map duplication found in retinal EphA knock-in mice. Nonetheless, it is based on a single constraint of topographic growth cone navigation: the balancing of ephrin-A/EphA forward and reverse signaling (Gebhardt, 2012).

Evidence for rigid mapping originates from classical regeneration studies carried out by Roger Sperry. When the optic nerve is transected and a part of the retina is deleted in adult teleosts or amphibians, the regenerating partial projection selectively targets its original destination, ignoring termination sites vacated by the deletion. Recent experiments in the zebrafish indicate that the leading tips of individual RGC axonal arbors map topographically even in the absence of any other retinal fibers. This evidence corroborates Sperry's hypothesis (Sperry, 1963), which postulates genetically encoded matching labels on fiber terminals and target sites [fiber/target (FT) chemospecificity]. Indeed, tectal cell membranes convey graded repulsive signals (a<p) to retinal fibers in vitro owing to the expression of ephrin-As sensed through counter-graded retinal EphA receptors. Eventually, individual ephrin-A/EphA expression patterns were found to sum up to counter-gradients along both the retinal n/t and the tectal a/p axis. Ephrin-A/EphA forward, as well as reverse, signaling exert repulsive actions on RGC growth cones in vitro and genetic deletions in mice suggest corresponding roles in vivo. Surprisingly, however, it has never been possible to reconstitute fully topographically appropriate growth of RGC fibers in vitro using the identified guidance molecules. Thus, in choice assays, in which retinal fibers are confronted with alternating stripes of ephrin-A and a neutral substrate (here called single-cue stripe assay), nasals never decide properly. The reasons for this failure have remained puzzling (Gebhardt, 2012 and references therein).

The concept of rigid chemospecificity was first challenged by other seminal regeneration experiments, which instead promoted the concept of adaptive mapping. When a regenerating half-retina innervates a full tectum, terminals only initially occupy their proper tectal half. After longer regeneration periods, the projection spreads to cover the whole target evenly (expansion). Conversely, the projection of a full retina properly scales to a half-tectum (compression) (. When a nasal half-retina is forced to innervate an emptied anterior half-tectum (mismatch), a normally oriented half-map develops on the foreign field. If in a similar experiment the occupied p-tectum is left in place when the a-tectum is de-afferentiated, a second nasal population again forms a half-map on the foreign anterior target, but now with reversed orientation (polarity reversal). These results have usually been taken to indicate guidance mechanisms based on fiber/fiber (FF) interactions, the molecular underpinnings of which, however, are unknown. Recently, FF interactions have gained renewed interest. When, in a scattered half-population of RGCs, a constant amount of EphA is added by transgenic expression, a map duplication occurs, in which knock-in terminals occupying the a-tectum displace wild-type terminals posteriorly (Gebhardt, 2012 and references therein).

These seemingly conflicting bodies of evidence have been accompanied by numerous attempts at computational modeling. Although many models addressed certain aspects, at least four were successful in conceptually reconciling rigid and adaptive mapping evidences. Neither of them, however, rigorously relies on experimentally observed ephrin-A/EphA-based guidance mechanisms. Significantly, none of them has successfully addressed the seminal results of stripe assay experiments (Gebhardt, 2012).

In this study, by introduction of novel receptor/ligand (double-cue) stripe assays, fabricated by a combination of microfluidic network and contact-printing techniques, the reconstitute was accomplished of topographically appropriate guidance of RGC growth cones in vitro, revealing that the simultaneous presence of forward and reverse FT signaling is sufficient for rigid topographic growth decisions. To additionally cover adaptive mapping, it is suggest that Sperry's model be extended to include FF chemospecificity, also based on ephrin-A/EphA bidirectional signaling. A comprehensive model is presented for the guidance of topographically projecting axons that is essentially based on the single constraint of balancing ephrin-A/EphA forward and reverse signaling. In computational simulations, it successfully replicates evidences for both, rigid and adaptive mapping (Gebhardt, 2012).

Developmental expression of Eph receptors and ligands

Receptor tyrosine kinases (RTKs) play important roles in cellular proliferation, differentiation, and survival. Reverse transcriptase-polymerase chain reactions (RT-PCR) from enriched embryonic day 5 (E5) chick motoneurons were performed by panning to identify RTKs involved in the early development of motoneuron. Cek8, a member of the eph family, is specifically expressed on motoneurons at the brachial and lumbar segments of the spinal cord that innervate limb muscles; Cek8 disappears after the naturally occurring cell death period (E6-E11). Immunohistochemistry using an anti-Cek8 monoclonal antibody showed the localization of Cek8 protein at the cell bodies and axonal fibers of motoneurons and muscles. The unique expression of Cek8 suggests its involvement in cellular survival or cell-cell interactions for specific subpopulations of developing motoneurons (Ohta, 1996).

Many Eph-related receptor tyrosine kinases, and their numerous membrane-bound ligands, can all be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan (Gale, 1996).

Recent studies have implicated Eph-related receptor tyrosine kinases and their membrane-bound ligands in restricting or stimulating the movement of cells and axons. Members of these large families of receptors and ligands fall into two major binding specificity classes, in which the GPI-anchored subgroup of ligands can each bind to all members of a subgroup of receptors, whereas the transmembrane ligands interact with a distinct subgroup of receptors. Analysis of expression patterns is therefore important in order to understand which receptor-ligand interactions occur in vivo. Mouse orthologs of five members of the ligand family have been cloned and their developmental expression has been analysed in detail, both in comparison with one other, and with the receptor specificity class with which they interact. B61, AL-1/RAGS, LERK4, and ELF-1, members of the GPI-anchored subgroup of ligands, have both distinct and overlapping aspects to their expression in early mesoderm, somites, and branchial arches; in complex, dynamic patterns in the limb, and in spatial domains and specific neurons in the CNS. Similarly, Elk-L is expressed in hindbrain segments, the roof plate, and floor plate, overlapping in these locations the expression of other transmembrane ligands, but Elk-L has distinct expression in somites. The expression domains of ligands are complementary to those of the corresponding receptors in a number of tissues, including the midbrain, hindbrain, and differentiating limbs, consistent with potential roles in restricting cell movement. There are some overlaps in the expression of receptors and ligands, for example, in somites and the early limb. Taken together with previous studies showing that Eph-related receptors also have distinct but overlapping expression patterns, these data indicate that each ligand may have stage- and tissue-specific interactions with an individual member or multiple members of the receptor family (Flenniken, 1996).

Neuron-target interaction is a key feature in the establishment of neuronal networks. However, the underlying mechanism remains unclear. At the time of target innervation, Bsk, an eph family receptor, is expressed at high levels in several brain regions, including the hippocampus, olfactory bulb, and retina. To study whether the ligands are expressed in the target tissues, the expression of Bsk ligands was examined using a ligand-affinity probe, Bsk-AP, which consisted of the extracellular domain of Bsk fused in frame with a human placental alkaline phosphatase. These analyses show that the ligands are expressed at high levels in the developing septum, hypothalamus, olfactory neural epithelium, and tectum. In situ hybridization studies reveal that at least three different factors are responsible for the Bsk-AP binding. In the septum, Elf-1, Lerk3 (Eff-2), and AL-1/Lerk7 are transcribed. In the hypothalamus, AL-1/Lerk7 is the ligand detected by Bsk-AP. In the olfactory system, high levels of Lerk3 are detected in the sensory neurons. Both Elf-1 and AL-1/Lerk7 are present in the tectum. These ligand-positive areas are known to be anatomically connected to Bsk-expressing regions. These observations strongly suggest that Bsk and the ligands participate in neuron-target interactions in multiple systems and provide support for their involvement in topographic projection (Zhang, 1996).

The task of organizing the vast array of longitudinal axons that constitute the ventral and lateral funiculi, which project alongside or near the vertebrate midline of the developing vertebrate spinal cord, is likely to be relegated to a number of discrete repellent guidance cues that act upon axons emanating from multiple populations of functionally distinct interneurons. The Eph family of receptor tyrosine kinases are particularly good candidates since they interact exclusively with membrane-associated ligands, exhibit dynamic and spatially restricted expression patterns in the developing central nervous system, and together with the ephrins, mediate axonal patterning and pathway selection through contact-dependent repulsion in a variety of neural systems. Given their transmembrane structure, B-class ephrins are especially suited to function as highly localized, contact-dependent repellents. Although previous studies have documented a role for these proteins in repulsive axon guidance, the function of the transmembrane ephrins in the spinal cord proper are presently unknown. mRNA encoding all three transmembrane ligands is expressed in the floor plate during commissural axon pathfinding. Furthermore, B-class ephrin protein is tightly localized to the lateral floor plate margins, in immediate proximity to longitudinal fiber tracts formed by decussated commissural axons. Strikingly, expression of B-class Eph receptors is detected on only those segments of commissural axons that have crossed the floor plate and turned into the longitudinal axis. It is proposed that the onset of EphB receptor expression subsequent to midline crossing plays an important role in maintaining the longitudinal trajectory of commissural axons through a repulsive interaction with transmembrane ephrins situated at the lateral floor plate boundaries. In support of this interpretation, it is shown that all three B-class ephrins can induce the collapse of a subset of commissural growth cones in vitro. This is the first identification of a membrane-associated factor that directly promotes the collapse of vertebrate commissural growth cones (Imondi, 2000).

To identify molecules involved in neurogenesis, monoclonal antibodies were raised against embryonic day 12.5 mouse telencephalon. One antibody, monoclonal antibody 25H11, stains predominantly the ventricular zone of the anterior and lateral telencephalon. Purification of the 25H11 antigen, a 47 kDa integral membrane protein, from mouse telencephali reveals its identity with ephrin B1. Ephrin B1 appears at the onset of neocortical neurogenesis, being first expressed in neuron-generating neuroepithelial cells and rapidly thereafter in virtually all neuroepithelial cells. Expression of ephrin B1 persists through the period of neocortical neurogenesis and is downregulated thereafter. Ephrin B1 is present on the ventricular as well as basolateral plasma membrane of neuroepithelial cells and exhibits a ventricular-high to pial-low gradient across the ventricular zone. Expression of ephrin B1 is also detected on radial glial cells, extending all the way to their pial endfeet, and on neurons in the mantle/intermediate zone but not in the cortical plate. These results suggest that ephrin B1, presumably via ephrin-Eph receptor signaling, has a role in neurogenesis. Given the ventricular-to-pial gradient of ephrin B1 on the neuroepithelial cell surface and its known role in cell migration in other systems mediated by its repulsive properties, it is proposed that ephrin B1 may be involved in the migration of newborn neurons out from the ventricular zone toward the neocortex (Stuckmann, 2001).

Development of the tectum and the cerebellum is induced by a reciprocal inductive signaling between their respective primordia, the midbrain and the midbrain/hindbrain boundary (MHB). It is of interest to identify molecules that function in and downstream of this reciprocal signaling. Overexpression of LIM domain of the transcription factor Islet-3 (LIM Isl-3) leads to inhibition of this reciprocal signaling and to resultant defects in tectal and cerebellar development. Genes were sought that may be either up- or down-regulated by overexpression of LIMIsl-3 by comparing the gene expression profiles in the midbrain and the MHB of normal embryos and embryos in which Islet-3 function is repressed, using a combination of ordered differential display and whole-mount in situ hybridization. Among genes identified in this search, two cDNA fragments encode Wnt1 and FGF8, which are already known to be essential for the reciprocal signaling between the midbrain and the MHB, confirming the effectiveness of this strategy. Four other partial cDNA clones were identified that were specifically expressed around the MHB, ten cDNAs specifically expressed in the tectum, and three cDNAs expressed in neural crest cells, including those derived from the midbrain level. The ephrin-A3 gene is specifically expressed in posterior tectum in a gradient that decreases anteriorly. Although ephrin-A2 and ephrin-A5 have been reported to be expressed in the corresponding region in mouse embryos (the superior/inferior colliculi), mouse ephrin-A3 is not expressed prominently in this region, suggesting that the role of ephrin-A3 in brain development may have been altered in the process of brain evolution (Hirate, 2001).

Regulation of expression of Eph receptors

Graded expression of the Eph receptor EphA3 in the retina and its two ligands, ephrin A2 and ephrin A5 in the optic tectum, the primary target of retinal axons, has been implicated in the formation of the retinotectal projection map. Two homeobox containing genes, SOHo1 and GH6, are expressed in a nasal-high, temporal-low pattern during early retinal development, and thus in opposing gradients to EphA3. SOHo1 and GH6 belong to the HMX family of related homeobox genes (Drosophila homolog: H6-like-homeobox). Hmx genes share a conserved homeobox and have been identified in a number of species. Hmx genes are predominantly, but not exclusively, expressed in sensory organs, branchial arches and the rostral central nervous system (CNS). Hmx3/Nkx5.1 is critical for inner ear development. Retroviral misexpression of SOHo1 or GH6 completely and specifically represses EphA3 expression in the neural retina, but not in other parts of the central nervous system, such as the optic tectum. Under these conditions, some temporal ganglion cell axons overshoot their expected termination zones in the rostral optic tectum, terminating aberrantly at more posterior locations. However, the majority of ganglion cell axons map to the appropriate rostrocaudal locations, although they form somewhat more diffuse termination zones. These findings indicate that other mechanisms, in addition to differential EphA3 expression in the neural retina, are required for retinal ganglion axons to map to the appropriate rostrocaudal locations in the optic tectum. They further suggest that the control of topographic specificity along the retinal nasal-temporal axis is already split into several independent pathways at a very early time in development (Schulte, 2000).

Mesenchymal patterning is an active process whereby genetic commands coordinate cell adhesion, sorting and condensation, and thereby direct the formation of morphological structures. In mice that lack the Hoxa13 gene, the mesenchymal condensations that form the autopod skeletal elements are poorly resolved, resulting in missing digit, carpal and tarsal elements. In addition, mesenchymal and endothelial cell layers of the umbilical arteries (UAs) are disorganized, resulting in their stenosis and in embryonic death. To further investigate the role of Hoxa13 in these phenotypes, a loss-of-function allele was generated in which the GFP gene was targeted into the Hoxa13 locus. This allele allows FACS isolation of mesenchymal cells from Hoxa13 heterozygous and mutant homozygous limb buds. Hoxa13GFP expressing mesenchymal cells from Hoxa13 mutant homozygous embryos are defective in forming chondrogenic condensations in vitro. Analysis of pro-adhesion molecules in the autopod of Hoxa13 mutants reveals a marked reduction in EphA7 expression in affected digits, as well as in micromass cell cultures prepared from mutant mesenchymal cells. Finally, antibody blocking of the EphA7 extracellular domain severely inhibits the capacity of Hoxa13GFP heterozygous cells to condense and form chondrogenic nodules in vitro, which is consistent with the hypothesis that reduction in EphA7 expression affects the capacity of Hoxa13–/– mesenchymal cells to form chondrogenic condensations in vivo and in vitro. EphA7 and EphA4 expression are also decreased in the mesenchymal and endothelial cells that form the umbilical arteries in Hoxa13 mutant homozygous embryos. These results suggest that an important role for Hoxa13 during limb and UA development is to regulate genes whose products are required for mesenchymal cell adhesion, sorting and boundary formation (Stadler, 2001).

Chick brain factor 1 (CBF1), a nasal retina-specific winged-helix transcription factor, is known to prescribe the nasal specificity that leads to the formation of the precise retinotectal map, especially along the anteroposterior (AP) axis. However, its downstream topographic genes and the molecular mechanisms by which CBF1 controls the expression of them have not been elucidated. Misexpression of CBF1 represses the expression of EphA3 and CBF2, and induces that of SOHo1, GH6, ephrin A2 and ephrin A5. CBF1 controls ephrin A5 by a DNA binding-dependent mechanism, ephrin A2 by a DNA binding-independent mechanism, and CBF2, SOHo1, GH6 and EphA3 by dual mechanisms. BMP2 expression begins double-gradiently (varying in both naso-temporal and ventral-dorsal axes) in the retina from E5 in a complementary pattern to Ventroptin expression. Ventroptin antagonizes BMP2 as well as BMP4. CBF1 interferes in BMP2 signaling and thereby induces expression of ephrin A2. These data suggest that CBF1 is located at the top of the gene cascade for the regional specification along the nasotemporal (NT) axis in the retina and distinct BMP signals play pivotal roles in the topographic projection along both axes (Takahashi, 2003).

As axons grow past intermediate targets, they change their responsiveness to guidance cues. Local upregulation of receptor expression is involved, but the mechanisms for this are not clear. Protein synthesis can be traced within individual axons by introducing RNAs encoding visualizable reporters. Individual severed axons and growth cones can translate proteins and also export them to the cell surface. As axons reach the spinal cord midline, EphA2 is among the receptors upregulated on at least some distal axon segments. Midline reporter upregulation is recapitulated by part of the EphA2 mRNA 3' untranslated region, which is highly conserved and includes known translational control sequences. These results show axons contain all the machinery for protein translation and cell surface expression, and they reveal a potentially general and flexible RNA-based mechanism for regulation localized within a subregion of the axon (Brittis, 2002).

Eph receptors and axonal pathfinding

Sek4 and Nuk are members of the Eph-related family of receptor protein-tyrosine kinases. These receptors interact with a set of cell surface ligands that have recently been implicated in axon guidance and fasciculation. The formation of the corpus callosum and anterior commissure, two major commissural axon tracts that connect the two cerebral hemispheres, is critically dependent on Sek4 and Nuk. While mice deficient in Nuk exhibit defects in pathfinding of the anterior commissure axons, sek4 mutants have defects in corpus callosum formation. The phenotype in both axon tracts is markedly more severe in sek4/nuk1 double mutants, indicating that the two receptors act in a partially redundant fashion. sek4/nuk1 double mutants also exhibit specific guidance and fasciculation defects of diencephalic axon tracts. Moreover, while mice singly deficient in either Sek4 or Nuk are viable, most sek4/nuk1 double mutants die immediately after birth primarily due to a cleft palate. These results demonstrate essential and cooperative functions for Sek4 and Nuk in establishing axon pathways in the developing brain, and during the development of facial structures (Orioli, 1996).

Tyro4 is a member of the eph family of receptor protein-tyrosine kinases. Tyro4 is the rat homolog of mouse Mek4 and chick Cek4. An evolutionarily conserved pattern of expression for Tyro4, Mek4, and Cek4 has been demonstrated. Most strikingly, this receptor is specifically expressed, in all three species, in a subset of motor neurons in the medial motor column and in a subset of axial, but not limb, muscles. Mek4 has previously been ascribed a role in guiding retinal axons to their targets in the optic tectum. These results extend the purported role of Mek4 in axon guidance to include motor neurons of the medial motor column (Kilpatrick, 1996).

Mice that are homozygous for a mutation that disrupts the gene encoding EphA8, a member of the Eph family of tyrosine protein kinase receptors, previously known as Eek. These mice develop to term, are fertile and do not display obvious anatomical or physiological defects. The mouse ephA8/eek gene is expressed primarily in a rostral to caudal gradient in the developing tectum. Axonal tracing experiments have revealed that in these mutant mice, axons from a subpopulation of tectal neurons located in the superficial layers of the superior colliculus do not reach targets located in the contralateral inferior colliculus. Moreover, ephA8/eek null animals display an aberrant ipsilateral axonal tract that projects to the ventral region of the cervical spinal cord. Retrograde labeling reveals that these abnormal projections originate from a small subpopulation of superior colliculus neurons that normally express the ephA8/eek gene. These results suggest that EphA8/Eek receptors play a role in axonal pathfinding during development of the mammalian nervous system (Park, 1997).

AL-1 is a glycosylphosphatidylinositol-linked ligand for the Eph-related receptor, REK7. It shows that a REK7-IgG fusion protein can block axon bundling in co-cultures of cortical neurons on astrocytes, suggesting a role for REK7 and AL-1 in axon fasciculation. Subsequent identification of RAGS, the chick homolog of AL-1, as a repellent axon guidance molecule in the developing chick visual system has led to speculation that AL-1, expressed on astrocytes, provides a repellent stimulus for cortical axons, inducing them to bundle as an avoidance mechanism. Using a growth cone collapse assay to test this hypothesis, it has been shown that a soluble AL-1-IgG fusion protein is a potent collapsing factor for embryonic rat cortical neurons. The response is strongly correlated with REK7 expression, implicating REK7 as a receptor mediating AL-1-induced collapse. Morphological collapse is preceded by an AL-1-IgG-induced reorganization of the actin cytoskeleton that resembles the effects of cytochalasin D. This suggests a pathway whereby REK7 activation by AL-1 leads to perturbation of the actin cytoskeleton, possibly by an effect on actin polymerization, followed by growth cone collapse. AL-1-IgG causes collapse of rat hippocampal neurons and rat retinal ganglion cells. These data suggest a role for REK7 and AL-1 in the patterning of axonal connections in the developing cortex, hippocampus and visual system (Meima, 1997a).

The transmembrane (TM) subfamily of Eph ligands and their receptors have been implicated in axon pathfinding and in pattern formation during embryogenesis. These functions are thought to involve repulsive interactions but this has not been demonstrated directly. A growth cone collapse assay has been used to determine if the TM ligands Lerk2 and HtkL have repellant guidance activity. Lerk2, but not HtkL, is a collapsing factor for a subset of embryonic cortical neurons. Analysis of the effects of Lerk2 on both the morphology and the cytoskeleton of cortical neurons suggests a mechanism of action different from that of AL-1, a GPI-linked Eph ligand having similar repellant activity. Treatment with Lerk2 disrupts the organization of both the actin cytoskeleton and the microtubules and induces the formation of swellings in the center of the growth cone and along the axon. Measurement of the relative F-actin concentrations in the neurites and soma indicate that F-actin levels in the neurites decrease while those in the soma increase, with the net F-actin content of the neuron remaining unchanged. In contrast, prolonged treatment with AL-1 leads to a net loss of F-actin, consistent with the hypothesis that AL-1 acts by perturbing actin polymerization. These results provide evidence that the ectodomain of Lerk2 functions as a repellant guidance cue and show that, despite overlapping specificities in vitro, the biological activities of related ligands are not necessarily overlapping. Further, TM and GPI-linked Eph ligands appear to exert repellant activity by different mechanisms, opening up the possibility that they may have different effects on growth cones in vivo (Meima, 1997b).

During the past few years, evidence has accumulated that activation of EphA kinases on the axons of retinal ganglion cells by ephrins-A on cells of the optic tectum (superior colliculus) plays a critical role in mapping the rostrocaudal axis of the retina onto the corresponding axis of the tectum. The similarities between topographic maps in the motor and visual systems has suggested that similar molecular mechanisms might underlie them. 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. Subfamily A ephrins are implicated in this topographic mapping because: (1) developing muscles express all five of the ephrin-A genes; (2) rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5; (3) the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles; (4) 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. If the conclusion that ephrins are directly involved in nerve-muscle matching is accepted, new questions arise about how they act. These can be divided into issues of mapping and synaptogenesis. In terms of mapping, one plausible model is that muscle fibers express different levels or combinations of ephrins in accordance with their positions and that motor axons express different levels or combinations of Eph kinases (the likely ephrin receptors) in accordance with their positions. For example, levels of expression might be graded along the rostrocaudal axis, as appears to be the case in the retinotectal system. Alternatively, patterns of ephrin and Eph kinase expression might describe a more complex combinatorial code, as suggested by the discrete identities of motor pools and muscles. Indeed, whereas Eph kinase and ephrin levels are graded along the rostrocaudal axis in the visual system, there is currently no evidence for graded expression of ligands or receptors in the motor system. Another possiblity is that interactions between ephrins and their receptors are permissive rather than instructive, enabling positional information to be transmitted by other molecules. It is important to emphasize that the data do not distinguish permissive versus instructive roles for ephrins (Feng, 2000).

In the embryonic visual system, EphA receptors are expressed on both temporal and nasal retinal ganglion cell axons. Only the temporal axons, however, are sensitive to the low concentrations of ephrin-A ligands found in the anterior optic tectum. The poor responsiveness of nasal axons to ephrin-A ligands, which allows them to traverse the anterior tectum and reach their targets in the posterior tectum, has been attributed to constitutive activation of the EphA4 receptor expressed in these axons. EphA4 is highly expressed throughout the retina, but is preferentially phosphorylated on tyrosine (activated) in nasal retina. In a screen for EphA4 ligands expressed in chicken embryonic retina, a novel ephrin, ephrin-A6, has been identified. Like ephrin-A5, ephrin-A6 has high affinity for EphA4 and activates this receptor in cultured retinal cells. In the embryonic day 8 (E8) chicken visual system, ephrin-A6 is predominantly expressed in the nasal retina and ephrin-A5 in the posterior tectum. Thus, ephrin-A6 has the properties of a ligand that activates the EphA4 receptor in nasal retinal cells. Ephrin-A6 binds with high affinity to several other EphA receptors as well and causes growth cone collapse in retinal explants, demonstrating that it can elicit biological responses in retinal neurons. Ephrin-A6 expression is high at E6 and E8, when retinal axons grow to their tectal targets, and gradually declines at later developmental stages. The asymmetric distribution of ephrin-A6 in retinal cells, and the time course of its expression, suggest that this new ephrin plays a role in the establishment of visual system topography (Menzel, 2001).

The role of the Eph family of receptor tyrosine kinases and their ligands in the establishment of the vomeronasal projection in the mouse has been investigated. The data show intriguing differential expression patterns of ephrin-A5 on vomeronasal axons and of EphA6 in the accessory olfactory bulb (AOB), such that axons with high ligand concentration project onto regions of the AOB with high receptor concentration and vice versa. These data suggest a mechanism for development of this projection that is the opposite of the repellent interaction between Eph receptors and ligands observed in other systems. In support of this idea, when given the choice of whether to grow on lanes containing EphA-Fc/laminin or Fc/laminin protein (in the stripe assay), vomeronasal axons prefer to grow on EphA-Fc/ laminin. Analysis of ephrin-A5 mutant mice reveal a disturbance of the topographic targeting of vomeronasal axons to the AOB. In summary, these data, which are derived from in vitro and in vivo experiments, indicate an important role of the EphA family in setting up the vomeronasal projection (Knoll, 2001a).

Growing axons follow highly stereotypical pathways, guided by a variety of attractive and repulsive cues, before establishing specific connections with distant targets. A particularly well-known example that illustrates the complexity of axonal migration pathways involves the axonal projections of motor neurons located in the motor cortex. These projections take a complex route during which they first cross the midline, then form the corticospinal tract, and ultimately connect with motor neurons in the contralateral side of the spinal cord. These obligatory contralateral connections account for why one side of the brain controls movement on the opposing side of the body. The netrins and slits provide well-known midline signals that regulate axonal crossings at the midline. A member of the ephrin family, ephrin-B3, also plays a key role at the midline to regulate axonal crossing. In particular, ephrin-B3 acts as the midline barrier that prevents corticospinal tract projections from recrossing when they enter the spinal gray matter. In ephrin-B3-/- mice, corticospinal tract projections freely recross in the spinal gray matter, such that the motor cortex on one side of the brain now provides bilateral input to the spinal cord. This neuroanatomical abnormality in ephrin-B3-/- mice correlates with loss of unilateral motor control, yielding mice that simultaneously move their right and left limbs and thus have a peculiar hopping gait quite unlike the alternate step gait displayed by normal mice. The corticospinal and walking defects in ephrin-B3-/- mice resemble those recently reported for mice lacking the EphA4 receptor, which binds ephrin-B3 as well as other ephrins, suggesting that the binding of EphA4-bearing axonal processes to ephrin-B3 at the midline provides the repulsive signal that prevents corticospinal tract projections from recrossing the midline in the developing spinal cord (Kullander, 2001a).

To investigate Eph-ephrin bidirectional signaling, a series of mutations were generated in the ephrin-B3 locus. The absence of both forward and reverse signaling results in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract is defective since axons fail to respect the midline bound high-fidelity connection between the motor cortex and contralateral and ipsilateral motor neuron populations. A second mutation that expresses a truncated ephrin-pathfinding protein lacking Ephrin-B3's cytoplasmic domain does not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal cortico-spinal neurons as their fibers pathfind down the contralateral spinal cord. These data indicate ephrin-B3 functions as a midline-anchored repellent to stimulate forward signaling in EphA4-expressing axons (Yokoyama, 2001).

In both invertebrate and lower vertebrate species, decussated commissural axons travel away from the midline and assume positions within distinct longitudinal tracts. In the developing chick and mouse spinal cord, most dorsally situated commissural neuron populations extend axons across the ventral midline and through the ventral white matter along an arcuate trajectory on the contralateral side of the floor plate. Within the dorsal (chick) and intermediate (mouse) marginal zone, commissural axons turn at a conserved boundary of transmembrane ephrin expression, adjacent to which they form a discrete ascending fiber tract. In vitro perturbation of endogenous EphB-ephrinB interactions results in the failure of commissural axons to turn at the appropriate dorsoventral position on the contralateral side of the spinal cord; consequently, axons inappropriately invade more dorsal regions of B-class ephrin expression in the dorsal spinal cord. Taken together, these observations suggest that B-class ephrins act locally during a late phase of commissural axon pathfinding to specify the dorsoventral position at which decussated commissural axons turn into the longitudinal axis (Imondi, 2001).

The development of connections between thalamic afferents and their cortical target cells occurs in a highly precise manner. Thalamic axons enter the cortex through deep cortical layers, then stop their growth in layer 4 and elaborate terminal arbors specifically within this layer. The mechanisms that underlie target layer recognition for thalamocortical projections are not known. The growth patterns have been compared of thalamic explants cultured on membrane substrates purified from cortical layer 4, the main recipient layer for thalamic axons, and cortical layer 5, a non-target layer. Thalamic axons exhibit a reduced growth rate and an increased branching density on their appropriate target membranes compared with non-target substrate. When confronted with alternating stripes of both membrane substrates, thalamic axons grow preferentially on their target membrane stripes. Enzymatic treatment of cortical membranes has revealed that growth, branching and guidance of thalamic axons are independently regulated by attractive and repulsive cues differentially expressed in distinct cortical layers. These results indicate that multiple membrane-associated molecules collectively contribute to the laminar targeting of thalamic afferents. Furthermore, it was found that interfering with the function of Eph tyrosine kinase receptors and their ligands (ephrins) abolishes the preferential branching of thalamic axons on their target membranes, and that recombinant ephrin-A5 ligand elicites a branch-promoting activity on thalamic axons. It is concluded that interactions between Eph receptors and ephrins mediate branch formation of thalamic axons and thereby may play a role in the establishment of layer-specific thalamocortical connections (Mann, 2002).

Motor neurons in the ventral neural tube project axons specifically to their target muscles in the periphery. Although many of the transcription factors that specify motor neuron cell fates have been characterized, less is understood about the mechanisms that guide motor axons to their correct targets. Ectopic expression of EphA4 receptor tyrosine kinase alters the trajectories of a specific population of motor axons in the avian hindlimb. Most motor neurons in the medial portion of the lateral motor column (LMC) extend their axons aberrantly in the dorsal nerve trunk at the level of the crural plexus, in the presence of ectopic EphA4. This misrouting of motor axons is not accompanied by alterations in motor neuron identity, settling patterns in the neural tube, or the fasciculation of spinal nerves. However, ectopic EphA4 axons do make errors in pathway selection during sorting in the plexus at the base of the hindlimb. These results suggest that EphA4 in motor neurons acts as a population-specific guidance cue to control the dorsal trajectory of their axons in the hindlimb (Eberhart, 2002).

Neural maps in the vertebrate central nervous system often show discontinuously segregated, domain-to-domain patterns. However, the molecular mechanism that establishes such maps is not well understood. In the chicken olivocerebellar system, EphA receptors and ephrin-As are expressed with distinct levels and combinations in mapping domains. When ephrin-A2 is retrovirally overexpressed in the cerebellum, the olivocerebellar map is disrupted, excluding axons with high receptor activity from ectopic expression domains. Conversely, overexpression of a truncated EphA3 receptor in the cerebellum reduces endogenous ligand activity to undetectable levels and causes aberrant mapping, with high receptor axons invading high ligand domains. In vitro, ephrin-A2 inhibits outgrowth of inferior olive axons in a region-specific manner. These results suggest that Eph receptors and ephrins constitute domain-specific positional information, and the spatially accurate receptor-ligand interaction is essential to guide inferior olive axons to their correct target domains (Nishida, 2002).

The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, in vivo evidence is provided that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. These results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas (Dufour, 2003).

Molecular mechanisms generating the topographic organization of corticothalamic (CT) circuits, which comprise more than three-quarters of the synaptic inputs onto sensory relay neurons, and their interdependence with thalamocortical (TC) axon development, are unknown. Using in utero electroporation-mediated gene transfer, EphA7-mediated signaling on neocortical axons was shown to control the within-nucleus topography of CT projections in the thalamus. Notably, CT axons that misexpress EphA7 do not shift the relative positioning of their pathway within the subcortical telencephalon (ST), indicating that they do not depend upon EphA7/ephrin-A signaling in the ST for establishing this topography. Moreover, misexpression of cortical EphA7 results in disrupted topography of CT projections, but unchanged inter- and intra-areal topography of TC projections. These results support a model in which EphA/ephrin-A signaling controls independently the precision with which CT and TC projections develop, yet is essential for establishing their topographic reciprocity (Torii, 2005).

How can TC and CT projections achieve their reciprocal organization without interdependent controls? Based on the present results and studies suggesting that the intra-areal topography of TC projections is regulated by an EphA gradient in thalamic nuclei and an ephrin-A gradient in target cortical areas, a model is proposed in which complementary gradients of EphA and ephrin-A activity, located between as well as within specific cortical areas and their corresponding thalamic nuclei, produce reciprocal signaling between these structures. In this model, however, the EphA/ephrin-A signaling controls the topography of TC and CT projections independently, yet establishes essential functional topographic reciprocity (Torii, 2005).

In this model, functional gradients of EphA receptors and ephrin-A ligands are required to be complementary both between and within each reciprocal target. Specific expression patterns of EphAs and ephrin-As appear before CT and TC axons reach their targets. Together with recent evidence for intrinsic regulation of EphA7 and ephrin-A5 expression within the neocortex by transcription factors or morphogens, gradients of EphAs and ephrin-As are likely established by intrinsic regulation within the neocortex and DT independently. Complementary patterns might be achieved by reciprocal transcriptional regulation for each by the same transcription factor. For example, in dorsoventral patterning of the retina, Tbx5 enhances transcription of ephrin-Bs and represses EphBs. In contrast, Vax2 enhances the transcription of EphBs and represses ephrin-Bs. In addition to transcriptional regulation, repressive modulation of EphA function by coexpressed ephrin-A, as shown on retinal ganglion cell axons, might sharpen their functional complementarity where their expression overlaps. Reciprocal and topographic neural connections are found in many other systems throughout the brain. It will be important to determine whether this independent, but reciprocal, EphA/ephrin-A signaling model is applicable generally to other systems (Torii, 2005).

Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 (Drosophila homolog: CG3799) interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, it was found that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1’s GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse (Sahin, 2005).

Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act to organize thalamic inputs. This study mapped the visual cortex (V1) in mice deficient for ephrin-A2, -A3, and -A5 functionally [ephrin-A triple knock out (TKO)], using intrinsic signal optical imaging and microelectrode recording, and structurally, by anatomical tracing of thalamocortical projections. V1 is shifted medially, rotated, and compressed and its internal organization is degraded. Expressing ephrin-A5 ectopically by in utero electroporation in the lateral cortex shifts the map of V1 medially, and expression within V1 disrupts its internal organization. These findings indicate that interactions between gradients of EphA/ephrin-A in the cortex guide map formation, but that factors other than redundant ephrin-As are responsible for the remnant map. Together with earlier work on the retinogeniculate map, the current findings show that the same molecular interactions may operate at successive stages of the visual pathway to organize maps (Cang, 2005).

The visual system has been a long-standing model in the study of topographic map development. EphA receptors and ephrin-A ligands have been shown to be necessary for the development of topographic maps from the retina to two of its subcortical targets, the superior colliculus (SC) in the midbrain and dorsal lateral geniculate nucleus (dLGN) in the thalamus. The dLGN then projects to the visual cortex. In this study, it was found that the topographic mapping of geniculocortical projections is disrupted both structurally and functionally in an ephrin-A TKO. The expression patterns of EphA receptors and ephrin-A ligands in the geniculocortical projection are consistent with EphA/ephrin-A interactions acting in thalamocortical (TC) projections in the same manner as that proposed for EphA/ephrin-A interactions in subcortical mapping. In the cases in which ephrin-A ligand was successfully misexpressed within the developing visual cortex, the functional topographic maps were defective, consistent with ephrin-A ligands acting as molecular labels in the cortex. Together, these experiments suggest that the same mapping mechanisms are used at subcortical and cortical stages, acting in each projection and target area to maintain the topographic order of visual information as it is transferred to the next stage (Cang, 2005).

Signaling by receptor tyrosine kinases (RTKs) is mediated by their intrinsic kinase activity. Typically, kinase-activating mutations result in ligand-independent signaling and gain-of-function phenotypes. Like other RTKs, Ephs require kinase activity to signal, but signaling by Ephs in vitro also requires clustering by their membrane bound ephrin ligands. The relative importance of Eph kinase activity and clustering for in vivo functions is unknown. Knockin mice expressing a mutant form of EphA4 (EphA4EE), whose kinase is constitutively activated in the absence of ephrinB ligands, are deficient in the development of thalamocortical projections and some aspects of central pattern generator rhythmicity. Surprisingly, other functions of EphA4 are regulated normally by EphA4EE, including midline axon guidance, hindlimb locomotion, in vitro growth cone collapse, and phosphorylation of ephexin1. These results suggest that signaling of Eph RTKs follows a multistep process of induced kinase activity and higher-order clustering that is different from RTKs responding to soluble ligands (Egea, 2005).

Among the diverse in vivo functions of EphA4, thalamocortical projections were not rescued by kinase-active EphA4EE. One of the possible explanations is that thalamocortical projections are controlled by ephrinA5, whereas midline guidance of CST and CPG axons is provided by ephrinB3. It is possible that the functional requirement for regulated kinase activity differs depending on whether the receptor is activated by ephrinA or ephrinB ligands. Also, the intrinsic response of thalamic neurons to EphA4 signaling may be different from that of corticospinal or spinal cord interneurons. The JM tyrosine residues that are mutated in ephA4EE, besides controlling the kinase activity of the receptor, have been shown to bind SH2 domain-containing effector proteins. It is conceivable that thalamic neurons require an EphA4 effector that binds to phosphorylated JM tyrosine residues (Egea, 2005).

A final possibility for the phenotype in thalamocortical projections may be the fact that they are topographic and EphA4 has to respond to a smooth gradient of ligand, whereas midline guidance is achieved by a step gradient of ligand. The ability to generate a graded response to ephrins likely requires a particularly tight regulation of Eph activation, including its phosphorylation state: in this context the dynamic range of sensitivity of EphA4 to ephrin gradients could be impaired in ephA4EE/EE mutants, hence leading to the topographic errors observed in vivo in the thalamocortical system. It would be interesting to set up in vitro assays challenging the EphA4EE receptor with either smooth or step gradients of the same ligand to investigate whether the regulation of kinase activity is more critical when the growth cone has to sense an ephrin gradient. In this respect, it is interesting to note that the physiology of the spinal central pattern generator (CPG) is not completely normal in the ephA4EE/EE mutants. In the normal spinal cord, EphA4 signaling is required for CPG neurons projecting ipsilaterally to an unknown target and responding to so far uncharacterized ephrins. A certain degree of drifting of firing patterns between L2 and L5 and aberrant synchrony at lumbar level 5 are in the ephA4EE/EE mutants. It is tempting to speculate that the EphA4-dependent ipsilateral projections are topographically organized and that they follow similar constraints as the thalamocortical projections. If this were true, then EphA4 would have two distinct functions in the spinal CPG: (1) to prevent aberrant midline crossing (rescued by EphA4EE) and (2) to provide ipsilateral topography (partially rescued by EphA4EE) (Egea, 2005).

The ephrin/Eph system plays a central role in neuronal circuit formation; however, its downstream effectors are poorly understood. α-chimerin Rac GTPase-activating protein mediates ephrinB3/EphA4 forward signaling. A spontaneous mouse mutation, miffy (mfy), was discovered that results in a rabbit-like hopping gait, impaired corticospinal axon guidance, and abnormal spinal central pattern generators. Using positional cloning, transgene rescue, and gene targeting, loss of α-chimerin was found to lead to mfy phenotypes similar to those of EphA4−/− and ephrinB3−/− mice. α-chimerin interacts with EphA4 and, in response to ephrinB3/EphA4 signaling, inactivates Rac, which is a positive regulator of process outgrowth. Moreover, downregulation of α-chimerin suppresses ephrinB3-induced growth cone collapse in cultured neurons. These findings indicate that ephrinB3/EphA4 signaling prevents growth cone extension in motor circuit formation via α-chimerin-induced inactivation of Rac. They also highlight the role of a Rho family GTPase-activating protein as a key mediator of ephrin/Eph signaling (Iwasato, 2007).

Eph receptors and synapses

During development, Eph receptors mediate the repulsive axon guidance function of ephrins, a family of membrane attached ligands with their own receptor-like signaling potential. In cultured glutamatergic neurons, EphB2 receptors associate with NMDA receptors at synaptic sites and have been suggested to play a role in synaptogenesis. Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways. Mice lacking EphB2 have normal hippocampal synapse morphology, but display defects in synaptic plasticity. In EphB2-/- hippocampal slices, protein synthesis-dependent long-term potentiation (LTP) is impaired, and two forms of synaptic depression are completely extinguished. Interestingly, targeted expression of a carboxy-terminally truncated form of EphB2 rescues the EphB2 null phenotype, indicating that EphB2 kinase signaling is not required for these EphB2-mediated functions (Grunwald, 2001).

Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain show wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor have reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current is reduced in dentate granule neurons in EphB2 null mice, as is synaptically localized NR1 as revealed by immunogold localization. EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function (Henderson, 2001).

The small GTPase Rac1 (see Drosophila Rac1) orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. This study, carried out in cultured mammalian cells, identified a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors (see Drosophila Eph) utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases (Um, 2014).

Eph receptors and targeting of retinal fibers

The expression of Cek5, a receptor-type tyrosine kinase of the Eph subclass, and its variant form Cek5+ were examined in the chick neural retina during development. Cek5 is present at high levels at all stages of retinal development examined, while Cek5+ is most abundant during differentiation. Cek5 mRNA expression and immunoreactivity are evenly distributed in the undifferentiated retina. With differentiation, Cek5 becomes concentrated in the inner and outer plexiform layers. While only moderate changes in Cek5 protein expression are observed throughout retinal development, Cek5 phosphorylation on tyrosine in vivo is dramatically increased during differentiation. This suggests that the Cek5 ligand is expressed at high levels and causes Cek5 activation. Thus, Cek5 is likely to play an active role in retinal morphogenesis, particularly during the establishment of interneuronal contacts (Pasquale, 1994).

In the retinotectal system, positional information has long been postulated to take the form of molecular gradients within both the retina and the tectum. Recent reports have implicated Mek4 in this process, as well as a member of the Eph (also named class V) family of tyrosine kinase receptors (RTKs), and two ligands: RAGS and ELF-1. QEK5, another member of the Eph family of RTKs, has been isolated from a quail cDNA library. During retinal differentiation, QEK5 transcripts accumulate in a ventral to dorsal gradient within the retinal neuroepithelium, where its expression becomes restricted to the ganglion and bipolar cell layers. Within the tectum, QEK5 transcripts are detectable in a posterior to anterior gradient in the ventricular layer and newly formed superficial layers. The pattern of QEK5 expression in the retina and tectum is distinct from that of Mek4, suggesting that complex patterns of Eph RTKs and their ligands may play a role in cell-cell interactions involved in retinotectal projections and differentiation of the central nervous system (Kenny, 1995).

Topographic maps with a defined spatial ordering of neuronal connections are a key feature of brain organization. Such maps are believed to develop in response to complementary position-specific labels in presynaptic and postsynaptic fields. However, the complementary labeling molecules are not known. In the well-studied visual map of retinal axons projecting to the tectum, the labels are hypothesized to be in gradients, without needing large numbers of cell-specific molecules. ELF-1 is a ligand for Eph family receptors. RNA hybridization shows matching expression gradients for ELF-1 in the tectum and its receptor Mek4 in the retina. Binding activity detected with alkaline phosphatase fusions of ELF-1 and Mek4 also reveals gradients and provides direct evidence for molecular complementarity of gradients in reciprocal fields. ELF-1 and Mek4 may therefore play roles in retinotectal development and have properties predicted of topographic mapping labels (Cheng, 1995).

Receptor protein tyrosine kinases of the Eph subfamily have been proposed to play roles in pattern formation based on their distribution during embryonic development. Cek5 (chicken embryo kinase 5) and Cek8 (chicken embryo kinase 8) are Eph-related kinases highly expressed in the chicken embryonic retina. To assess their potential roles in the development of the visual pathway, their distribution was examined by immunoperoxidase labeling. Cek8 is expressed throughout the pathway of the retinal ganglion cell axons, including the nerve fiber layer of the retina, optic nerve, optic chiasm, and stratum opticum of the tectum. Cek5 immunoreactivity is highly concentrated in only a portion of the optic nerve and optic chiasm; in retinal cultures, Cek5 is detected in neurons. This prompted an examination of the regional distribution of Cek5 in the developing retina and led to the observation that Cek5 is most concentrated in the ventral aspect. RT-PCR established that the differential regulation of Cek5 expression in different portions of the retina occurs at the transcriptional level. Immunoblotting analysis reveals that this unusual expression pattern is distinctive for Cek5, since three other members of the Eph subfamily, Cek4, Cek8, and Cek9, are evenly expressed across the dorsal-ventral axis of the retina. Both Cek5 and Cek8 are distributed in ways that are consistent with their regulating the outgrowth of retinal ganglion cell axons to the tectum. Furthermore, Cek5 represents the first signal transduction molecule found to exhibit the polarized pattern of expression predicted for proteins that control the specificity of the retinotectal projections (Holash, 1995).

The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Eck- and Elk-related receptors in the retina distribute in high temporal-low nasal and high ventral-low dorsal gradients, respectively. Ligands from each subclass distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members, in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes (Marcus, 1996).

Visual connections to the mammalian forebrain are known to be patterned by neural activity, but it remains unknown whether the map topography of such higher sensory projections depends on axon guidance labels. Complementary expression and binding are shown for the receptor EphA5 in mouse retina and its ligands ephrin-A2 and ephrin-A5 in multiple retinal targets, including the major forebrain target, the dorsal lateral geniculate nucleus (dLGN). These ligands can act in vitro as topographically specific repellents for mammalian retinal axons and are necessary for normal dLGN mapping in vivo. The results suggest a general and economic modular mechanism for brain mapping whereby a projecting field is mapped onto multiple targets by repeated use of the same labels. They also indicate the nature of a coordinate system for the mapping of sensory connections to the forebrain (Feldheim, 1999).

The retinotectal projection map is organized in a precise retinotopic order, so that the temporo-nasal axis of the retina corresponds to the rostro-caudal axis of the tectum. en-1 and en-2, homologues of the Drosophila segment polarity gene engrailed, are expressed in a gradient along the rostro-caudal axis of the tectal anlage, and are suggested to confer caudal characteristics as the results of transplantation and ectopic engrailed (en) expression. The ligands for Eph type receptor tyrosine kinases are expressed strongly at the caudal tectum and play a role in retinotectal map formation by repulsing the temporal retinal fibers. Using the system of replication competent retroviral vector, en-2 RCAS (A/B), en-2 was misexpressed on the tectum. Elf-1 or RAGS is induced at the ectopic En-2 sites. The present results show that En-2 can regulate expression of both Elf-1 and RAGS. This suggests that the cells that express en at the early stage of tectum development acquire positional specificity as 'caudal' tectum, and these cells may later express the ligands for Eph type receptor tyrosine kinases. Therefore the temporal retinal fibers which have the receptors are repelled when they meet the ligands on the tectum (Shigetani, 1997).

In the retinotectal projection, the Eph receptor tyrosine kinase ligands ephrinA2 and ephrinA5 are differentially expressed, not only in the tectum, but also in a high-nasal-to-low-temporal pattern in the retina. Recently, retrovirally driven overexpression of ephrinA2 on retinal axons leads to topographic targeting errors of temporal axons in that they overshoot their normal termination zones in the rostral tectum and project onto the mid- and caudal-tectum. The behavior of nasal axons, however, is only marginally affected. Overexpression of ephrinA5 affects the topographic targeting behavior of both temporal and nasal axons. These data reinforce the idea that differential ligand expression on retinal axons contributes to topographic targeting in the retinotectal projection. Additionally, it has been found that ectopic expression of ephrinA2 and ephrinA5 frequently leads to pathfinding errors at the chiasm, resulting in an increased stable ipsilateral projection (Dutting, 1999).

Optic nerve formation requires precise retinal ganglion cell (RGC) axon pathfinding within the retina to the optic disc, the molecular basis of which is not well understood. At CNS targets, interactions between Eph receptor tyrosine kinases on RGC axons and ephrin ligands on target cells have been implicated in formation of topographic maps. However, studies in chick and mouse have shown that both Eph receptors and ephrins are also expressed within the retina itself, raising the possibility that this receptor-ligand family mediates aspects of retinal development. The presence of specific EphB receptors and B-ephrins in embryonic mouse retina is fully documented in this study, and evidence is provided that EphB receptors are involved in RGC axon pathfinding to the optic disc. As RGC axons begin this pathfinding process, EphB receptors are uniformly expressed along the dorsal-ventral retinal axis. This is in contrast to the previously reported high ventral-low dorsal gradient of EphB receptors later in development when RGC axons map to CNS targets. Mice lacking both EphB2 and EphB3 receptor tyrosine kinases, but not each alone, exhibit increased frequency of RGC axon guidance errors to the optic disc. In these animals, major aspects of retinal development and cellular organization appear normal, as do the expression of other RGC guidance cues netrin, DCC, and L1. Unexpectedly, errors occur in dorsal but not ventral retina despite early uniform or later high ventral expression of EphB2 and EphB3. Furthermore, embryos lacking EphB3 and the kinase domain of EphB2 do not show increased errors, consistent with a guidance role for the EphB2 extracellular domain. Thus, while Eph kinase function is involved in RGC axon mapping in the brain, RGC axon pathfinding within the retina is partially mediated by EphB receptors acting in a kinase-independent manner (Birgbauer, 2000).

A role of Eph receptor molecules independent of their kinase domain for pathfinding within the retina is in contrast to the proposed kinase-dependent function of Eph receptors in mediating RGC axon mapping onto CNS targets. However, since retinotopic mapping at the target has thus far only been demonstrated with EphA receptors, and the results here involve EphB receptors, it is formally possible that a difference exists such that EphA receptor function depends on kinase activity while EphB function is kinase independent. However, another possibility is that Eph receptors mediate RGC axon pathfinding at multiple sites along the visual pathway by different mechanisms. Thus, Eph function in the retina itself may be kinase independent, but Eph kinase activity may be essential later during RGC axon topographic mapping within CNS targets. It may be possible to differentiate between these two models by examining the role of EphA receptors in intra-retinal axon pathfinding and the involvement of the kinase domain of EphB receptors in RGC axon mapping within the superior colliculus (Birgbauer, 2000).

Axon pathfinding relies on cellular signaling mediated by growth cone receptor proteins responding to ligands, or guidance cues, in the environment. Eph proteins are a family of receptor tyrosine kinases that govern axon pathway development, including retinal axon projections to CNS targets. Recent examination of EphB mutant mice, however, has shown that axon pathfinding within the retina to the optic disc is dependent on EphB receptors, but independent of their kinase activity. This study shows a function for EphB1, B2 and B3 receptor extracellular domains (ECDs) in inhibiting mouse retinal axons when presented either as substratum-bound proteins or as soluble proteins directly applied to growth cones via micropipettes. In substratum choice assays, retinal axons have tended to avoid EphB-ECDs, while time-lapse microscopy shows that exposure to soluble EphB-ECD leads to growth cone collapse or other inhibitory responses. These results demonstrate that, in addition to the conventional role of Eph proteins signaling as receptors, EphB receptor ECDs can also function in the opposite role as guidance cues to alter axon behavior. Furthermore, the data support a model in which dorsal retinal ganglion cell axons heading to the optic disc encounter a gradient of inhibitory EphB proteins which helps maintain tight axon fasciculation and prevents aberrant axon growth into ventral retina. In conclusion, development of neuronal connectivity may involve the combined activity of Eph proteins serving as guidance receptors and as axon guidance cues (Birgbauer, 2001).

In Xenopus tadpoles, all retinal ganglion cells (RGCs) send axons contralaterally across the optic chiasm. At metamorphosis, a subpopulation of EphB-expressing RGCs in the ventrotemporal retina begin to project ipsilaterally. However, when these metamorphic RGCs are grafted into embryos, they project contralaterally, suggesting that the embryonic chiasm lacks signals that guide axons ipsilaterally. Ephrin-B is expressed discretely at the chiasm of metamorphic but not premetamorphic Xenopus. When expressed prematurely in the embryonic chiasm, ephrin-B causes precocious ipsilateral projections from the EphB-expressing RGCs. Ephrin-B is also found in the chiasm of mammals, which have ipsilateral projections, but not in the chiasm of fish and birds, which do not. These results suggest that ephrin-B/EphB interactions play a key role in the sorting of axons at the vertebrate chiasm (Nakagawa, 2000).

Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinal axonal projection to the tectum or to its mammalian equivalent, the superior colliculus (SC). In this paper, gene disruptions for both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes (Feldheim, 2000).

Currently, a leading model is that both attractant and repellent gradients may be prespecified within the target. Each axon then identifies its correct termination zone as the point where repellent and attractant forces cancel out. In this model, wild-type nasal axons map to posterior SC because they are attracted there. A prediction of this model is that in the ephrin-A2-/-; ephrin-A5-/- double mutant, axons would tend to map more posteriorly than normal, or in the case of extreme nasal axons might show no shift. This appears to be contrary to the results obtained in this study, especially considering that the opposite shifts in the nasal and temporal axons are of similar magnitudes. For similar reasons these results do not seem consistent with a variant of the counterbalanced gradient model, where a posterior to anterior ephrin gradient would be balanced by a prespecified repellent in an anterior to posterior gradient. An alternative possibility that could perhaps be reconciled with these results would be for ephrins in the target to act as either repellents or attractants at different concentrations, although there is currently no direct evidence to support such an effect from axon guidance assays (Feldheim, 2000 and references therein).

As an alternative to models with two counterbalanced gradients prespecified in the target, these results could be explained by a model involving a repellent gradient of ephrins, in combination with axon-axon competition. This competition could be for limiting positive factors in the target or could also involve direct axon-axon interactions. A model of this type has been suggested in an analysis of the LGN in ephrin-A5-/- mice, and the model now receives strong support from the more comprehensive analysis in the SC presented here. The repulsion/competition model would account for normal mapping as follows. Temporal axons would be unable to terminate in the posterior SC, because they are repelled by the ephrins, so they would be forced to arborize in the anterior SC. Nasal axons are less sensitive to ephrin repulsion and so would be able to terminate in the posterior SC. In the anterior SC, nasal axons face greater competition for limiting amounts of permissive factor(s), so they prefer to avoid this competition and arborize only in the posterior SC (Feldheim, 2000).

Incorporating competition in the model can explain several aspects of the data. (1) Nasal axons shift anteriorly in the ephrin-A5-/- single mutant (although this could be explained by the axon sensitivity model outlined above), and shift even further to the anterior in the ephrin-A2-/-; ephrin-A5-/- double mutant. According to the competition model, posterior repellents are removed, so axons from temporal or central retina are now able to compete more effectively in posterior SC; nasal axons therefore face increased competition in the posterior SC, so they lose their strong preference for this region and spread out into more anterior regions. The model thus seems to fit well with the opposite and quantitatively similar shifts of axons from temporal and nasal extremes of the retina. Labelings at intermediate retinal positions are more difficult to characterize because of the lack of fixed landmarks but, consistent with the competition model, such labelings revealed multiple arborizations, seemingly shifted in both anterior and posterior directions. (2) Axons are not respecified to a specific ectopic position. Instead, arborizations are scattered over an abnormally broad zone in the mutants, including both normal and abnormal regions. This is the result predicted by the competition model: as the topographically specific repellents are removed, axons would spread into abnormal regions, but there is no reason for them to disfavor the correct region. (3) Even in the ephrin-A2-/-; ephrin-A5-/- mutant, retinal axons fill the entire SC and axons from both nasal and temporal extremes of the retina form connections within the target. Models that involve a strict matching of values in the projecting and target field would predict that the mutant should have unmatched areas, whereas the competition model predicts that the entire projecting and target fields should still match up (Feldheim, 2000).

In the mammalian visual system, retinal axons undergo temporal and spatial rearrangements as they project bilaterally to targets on the brain. Retinal axons cross the neuraxis to form the optic chiasm on the hypothalamus in a position defined by overlapping domains of regulatory gene expression. However, the downstream molecules that direct these processes remain largely unknown. A novel in vitro paradigm was used to study possible roles of the Eph family of receptor tyrosine kinases in chiasm formation. In vivo, Eph receptors and their ligands distribute in complex patterns in the retina and hypothalamus. In vitro, retinal axons are inhibited by reaggregates of isolated hypothalamic, but not dorsal diencephalic or cerebellar cells. Furthermore, temporal retinal neurites are more inhibited than nasal neurites by hypothalamic cells. Addition of soluble EphA5-Fc to block Eph 'A' subclass interactions decreases both the inhibition and the differential response of retinal neurites by hypothalamic reaggregates. These data show that isolated hypothalamic cells elicit specific, position-dependent inhibitory responses from retinal neurites in culture. Moreover, these responses are mediated, in part, by Eph interactions. Together with the in vivo distributions, these data suggest possible roles for Eph family members in directing retinal axon growth and/or reorganization during optic chiasm formation (Marcus, 2000).

Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. The distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon has been examined during the period when visual connections are being established. Both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor-ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph-ephrin molecules is observed. A similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway (Marin, 2001).

The idea has been put forward that molecules and mechanisms acting during development are re-used during regeneration in the adult, for example in response to traumatic injury in order to re-establish the functional integrity of neuronal circuits. Members of the Eph family of receptor tyrosine kinases and their 'ligands', the ephrins, play a prominent role during development of the retinocollicular projection in rodents, where EphA receptors and ephrin-As are expressed in gradients in both the retina and the superior colliculi (SC). Whether EphA family members are also expressed or re-expressed in the adult after optic nerve lesion was examined, since the presence of axon guidance information is an important prerequisite for a topographically appropriate re-connection by retinal ganglion cell (RGC) axons. This analysis was encouraged by results showing that RGC axons do not exert guidance preferences in response to membranes from adult unlesioned SC, but in response to membranes from the adult deafferented SC. A graded expression pattern of ephrin-As was found in the SC both before and after deafferentation, which is remarkably similar to those found during development. EphA receptor levels are reduced in the SC after deafferentation and the expression patterns of the EphB family are not changed. In particular, the presence of a graded ephrin-A expression in the deafferented SC suggests that -- if robust regeneration of RGC axons can be achieved -- topographic guidance information as a likely requirement for a functionally successful re-establishment of the retinocollicular projection is available (Knoll, 2001b).

The retinotectal projection is the predominant model for studying molecular mechanisms controlling development of topographic axonal connections. Analyses of topographic mapping of retinal ganglion cell (RGC) axons in chick optic tectum indicate that a primary role for guidance molecules is to regulate topographic branching along RGC axons, a process that imposes unique requirements on the molecular control of map development. Topographically appropriate connections are established exclusively by branches that form along the axon shaft. Initially, RGC axons overshoot their appropriate termination zone (TZ) along the anterior-posterior (A-P) tectal axis; temporal axons overshoot the greatest distance and nasal axons the least: this correlates with the nonlinear increasing A-P gradient of ephrin-A repellents. In contrast, branches form along the shaft of RGC axons with substantial A-P topographic specificity. Topography is enhanced through the preferential arborization of appropriately positioned branches and elimination of ectopic branches. Using a membrane stripe assay and time-lapse microscopy, it has been shown that branches form de novo along retinal axons. Temporal axons preferentially branch on their topographically appropriate anterior tectal membranes. After the addition of soluble EphA3-Fc, which blocks ephrin-A function, temporal axons branch equally on anterior and posterior tectal membranes, indicating that the level of ephrin-As in posterior tectum is sufficient to inhibit temporal axon branching and generate branching specificity in vitro. These findings indicate that topographic branch formation and arborization along RGC axons are critical events in retinotectal mapping. Ephrin-As inhibit branching along RGC axons posterior to their correct TZ, but alone cannot account for topographic branching and must cooperate with other molecular activities to generate appropriate mapping along the A-P tectal axis (Yates, 2001).

In animals with binocular vision, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. Ephrin-Bs in the chiasm region direct the divergence of retinal axons through the selective repulsion of a subset of RGCs that express EphB1. Ephrin-B2 is expressed in radial glia at the mouse chiasm midline as the ipsilateral projection is generated and is selectively inhibitory to axons from ventrotemporal (VT) retina, where ipsilaterally projecting RGCs reside. Moreover, blocking ephrin-B2 function in vitro rescues the inhibitory effect of chiasm cells and eliminates the ipsilateral projection in the semiintact mouse visual system. A receptor for ephrin-B2, EphB1, is found exclusively in regions of retina that give rise to the ipsilateral projection. EphB1 null mice exhibit a dramatically reduced ipsilateral projection, suggesting that this receptor contributes to the formation of the ipsilateral retinal projection, most likely through its repulsive interaction with ephrin-B2. This study provides the first direct evidence that ephrin-B2 is more inhibitory to ipsilateral than contralateral retinal axons and demonstrates that ephrin-B2 is necessary for the ipsilateral projection to form. Using in situ hybridization and analyses of mutant mice, EphB1, a receptor for ephrin-B2, is shown to be expressed specifically in regions of the retina that give rise to the ipsilateral projection, and in mice lacking EphB1. Together these data identify ephrin-B2 and EphB1 as key players in retinal axon divergence and suggest that the function of B-class Ephs and ephrins in patterning binocular vision is conserved between species (Williams, 2003).

During development of the retinocollicular projection in mouse, retinal axons initially overshoot their future termination zones (TZs) in the superior colliculus (SC). The formation of TZs is initiated by interstitial branching at topographically appropriate positions. Ephrin-As are expressed in a decreasing posterior-to-anterior gradient in the SC, and they suppress branching posterior to future TZs. This study investigates the role of an EphA7 gradient in the SC, which has the reverse orientation compared to the ephrin-A gradient. In EphA7 mutant mice the retinocollicular map is disrupted, with nasal and temporal axons forming additional or extended TZs, respectively. In vitro, retinal axons are repelled from growing on EphA7-containing stripes. These data support the idea that EphA7 is involved in suppressing branching anterior to future TZs. These findings suggest that opposing ephrin-A and EphA gradients are required for the proper development of the retinocollicular projection (Rashid, 2005).

Eph receptors and dendritic spine morphogenesis

The cell surface proteoglycan syndecan-2 can induce dendritic spine formation in hippocampal neurons. The EphB2 receptor tyrosine kinase phosphorylates syndecan-2 and this phosphorylation event is crucial for syndecan-2 clustering and spine formation. Syndecan-2 is tyrosine phosphorylated and forms a complex with EphB2 in mouse brain. Dominant-negative inhibition of endogenous EphB receptor activities blocks clustering of endogenous syndecan-2 and normal spine formation in cultured hippocampal neurons. This is the first evidence that Eph receptors play a physiological role in dendritic spine morphogenesis. These observations suggest that spine morphogenesis is triggered by the activation of Eph receptors: this causes tyrosine phosphorylation of target molecules, such as syndecan-2, in presumptive spines (Ethell, 2001).

Dendritic spines are the principal postsynaptic targets for excitatory synapses. In recent years, these small protrusions on the surface of dendrites have attracted significant interest because changes in their morphology are implicated in synaptic plasticity and long-term memory. Dendritic spines undergo morphological changes in a developmentally regulated and activity-dependent manner. Abnormal spine morphologies have been reported in several neurodevelopmental disorders, including fragile X syndrome. The molecular mechanisms that govern spine morphogenesis are not completely understood, but several different physiological and molecular factors have been shown to affect spine morphology. These factors include synaptic activity and plasticity, actin filament reorganization, calcium dynamics, and protein phosphorylation (Ethell, 2001 and references therein).

Dendritic spine formation occurs during the late stages of development after neuronal connectivity has been established. Before the appearance of mature spines, dendrites exhibit long, thin filopodia-like protrusions without a bulbous head. As the brain matures, these dendritic filopodia disappear, and spines, which typically have mushroom-like and stubby shapes, begin to appear. Primary cultures of rat hippocampal neurons provide an excellent system in which the process of spine formation can be studied in vitro. At 1 week in vitro, these neurons possess predominantly filopodia-like protrusions. Over the next few weeks, these dendritic filopodia gradually decrease in number and are progressively replaced by protrusions that have mushroom-like and stubby shapes. After 3-4 weeks, the majority of the protrusions exhibit mature spine morphologies. The cell surface proteoglycan syndecan-2 has been shown to play a role in spine formation (Ethell, 2001 and references therein).

Syndecan-2 is a member of the syndecan family of transmembrane heparan sulfate proteoglycans. There is increasing evidence that syndecans are involved in transmembrane signaling by interacting with cytoskeletal and signaling molecules. The cytoplasmic domain of syndecans can be subdivided into a highly conserved juxtamembrane segment (C1 region), another conserved segment at the C terminus (C2 region), and a variable segment (V region) located between the C1 and C2 regions. The EFYA (Glu-Phe-Tyr-Ala) sequence at the C terminus serves as the binding site for at least four cytoplasmic proteins, namely syntenin, CASK, synectin, and synbindin. Moreover, the cytoplasmic domain contains 4 tyrosine residues that are conserved among all syndecans. Some of these tyrosine residues are phosphorylated in vitro, and tyrosine phosphorylation has been speculated to play important roles in syndecan-mediated signal transduction (Ethell, 2001 and references therein).

Potential functional roles of syndecan-2 in synapses were first suggested by its interaction with the synaptic PDZ domain protein CASK. Syndecan-2 is clustered at dendritic spines of mature hippocampal neurons in culture and its accumulation occurs concomitant with the morphological maturation of spines. More importantly, transfection of syndecan-2 induces the formation of morphologically mature dendritic spines in immature (8 days in vitro [DIV]) hippocampal neurons. Deletion studies have demonstrated that the C1 and V regions of the syndecan-2 cytoplasmic domain (which contains two potential tyrosine phosphorylation sites) are required for syndecan-2 clustering on dendrites and the induction of mature spines. Based on these data, it is hypothesized that tyrosine phosphorylation of syndecan-2 is the crucial upstream event that leads to dendritic spine formation. This premise then suggests that tyrosine kinase(s) present in dendritic spines play a role in spine formation by phosphorylating syndecan-2 (Ethell, 2001 and references therein).

The Eph family is a large family of receptor tyrosine kinases. Upon stimulation by ephrin ligands, Eph receptors activate signaling cascades in various biological systems. While Eph receptors have been studied primarily in the context of axon guidance during development, there have been suggestions that they may play some roles in synapses in the adult brain. It is speculated that Eph receptors are the kinases involved in the syndecan-2-induced spine formation for several reasons.: (1) syndecan-3, another member of the syndecan family, is tyrosine phosphorylated by recombinant EphB1 in vitro; (2) some Eph receptors interact with syntenin, a syndecan-2 binding PDZ domain protein, and (3) most importantly, some Eph receptors, including EphB2, are present in dendritic spines. These observations have led to this investigation of the possibility that Eph receptors are involved in syndecan-2 phosphorylation during dendritic spine formation (Ethell, 2001 and references therein).

In this paper, it is demonstrated that EphB2 is a crucial tyrosine kinase that phosphorylates syndecan-2 during dendritic spine formation. Furthermore, inhibition of endogenous EphB receptor activities by dominant-negative EphB2 blocks endogenous syndecan-2 clustering and normal spine formation. These results demonstrate a physiological role for EphB2/syndecan-2 signaling in dendritic spine morphogenesis. These findings provide a basis for the role of cell surface ligand-receptor interactions in spine morphogenesis and suggest that the signaling cascade leading to the formation of mature spines is triggered by the activation of Eph receptors by their extracellular ligands (Ethell, 2001).

The morphogenesis of dendritic spines, the major sites of excitatory synaptic transmission in the brain, is important in synaptic development and plasticity. An ephrinB-EphB receptor trans-synaptic signaling pathway has been identified that regulates the morphogenesis and maturation of dendritic spines in hippocampal neurons. Activation of the EphB receptor induces translocation of the Rho-GEF kalirin (Drosophila ortholog: Trio) to synapses and activation of Rac1 and its effector PAK. Overexpression of dominant-negative EphB receptor, catalytically inactive kalirin or dominant-negative Rac1, or inhibition of PAK each eliminates ephrin-induced spine development. This novel signal transduction pathway may be critical for the regulation of the actin cytoskeleton controlling spine morphogenesis during development and plasticity (Penzes, 2003).

The role of the Rac1 effector p21-activated kinase PAK was examined. Several PAK proteins are expressed in the brain, and previous studies have shown that some of the effects of Rac1 on the cytoskeleton are mediated by PAK. In addition, genetic analysis in Drosophila has shown that PAK1 is genetically associated with Trio, the fly ortholog of kalirin, in the pathway through which Trio affects axon growth and guidance. Binding of activated Rac1 to PAK induces PAK autophosphorylation, which strongly correlates with its activation. To test whether ephrinB treatment induces activation of PAKs, an antibody detecting autophosphorylated PAK (P-PAK) was used. In addition, this experiment can be regarded as a way to visualize endogenous Rac1 activation. Treatment of hippocampal neurons with clustered ephrinB1 induce a dramatic increase in the number and size of clusters stained with the P-PAK antibody. This effect was confirmed by Western analysis with the P-PAK antibody of extracts of 4-week-old high-density cortical neurons treated with ephrinB1. Moreover, in hippocampal neurons, ephrinB1 treatment induces activation of PAK at synapses, as shown by P-PAK immunostaining coincident with synaptophysin (Penzes, 2003).

To test whether kalirin-7 was required for ephrinB1-induced PAK phosphorylation, the effect was examined of overexpressing the GEF inactive kal7-mut in hippocampal neurons on the ability of clustered ephrinB1 to induce phosphorylation of PAK. Therefore, DIV7 hippocampal neurons were transfected with myc-kal7-mut, and 2 days later the neurons were treated with clustered ephrinB1 for 2 hr, followed by fixation and immunostaining for myc and P-PAK. While ephrinB1 treatment induces an increased phosphorylation of PAK in nontransfected neurons, in neurons expressing kal7-mut, the level of P-PAK is visibly reduced compared to adjacent nontransfected neurons. Quantification of the ratios of P-PAK fluorescence intensities to total cell areas of nontransfected control neurons relative to the same ratios for neurons expressing myc-kal7-mut confirmed this observation (Penzes, 2003).

PAKs phosphorylate proteins involved in regulating the actin cytoskeleton and gene expression. To test whether PAK is an essential downstream component of ephrinB signaling in spine morphogenesis, GFP-transfected hippocampal neurons were treated with a fusion protein of the PAK1 inhibitory domain (PID) fused with the cell-penetrating peptide (TAT-PID) along with ephrinB1. These neurons exhibit a reduction in the number and size of spines, compared to the ephrinB1-treated neurons, while also showing a reduced phosphorylation level of PAK, confirming its inhibition by PID. Together, these data demonstrate that Rac1 and PAK are key downstream components of ephrinB regulation of spine morphogenesis (Penzes, 2003).

During development, it is necessary to coordinate accurately the formation and location of presynaptic active zones with those of the postsynaptic structures. This could be achieved by signaling from presynaptic ephrinB, clustered at active zones on axons, to activate postsynaptic EphB2, resulting in synaptogenesis on the apposing dendrites. Even in mature neurons, dendritic spines are very dynamic structures, and recent studies have demonstrated that LTP induces morphological changes in spines, which may contribute to plasticity in adult neurons. The rapid and dramatic effect of ephrinB on spine maturation suggests that ephrinB-EphB2 signaling may be a key component in the regulation of spine morphogenesis during plasticity. Other extracellular signals have been shown to regulate spine morphogenesis, such as K+ depolarization, glutamate action on NMDA receptors, and BDNF. It is possible that kalirin mediates the intracellular effects of these signals as well (Penzes, 2003).

Eph receptors and the segmentation of the hindbrain

Pattern formation in the hindbrain involves a segmentation process leading to the formation of metameric units, manifested as successive swellings known as rhombomeres (r). In search for genes involved in cell-cell interactions during hindbrain segmentation, a screen was carried out for protein kinase genes with restricted expression patterns in this region of the CNS. Three novel mouse genes, Sek-2, Sek-3 and Sek-4 (members of the Eph subfamily of putative transmembrane receptor protein tyrosine kinases [RTKs]) were cloned, their chromosomal locations were identified, and their expression between 7.5 and 10.5 days of development was analyzed. Before morphological segmentation, Sek-2 is transcribed in a transverse stripe corresponding to prospective r4 and the adjacent mesoderm, suggesting possible roles both in hindbrain segmentation and signaling between neuroepithelium and mesoderm. Sek-3 and Sek-4 have common domains of expression, including r3, r5 and part of the midbrain, as well as specific domains in the diencephalon, telencephalon, spinal cord and in mesodermal and neural crest derivatives. Together with the finding that Sek (Sek-1) is expressed in r3 and r5, these data indicate that members of the Eph family of RTKs may co-operate in the segmental patterning of the hindbrain (Becker, 1994).

Described here is the isolation and characterization of two zebrafish Eph receptor ligand cDNAs that have been called zfEphL3 and zfEphL4. These genes are expressed in the presumptive midbrain of developing embryos from 6 somites. By 24 hours, L3 is expressed throughout the midbrain including the region of the presumptive tectum, whereas L4 is strongly expressed in the midbrain caudal to the presumptive tectum. At later stages of development L3 is expressed in a graded fashion throughout the tectum and L4 is maintained at its posterior margin. Growth cone collapse and pathway selection assays demonstrate that both these proteins have a collapse activity for retinal ganglion cells. When faced with a choice of substrate on which to grow, temporal axons from chick retinal ganglion cells selectively avoid membranes from Cos cells transfected with L3, whereas nasal axons do not. Both temporal and nasal axons avoid membranes from Cos cells transfected with L4. The expression patterns, together with the functional data, suggest that although both ligands may be able to guide retinal ganglion cells axons in vitro, they have different roles in the guidance of retinotectal projections in vivo. The expression of L3 is consistent with a role in the guidance of retinal ganglion cells to their targets on the tectum, whereas that of L4 suggests a role in delineating the posterior boundary of the optic tectum (Brennan, 1997).

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres (r) with distinct identities. Recent studies have uncovered regulatory links between transcription factors governing this process, but little is known of how these relate to molecules mediating cell-cell signaling. The Eph receptor tyrosine kinase gene EphA4 (Sek-1) is expressed in r3 and r5, and function-blocking experiments suggest that it is involved in restricting intermingling of cells between odd- and even-numbered rhombomeres. The cis-acting regulatory sequences of the EphA4 gene have been analyzed in transgenic mice and a 470 bp enhancer element that drives specific expression in r3 and r5 has been identified. Within this element, eight binding sites have been identified for the Krox-20 transcription factor that is also expressed in r3 and r5. Mutation of these binding sites abolishes r3/r5 enhancer activity and ectopic expression of Krox-20 leads to ectopic activation of the enhancer. These data indicate that Krox-20 is a direct transcriptional activator of EphA4. Together with evidence that Krox-20 regulates Hox gene expression, these findings reveal a mechanism by which the identity and movement of cells are coupled such that sharply restricted segmental domains are generated (Theil, 1998).

The restriction of intermingling between specific cell populations is crucial for the maintenance of organized patterns during development. A striking example is the restriction of cell mixing between segments in the insect epidermis and the vertebrate hindbrain that may enable each segment to maintain a distinct identity. In the hindbrain, this is a result of different adhesive properties of odd- and even-numbered segments (rhombomeres), but an adhesion molecule with alternating segmental expression has not been found. However, blocking experiments suggest that Eph-receptor tyrosine kinases may be required for the segmental restriction of cells. Eph receptors and their membrane-bound ligands, ephrins, are expressed in complementary rhombomeres and, by analogy with their roles in axon pathfinding, could mediate cell repulsion at boundaries. Remarkably, transmembrane ephrins can themselves transduce signals, raising the possibility that bi-directional signaling occurs between adjacent ephrin- and Eph-receptor-expressing cells. Mosaic activation of Eph receptors leads to sorting of cells to boundaries in odd-numbered rhombomeres, whereas mosaic activation of ephrins results in sorting to boundaries in even-numbered rhombomeres. These data implicate Eph receptors and ephrins in the segmental restriction of cell intermingling (Xu, 1999).

Rhombomeres are segmental units of the developing vertebrate hindbrain that underlie the reiterated organization of cranial neural crest migration and neuronal differentiation. valentino (val), a zebrafish homolog of the mouse bzip transcription factor-encoding gene, kreisler (potential Drosophila homolog: CG10034), is required for segment boundary formation caudal to rhombomere 4 (r4). val is normally expressed in r5/6 and is required for cells to contribute to this region. In val minus mutants, rX, a region one rhombomere in length and of mixed identity, lies between r4 and r7. While a number of genes involved in establishing rhombomeric identity are known, it is still largely unclear how segmental integrity is established and boundaries are formed. Members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, are candidates for functioning in rhombomere boundary formation. Indeed, expression of the receptor ephB4a coincides with val in r5/6, while ephrin-B2a, which encodes a ligand for EphB4a, is expressed in r4 and r7, complementary to the domain of val expression (Cooke, 2001).

In val minus embryos, ephB4a expression is downregulated and ephrin-B2a expression is upregulated between r4 and r7, indicating that Val is normally required to establish the mutually exclusive expression domains of these two genes. Juxtaposition of ephB4a-expressing cells and ephrin-B2a-expressing cells in the hindbrain leads to boundary formation. Loss of the normal spatial regulation of eph/ephrin expression in val mutants correlates not only with absence of boundaries but also with the inability of mutant cells to contribute to wild-type r5/6. Using a genetic mosaic approach, it has been shown that spatially inappropriate Eph signaling underlies the repulsion of val minus cells from r5/6. It is proposed that Val controls eph expression and that interactions between EphB4a and Ephrin-B2a mediate cell sorting and boundary formation in the segmenting caudal hindbrain (Cooke, 2001).

Eph receptors and neural crest patterning

During vertebrate head development, neural crest cells migrate from hindbrain segments to specific branchial arches, where they differentiate into distinct patterns of skeletal structures. The rostrocaudal identity of branchial neural crest cells appears to be specified prior to migration, so it is important that they are targeted to the correct destination. In Xenopus embryos, branchial neural crest cells segregate into four streams that are adjacent during early stages of migration. It is not known what restricts the intermingling of these migrating cell populations and targets them to specific branchial arches. Xenopus EphA4 and EphB1 are expressed in migrating neural crest cells and mesoderm of the third arch, and third plus fourth arches, respectively. The ephrin-B2 ligand, which interacts with these receptors, is expressed in the adjacent second arch neural crest and mesoderm. Using truncated receptors, it has been shown that the inhibition of EphA4/EphB1 function leads to abnormal migration of third arch neural crest cells into second and fourth arch territories. Furthermore, ectopic activation of these receptors by overexpression of ephrin-B2 leads to scattering of third arch neural crest cells into adjacent regions. Similar disruptions occur when the expression of ephrin-B2 or truncated receptors is targeted to the neural crest. These data indicate that the complementary expression of EphA4/EphB1 receptors and ephrin-B2 is involved in restricting the intermingling of third and second arch neural crest and in targeting third arch neural crest to the correct destination. Together with previous work showing that Eph receptors and ligands mediate neuronal growth cone repulsion, these findings suggest that similar mechanisms are used for neural crest and axon pathfinding (Smith, 1997).

In the trunk of avian embryos, neural crest migration through the somites is segmental, with neural crest cells entering the rostral half of each somitic sclerotome but avoiding the caudal half. Little is known about the molecular nature of the cues (intrinsic to the somites) that are responsible for this segmental migration of neural crest cells. Eph-related receptor tyrosine kinases and their ligands are essential for the segmental migration of avian trunk neural crest cells through the somites. EphB3 localizes to the rostral half-sclerotome, including the neural crest, and the ligand ephrin-B1 has a complementary pattern of expression in the caudal half-sclerotome. To test the functional significance of this striking asymmetry, soluble ligand ephrin-B1 was added to interfere with receptor function in either whole trunk explants or neural crest cells cultured on alternating stripes of ephrin-B1 versus fibronection. Neural crest cells in vitro avoid migrating on lanes of immobilized ephrin-B1; the addition of soluble ephrin-B1 blocks this inhibition. Similarly, in whole trunk explants, the metameric pattern of neural crest migration is disrupted by the addition of soluble ephrin-B1, allowing entry of neural crest cells into caudal portions of the sclerotome. Thus, both in vivo and in vitro, the addition of soluble ephrin-B1 results in a loss of the metameric migratory pattern and a disorganization of neural crest cell movement. These results demonstrate that Eph-family receptor tyrosine kinases and their transmembrane ligands are involved in interactions between neural crest and sclerotomal cells, mediating an inhibitory activity necessary to constrain neural precursors to specific territories in the developing nervous system (Krull, 1997).

Little is known about the mechanisms that direct neural crest cells to the appropriate migratory pathways. This study set out to determine how neural crest cells that are specified as neurons and glial cells migrate only ventrally and are prevented from migrating dorsolaterally into the skin, whereas neural crest cells specified as melanoblasts are directed into the dorsolateral pathway. Eph receptors and their ephrin ligands have been shown to be essential for migration of many cell types during embryonic development. Consequently, it was asked if ephrin-B proteins participate in the guidance of melanoblasts along the dorsolateral pathway, and prevent early migratory neural crest cells from invading the dorsolateral pathway. Using Fc fusion proteins, the expression of ephrin-B ligands was detected in the dorsolateral pathway at the stage when neural crest cells are migrating ventrally. Furthermore, ephrins block dorsolateral migration of early-migrating neural crest cells because when the Eph-ephrin interactions are disrupted by addition of soluble ephrin-B ligand to trunk explants, early neural crest cells migrate inappropriately into the dorsolateral pathway. Surprisingly, the ephrin-B ligands continue to be expressed along the dorsolateral pathway during melanoblast migration. RT-PCR analysis, in situ hybridization, and cell surface-labelling of neural crest cell cultures demonstrate that melanoblasts express several EphB receptors. In adhesion assays, engagement of ephrin-B ligands to EphB receptors increases melanoblast attachment to fibronectin. Cell migration assays demonstrate that ephrin-B ligands stimulate the migration of melanoblasts. Furthermore, when Eph signaling is disrupted in vivo, melanoblasts are prevented from migrating dorsolaterally, suggesting ephrin-B ligands promote the dorsolateral migration of melanoblasts. Thus, transmembrane ephrins act as bifunctional guidance cues: they first repel early migratory neural crest cells from the dorsolateral path, and then later stimulate the migration of melanoblasts into this pathway. The mechanisms by which ephrins regulate repulsion or attraction in neural crest cells are unknown. One possibility is that the cellular response involves signaling to the actin cytoskeleton, potentially involving the activation of the Cdc42/Rac family of GTPases. In support of this hypothesis, the adhesion of early migratory cells to an ephrin-B-derivatized substratum has been shown to result in cell rounding and disruption of the actin cytoskeleton, whereas plating of melanoblasts on an ephrin-B substratum induces the formation of microspikes filled with F-actin (Santiago, 2002).

Neural crest cells migrate segmentally through the rostral half of each trunk somite due to inhibitory influences of ephrins and other molecules present in the caudal-half of somites. To examine the potential role of Notch/Delta signaling in establishing the segmental distribution of ephrins, neural crest migration and ephrin expression were examined in Delta-1 mutant mice. Using Sox-10 as a marker, it was noted that neural crest cells moved through both rostral and caudal halves of the somites in mutants, consistent with the finding that ephrinB2 levels are significantly reduced in the caudal-half somites. Later, mutant embryos had aberrantly fused and/or reduced dorsal root and sympathetic ganglia, with a marked diminution in peripheral glia. These results show that Delta-1 is essential for proper migration and differentiation of neural crest cells. Interestingly, absence of Delta-1 leads to diminution of both neurons and glia in peripheral ganglia, suggesting a general depletion of the ganglion precursor pool in mutant mice (De Bellard, 2002).

The segmental pattern of neural-crest-derived sympathetic ganglia arises as a direct result of signals that restrict neural crest cell migratory streams through rostral somite halves. The spatiotemporal pattern of chick sympathetic ganglia formation is a two-phase process. Neural crest cells migrate laterally to the dorsal aorta, then surprisingly spread out in the longitudinal direction, before sorting into discrete ganglia. This study investigated the function of two families of molecules that are thought to regulate cell sorting and aggregation. By blocking Eph/ephrins or N-cadherin function, changes were measured in neural crest cell migratory behaviors that lead to alterations in sympathetic ganglia formation using a sagittal slice explant culture and 3D confocal time-lapse imaging. The results demonstrate that local inhibitory interactions within inter-ganglionic regions, mediated by Eph/ephrins, and adhesive cell-cell contacts at ganglia sites, mediated by N-cadherin, coordinate to sculpt discrete sympathetic ganglia (Kasemeier-Kulesa, 2006).

Other roles of Eph receptors in brain development

During development of the vertebrate hindbrain, regulatory gene expression is confined to precise segmental domains. Studies of cell lineage and gene expression suggest that establishment of these domains may involve a dynamic regulation of cell identity and restriction of cell movement between segments. A dominant negative approach has been taken to interfere with the function of Sek-1, a member of the Eph-related receptor tyrosine kinase family expressed in rhombomeres r3 and r5. In Xenopus and zebrafish embryos expressing truncated Sek-1, which lack kinase sequences, expression of r3/r5 markers occurs in adjacent even-numbered rhombomeres, in domains contiguous with r3 or r5. This disruption is rescued by full-length Sek-1, indicating a requirement for the kinase domain in the segmental restriction of gene expression. These data suggest that Sek-1, perhaps with other Eph-related receptors, is required for interactions that regulate the segmental identity or movement of cells (Xu, 1995).

A dominant-negative approach was taken in the zebrafish embryo to interfere with the function of Rtk1, an Eph-related RTK expressed in the developing diencephalon. Expression of a truncated receptor leads to expansion of the eye field into diencephalic territory and loss of diencephalic structures, indicating a role for Rtk1 in patterning the developing forebrain (Xu, 1996).

Eph family receptor tyrosine kinases have been proposed to control axon guidance and fasciculation. To address the biological functions of the Eph family member Nuk, two mutations in the mouse germline have been generated: a protein null allele (Nuk1) and an allele that encodes a Nuk-beta gal fusion receptor lacking the tyrosine kinase and C-terminal domains (Nuk[lacZ]). In Nuk1 homozygous brains, the majority of axons forming the posterior tract of the anterior commissure migrate aberrantly to the floor of the brain, resulting in a failure of cortical neurons to link the two temporal lobes. These results indicate that Nuk, a receptor that binds transmembrane ligands, plays a critical and unique role in the pathfinding of specific axons in the mammalian central nervous system (Henkemeyer, 1996).

The hippocampus and septum play central roles in one of the most important spheres of brain function: learning and memory. Although their topographic connections have been known for two decades and topography may be critical for cognitive functions, the basis for hippocamposeptal topographic projection is unknown. Elf-1, a membrane-bound eph family ligand, is a candidate molecular tag for the genesis of the hippocamposeptal topographic projection. Elf-1 is expressed in an increasing gradient from dorsal to ventral septum. Furthermore, Elf-1 selectively allows growth of neurites from topographically appropriate lateral hippocampal neurons, while inhibiting neurite outgrowth by medial hippocampal neurons. Complementary to the expression of Elf-1, an eph family receptor, Bsk, is expressed in the hippocampus in a lateral to medial gradient, consistent with a function as a receptor for Elf-1. Further, Elf-1 specifically binds Bsk, eliciting tyrosine kinase activity. It is concluded that the Elf-1/Bsk ligand-receptor pair exhibits traits of a chemoaffinity system for the organization of hippocamposeptal topographic projections (Gao, 1996).

Neuronal connections are arranged topographically such that the spatial organization of neurons is preserved by their termini in the targets. During the development of topographic projections, axons initially explore areas much wider than the final targets, and mistargeted axons are pruned later. The molecules regulating these processes are not known. The ligands of the Eph family tyrosine kinase receptors may regulate both the initial outgrowth and the subsequent pruning of axons. In the presence of ephrins, the outgrowth and branching of the receptor-positive hippocampal axons are enhanced. However, these axons are induced later to degenerate. These observations suggest that the ephrins and their receptors may regulate topographic map formation by stimulating axonal arborization and by pruning mistargeted axons (Gao, 1999).

Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens/olfactory tubercle, respectively, constituting mesostriatal and mesolimbic pathways. The molecular signals that confer target specificity of different dopaminergic neurons are not known. EphB1 and ephrin-B2, a receptor and ligand of the Eph family, are candidate guidance molecules for the development of these distinct pathways. EphB1 and ephrin-B2 are expressed in complementary patterns in the midbrain dopaminergic neurons and their targets, and the ligand specifically inhibits the growth of neurites and induces the cell loss of substantia nigra, but not ventral tegmental, dopaminergic neurons. These studies suggest that the ligand-receptor pair may contribute to the establishment of distinct neural pathways by selectively inhibiting the neurite outgrowth and cell survival of mistargeted neurons. Ephrin-B2 expression is upregulated by cocaine and amphetamine in adult mice, suggesting that ephrin-B2/EphB1 interaction may play a role in drug-induced plasticity in adults as well (Yue, 1999).

Eph tyrosine kinase receptors and their membrane-bound ephrin ligands mediate cell interactions and participate in several developmental processes. Ligand binding to an Eph receptor results in tyrosine phosphorylation of the kinase domain, and repulsion of axonal growth cones and migrating cells. A subpopulation of ephrin-A5 null mice display neural tube defects resembling anencephaly in man. This is caused by the failure of the neural folds to fuse in the dorsal midline, suggesting that ephrin-A5, in addition to its involvement in cell repulsion, can participate in cell adhesion. During neurulation, ephrin-A5 is co-expressed with its cognate receptor EphA7 in cells at the edges of the dorsal neural folds. Three different EphA7 splice variants, a full-length form and two truncated versions lacking kinase domains, are expressed in the neural folds. Co-expression of an endogenously expressed truncated form of EphA7 suppresses tyrosine phosphorylation of the full-length EphA7 receptor and shifts the cellular response from repulsion to adhesion in vitro. It is concluded that alternative usage of different splice forms of a tyrosine kinase receptor can mediate cellular adhesion or repulsion during embryonic development (Holmberg, 2000).

Because both ephrins and Eph receptors are membrane anchored, suppression of repulsive signal transduction could hypothetically turn these proteins into cell-adhesion molecules. Alternatively, the adhesive effect may be indirect where the ligand-receptor interaction may result in kinase-independent signal transduction that could affect other molecules with adhesive properties. Notably, mutations in the gene encoding the C. elegans Eph receptor, VAB-1, result in defective ventral enclosure, a process resembling neurulation. Analysis of different vab-1 mutants reveals that this process is independent of VAB-1 kinase activity, suggesting that the kinase-independent adhesive functions of Eph family receptors may be evolutionarily conserved. Over the past few years it has become clear that several molecules involved in axon guidance and cell migration, including netrins, semaphorins and slits, have both attractive and repulsive actions. The dual function of these molecules is regulated by interaction with different receptors or by modulation of cyclic-nucleotide-dependent signal transduction pathways. The differential response to ephrin-A5 is quite possibly the first example of how the response to a ligand can be modulated between repulsion or adhesion by alternative use of different splice forms of a receptor (Holmberg, 2000 and references therein).

The visual cortex in primates is distributed into distinct areas: cytoarchitectonic, physiologic, and connectional and include the striate cortex (V1) and the extrastriate cortex, consisting of V2 and numerous higher association areas. The innervation of distinct visual cortical areas by the thalamus is especially segregated in primates, such that the lateral geniculate (LG) nucleus specifically innervates striate cortex, whereas pulvinar projections are confined to extrastriate cortex. The molecular bases for the parcellation of the visual cortex and thalamus, as well as the establishment of reciprocal connections between distinct compartments within these two structures, are largely unknown. Prospective visual cortical areas and corresponding thalamic nuclei in the embryonic rhesus monkey can be defined by combinatorial expression of genes encoding Eph receptor tyrosine kinases and their ligands, the ephrins, prior to obvious cytoarchitectonic differentiation within the cortical plate and before the establishment of reciprocal connections between the cortical plate and thalamus. These results indicate that molecular patterns of presumptive visual compartments in both the cortex and thalamus can form independently of one another and suggest a role for EphA family members in both compartment formation and axon guidance within the visual thalamocortical system (Sestan, 2001).

To evaluate the influence of reciprocal connections between the cortex and thalamus on differential gene expression in these structures, Eph family expression was evaluated at embryonic day 65 (E65), before all of the neurons that eventually receive input from the thalamus have been generated (prospective layer IV), and, thus, before patterned thalamocortical connectivity has been established. Patterning of expression within the CP is obvious at E65: EphA6 and EphA7 are expressed in overlapping but distinct gradients that selectively label the posterior half of the cortical plate (CP), a region that corresponds spatially to future visual cortex. Furthermore, the expression of another receptor, EphA3, and a ligand, ephrin-A5, demarcates distinct compartments within the EphA6- and EphA7-positive prospective visual CP: EphA3 expression comprises a plus-minus pattern, with high anterior levels, being absent in the posterior, whereas ephrin-A5 expression respects the same border, but its expression varies according to prospective laminae. At this stage, levels of ephrin-A5 expression are constant within the most superficial CP along the anteroposterior axis but increase selectively in the deepest strata of the posterior CP. AChE historeactivity was used to visualize the location of pulvinar fibers, which have entered the intermediate zone but have not yet invaded the CP at E65. Ephrin-A5 is uniformly expressed in the CP overlying pulvinar fibers but is restricted to the deepest strata of the CP in AChE-poor cerebral wall (prospective layers V and VI). This data, in combination with extrapolation from localization at older ages, led to the conclusion that differences in expression of ephrin-A5 and EphA3 at E65 correspond to distinctions between presumptive striate and extrastriate cortex. Thus, Eph family gene expression distinguish between the two regions at a time when there are no other known areal landmarks within the visual CP. In addition, these molecular differences exist in the absence of reciprocal synaptic connections between the CP and thalamus, indicating that they emerge independently (Sestan, 2001).

Analyzed next was whether corresponding patterns of Eph family expression exist in the thalamus at E65. Despite the lack of reciprocal connections between the CP and thalamus at this age, patterning of ephrin-A5 and select Eph receptors was observed within the developing thalamus. Ephrin-A5 is predominantly expressed within the ventrolateral nucleus that eventually innervates somatosensory CP, whereas EphA3, EphA6, and EphA7 are most abundant in the pulvinar and to a lesser extent in the geniculate body that eventually innervate visual CP. Moreover, distinct gradients of EphA6 and EphA7 expression are present in the pulvinar. This patterned expression within the thalamus, in conjunction with the complex neocortical expression that has been documented, supports a model in which combinatorial Eph family expression independently establishes cellular groupings within the thalamus and cortex and then influences reciprocal innervation between these structures (Sestan, 2001).

Eph receptors have been implicated in cell-to-cell interaction during embryogenesis. EphA2 mutant mice were generated using a gene trap method. Homozygous mutant mice develop short and kinky tails. In situ hybridization using a Brachyury probe found the notochord to be abnormally bifurcated at the caudal end between 11.5 and 12.5 days post coitum. EphA2 is expressed at the tip of the tail notochord, while one of its ligands, ephrinA1, is expressed at the tail bud in normal mice. In contrast, EphA2-deficient notochordal cells are spread broadly into the tail bud. These observations suggest that EphA2 and its ligands are involved in the positioning of the tail notochord through repulsive signals between cells expressing these molecules on the surface (Naruse-Nakajima, 2001).

EphB receptor tyrosine kinases and ephrin-B ligands regulate several types of cell-cell interactions during brain development, generally by modulating the cytoskeleton. EphB/ephrinB genes are expressed in the developing neural tube of early mouse embryos with distinct overlapping expression in the ventral midbrain. To test EphB function in midbrain development, mouse embryos compounds homozygous for mutations in the EphB2 and EphB3 receptor genes were examined for early brain phenotypes. These mutants display a morphological defect in the ventral midbrain, specifically an expanded ventral midline evident by embryonic day E9.5-10.5, which forms an abnormal protrusion into the cephalic flexure. The affected area is comprised of cells that normally express EphB2 and ephrin-B3. A truncated EphB2 receptor causes a more severe phenotype than a null mutation, implying a dominant negative effect through interference with EphB forward (intracellular) signaling. In mutant embryos, the overall number, size, and identity of the ventral midbrain cells are unaltered. Therefore, the defect in ventral midline morphology in the EphB2;EphB3 compound mutant embryos appears to be caused by cellular changes that thin the tissue, forcing a protrusion of the ventral midline into the cephalic space. These data suggests a role for EphB signaling in morphological organization of specific regions of the developing neural tube (Altick, 2005).

The cerebral cortex is a laminated sheet of neurons composed of the arrays of intersecting radial columns. During development, excitatory projection neurons originating from the proliferative units at the ventricular surface of the embryonic cerebral vesicles migrate along elongated radial glial fibres to form a cellular infrastructure of radial (vertical) ontogenetic columns in the overlaying cortical plate. However, a subpopulation of these clonally related neurons also undergoes a short lateral shift and transfers from their parental to the neighbouring radial glial fibres, and intermixes with neurons originating from neighbouring proliferative units. This columnar organization acts as the primary information processing unit in the cortex. The molecular mechanisms, role and significance of this lateral dispersion for cortical development are not understood. This study shows that an Eph receptor A (EphA) and ephrin A (Efna) signalling-dependent shift in the allocation of clonally related neurons is essential for the proper assembly of cortical columns. In contrast to the relatively uniform labelling of the developing cortical plate by various molecular markers and retrograde tracers in wild-type mice, alternating labelling of columnar compartments was found in Efna knockout mice that are caused by impaired lateral dispersion of migrating neurons rather than by altered cell production or death. Furthermore, in utero electroporation showed that lateral dispersion depends on the expression levels of EphAs and ephrin-As during neuronal migration. This so far unrecognized mechanism for lateral neuronal dispersion seems to be essential for the proper intermixing of neuronal types in the cortical columns, which, when disrupted, might contribute to neuropsychiatric disorders associated with abnormal columnar organization (Torii, 2009).

Neurons of the cerebral cortex are organized in layers and columns. Unlike laminar patterning, the mechanisms underlying columnar organization remain largely unexplored. This study shows that ephrin-B1 plays a key role in this process through the control of nonradial steps of migration of pyramidal neurons. In vivo gain of function of ephrin-B1 results in a reduction of tangential motility of pyramidal neurons, leading to abnormal neuronal clustering. Conversely, following genetic disruption of ephrin-B1, cortical neurons display a wider lateral dispersion, resulting in enlarged ontogenic columns. Dynamic analyses revealed that ephrin-B1 controls the lateral spread of pyramidal neurons by limiting neurite extension and tangential migration during the multipolar phase. Furthermore, P-Rex1, a guanine-exchange factor for Rac3, was identified as a downstream ephrin-B1 effector required to control migration during the multipolar phase. These results demonstrate that ephrin-B1 inhibits nonradial migration of pyramidal neurons, thereby controlling the pattern of cortical columns (Dimidschstein, 2013).

During forebrain morphogenesis, there is extensive reorganisation of the cells destined to form the eyes, telencephalon and diencephalon. Little is known about the molecular mechanisms that regulate region-specific behaviours and that maintain the coherence of cell populations undergoing specific morphogenetic processes. his study shows that the activity of the Eph/Ephrin signalling pathway maintains segregation between the prospective eyes and adjacent regions of the anterior neural plate during the early stages of forebrain morphogenesis in zebrafish. Several Ephrins and Ephs are expressed in complementary domains in the prospective forebrain and combinatorial abrogation of their activity results in incomplete segregation of the eyes and telencephalon and in defective evagination of the optic vesicles. Conversely, expression of exogenous Ephs or Ephrins in regions of the prospective forebrain where they are not usually expressed changes the adhesion properties of the cells, resulting in segregation to the wrong domain without changing their regional fate. The failure of eye morphogenesis in rx3 mutants is accompanied by a loss of complementary expression of Ephs and Ephrins, suggesting that this pathway is activated downstream of the regional fate specification machinery to establish boundaries between domains undergoing different programmes of morphogenesis (Cavodeassi, 2013).

EphA/ephrin A reverse signaling promotes the migration of cortical interneurons from the medial ganglionic eminence

Inhibitory interneurons control the flow of information and synchronization in the cerebral cortex at the circuit level. During embryonic development, multiple subtypes of cortical interneurons are generated in different regions of the ventral telencephalon, such as the medial and caudal ganglionic eminence (MGE and CGE), as well as the preoptic area (POA). These neurons then migrate over long distances towards their cortical target areas. Diverse families of diffusible and cell-bound signaling molecules, including the Eph/ephrin system, regulate and orchestrate interneuron migration. Ephrin A3 and A5, for instance, are expressed at the borders of the pathway of MGE-derived interneurons and prevent these cells from entering inappropriate regions via EphA4 forward signaling. This study found that MGE-derived interneurons, in addition to EphA4, also express ephrin A and B ligands, suggesting Eph/ephrin forward and reverse signaling in the same cell. In vitro and in vivo approaches showed that EphA4-induced reverse signaling in MGE-derived interneurons promotes their migration and that this effect is mediated by ephrin A2 ligands. In EphA4 mutant mice, as well as after ephrin A2 knockdown using in utero electroporation, delayed interneuron migration was found at embryonic stages. Thus, besides functions in guiding MGE-derived interneurons to the cortex through forward signaling, this study describes a novel role of the ephrins in driving these neurons to their target via reverse signaling (Steinecke, 2014).

Eph receptors and limb innervation and patterning

The Eph-related receptor tyrosine kinase gene, Cek-8, is expressed in mesenchyme at the tip of chick limb buds, with high levels of transcripts posteriorly and apically but fading out anteriorly. Expression of Cek-8 in distal mesenchyme is regulated by apical ridge- and FGF-polarizing signals and retinoic acid, and is uniform across the anteroposterior axis in talpid3 mutants. These data indicate that Cek-8 expression responds to regulatory signals during limb patterning and suggest that this receptor tyrosine kinase may have a role in coordinating responses to signals in the progress zone of early buds. Later on in limb development, Cek-8 expression is associated with cell condensations that form tendons and their attachments to cartilage rudiments and then in developing feather buds (Patel, 1996).

A highly dynamic expression pattern of the chick EphA7 gene occurs in the developing limb. Expression is detected in discrete domains of the dorsal mesenchyme from 3 days of incubation. The expressing cells are adjacent to the routes where axons grow to innervate the limb at several key points: the region of plexus formation, the bifurcation between dorsal and ventral fascicles, and the pathway followed by axons innervating the dorsal muscle mass. These results suggested a role for EphA7 in cell-cell contact-mediated signaling in dorsal limb patterning and/or axon guidance. Experimental manipulations were carried in the chick embryo wing bud to alter the dorsoventral patterning of the limb. The analyses of EphA7 expression and innervation in the operated wings indicate that a signal emanating from the dorsal ectoderm regulates EphA7 in such a way that, in its absence, the wing bud lacks EphA7 expression and shows innervation defects at the regions where the gene is downregulated. EphA7 downregulation in the dorsal mesenchyme after dorsal ectoderm removal is more rapid than that of Lmx-1, the gene known to mediate dorsalization in response to the ectodermal signal. These results add a new gene to the dorsalization signaling pathway in the limb. Moreover, they implicate the Eph receptor family in the patterning and innervation of the developing limb, extending its role in axon pathfinding to the distal periphery (Araujo, 1998).

The distribution of members of the Eph family were examined during muscle precursor cell development. The EphA4 receptor tyrosine kinase and its ligand, ephrin-A5, are expressed by muscle precursor cells and forelimb mesoderm in unique spatiotemporal patterns during the period when muscle precursors delaminate from the dermomyotome and migrate into the limb. To test the function of EphA4/ephrin-A5 interactions in muscle precursor migration, targeted, in ovo electroporation was used to express ephrin-A5 ectopically, specifically in the presumptive limb mesoderm. In the presence of ectopic ephrin-A5, Pax7-positive muscle precursor cells are significantly reduced in number in the proximal limb, compared with controls, and congregate abnormally near the lateral dermomyotome. In stripe assays, isolated muscle precursor cells avoid substrate-bound ephrin-A5 and this avoidance is abolished by addition of soluble ephrin-A5. These data suggest that ephrin-A5 normally restricts migrating, EphA4-positive muscle precursor cells to their appropriate territories in the forelimb, disallowing entry into abnormal embryonic regions (Swartz, 2001).

Eph receptors and vascular system development

B61, a cytokine-inducible endothelial gene product, is the ligand for the Eck receptor protein tyrosine kinase (RPTK). Expression of a B61-immunoglobulin chimera shows that B61 could act as an angiogenic factor in vivo and a chemoattractant for endothelial cells in vitro. The Eck RPTK is activated by tumor necrosis factor-alpha (TNF-alpha) through induction of B61, and an antibody to B61 attenuates angiogenesis induced by TNF-alpha but not by basic fibroblast growth factor. This finding suggests the existence of an autocrine or paracrine loop involving activation of the Eck RPTK by its inducible ligand B61 after an inflammatory stimulus, the net effect of which would be to promote angiogenesis, a hallmark of chronic inflammation (Pandey, 1995a) .

The vertebrate circulatory system is composed of arteries and veins. The functional and pathological differences between these vessels have been assumed to reflect physiological differences such as oxygenation and blood pressure. Ephrin-B2, an Eph family transmembrane ligand, marks arterial but not venous endothelial cells from the onset of angiogenesis. Conversely, Eph-B4, a receptor for ephrin-B2, marks veins but not arteries. ephrin-B2 knockout mice display defects in angiogenesis by both arteries and veins in the capillary networks of the head and yolk sac as well as in myocardial trabeculation. These results provide evidence that differences between arteries and veins are in part genetically determined and suggest that reciprocal signaling between these two types of vessels is crucial for morphogenesis of the capillary beds (Wang, 1998).

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. A phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicates a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system (Adams, 1999).

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of vascular morphogenesis. EphrinB2 may have an active signaling role, resulting in bi-directional signal transduction downstream of both ephrinB2 and Eph receptors. To separate the ligand and receptor-like functions of ephrinB2 in mice, the endogenous gene was replaced by cDNAs encoding either carboxyterminally truncated (ephrinB2DeltaC) or, as a control, full-length ligand (ephrinB2WT). While homozygous ephrinB2WT/WT animals are viable and fertile, loss of the ephrinB2 cytoplasmic domain results in midgestation lethality similar to ephrinB2 null mutants (ephrinB2KO). The truncated ligand is sufficient to restore guidance of migrating cranial neural crest cells, but ephrinB2DeltaC/DeltaC embryos show defects in vasculogenesis and angiogenesis very similar to those observed in ephrinB2KO/KO animals. These results indicate distinct requirements of functions mediated by the ephrinB carboxyterminus for developmental processes in the vertebrate embryo (Adams, 2001).

The cues and signaling systems that guide the formation of embryonic blood vessels in tissues and organs are poorly understood. Members of the Eph family of receptor tyrosine kinases and their cell membrane-anchored ligands, the ephrins, have been assigned important roles in the control of cell migration during embryogenesis, particularly in axon guidance and neural crest migration. The role of EphB receptors and their ligands during embryonic blood vessel development has been investigated in Xenopus laevis. In a survey of tadpole-stage Xenopus embryos for EphB receptor expression, expression of EphB4 receptors was detected in the posterior cardinal veins and their derivatives, the intersomitic veins. However, vascular expression of other EphB receptors, including EphB1, EphB2 or EphB3, could not be observed, suggesting that EphB4 is the principal EphB receptor of the early embryonic vasculature of Xenopus. Ephrin-B ligands are expressed complementary to EphB4 in the somites adjacent to the migratory pathways taken by intersomitic veins during angiogenic growth. RNA injection experiments were performed to study the function of EphB4 and its ligands in intersomitic vein development. Disruption of EphB4 signaling by dominant negative EphB4 receptors or misexpression of ephrin-B ligands in Xenopus embryos results in intersomitic veins growing abnormally into the adjacent somitic tissue. These findings demonstrate that EphB4 and B-class ephrins act as regulators of angiogenesis, possibly by mediating repulsive guidance cues to migrating endothelial cells (Helbling, 2000).

Angiogenesis can be divided into three phases: initiation (induction of sprouting); invasion (cell proliferation, migration and matrix degradation), and maturation (remodeling, lumen formation and differentiation of endothelial cells). To date, three growth factor systems (VEGF, angiopoietins and ephrins) have been identified as critical players of angiogenesis. As revealed by the vascular phenotypes obtained from gene inactivation and gain-of-function experiments, each signaling system appears to have an essential role during at least one particular phase of angiogenic growth of intersomitic veins. A tentative hierarchy of the signaling systems essential for intersomitic vein development may therefore be deduced. Analysis of heterozygous VEGF mutant embryos, which are less affected than those homozygously deficient for VEGF, has revealed a strong decrease in intersomitic vein sprouts. VEGF is therefore involved in the initiation of intersomitic vein sprouting. Angiopoietin-1 and its receptor Tie-2 appear to be critical later during maturation and stabilization of the intersomitic veins. Indeed, mutant mice initially form intersomitic veins, which in case of Ang-1 mutants undergo subsequent regression in older embryos. Finally, analysis of ephrin-B2- and double EphB2/EphB3-deficient mice as well Xenopus embryos disrupted in EphB4 signaling indicate a requirement for EphB receptors and their ligands during the invasion phase of intersomitic vein development. Therefore, EphB signaling appears to act downstream of VEGF and its receptors, but upstream of the angiopoietin signaling system (Helbling, 2000 and references therein).

EphrinB2, a transmembrane ligand of EphB receptor tyrosine kinases, is specifically expressed in arteries. In ephrinB2 mutant embryos, there is a complete arrest of angiogenesis. However, ephrinB2 expression is not restricted to vascular endothelial cells, and it has been proposed that its essential function may be exerted in adjacent mesenchymal cells. Mice have been generated in which ephrinB2 is specifically deleted in the endothelium and endocardium of the developing vasculature and heart. Such a vascular-specific deletion of ephrinB2 results in angiogenic remodeling defects identical to those seen in the conventional ephrinB2 mutants. These data indicate that ephrinB2 is required specifically in endothelial and endocardial cells for angiogenesis, and that ephrinB2 expression in perivascular mesenchyme is not sufficient to compensate for the loss of ephrinB2 in these vascular cells (Gerety, 2002).

Other roles of Eph receptors in development

Mutations in the C. elegans vab-1 gene disrupt the coordinated movements of cells during two periods of embryogenesis. vab-1 mutants are defective in the movement of neuroblasts during closure of the ventral gastrulation cleft and in the movements of epidermal cells during ventral enclosure of the embryo by the epidermis. vab-1 encodes a receptor tyrosine kinase of the Eph family. Disruption of the kinase domain of VAB-1 causes weak mutant phenotypes, indicating that VAB-1 may have both kinase-dependent and kinase-independent activities. VAB-1 is expressed in neurons during epidermal enclosure and is required in these cells for normal epidermal morphogenesis, demonstrating that cell-cell interactions are required between neurons and epidermal cells for epidermal morphogenesis (George, 1998).

The epidermis of the nematode C. elegans is an epithelium that undergoes epiboly during embryogenesis, where it encloses the embryo ventrally. Ventral enclosure is the result of epidermal cell shape changes and involves two steps: (1) the extension of four anterior leading cells to the ventral midline, and (2) enclosure of the posterior epidermis. A catenin/cadherin system is required for normal movement of the anterior epidermal cells, and likely functions within epidermal cells to modulate cytoskeletal behavior. The Eph receptor VAB-1 is required in neurons for epidermal morphogenesis during C. elegans embryogenesis. Two models have been proposed for the nonautonomous role of VAB-1: neuronal VAB-1 might signal directly to epidermis, or VAB-1 signaling between neurons might be required for epidermal development. The ephrin VAB-2 (also known as EFN-1) is a ligand for VAB-1 and can function in neurons to regulate epidermal morphogenesis. In the absence of VAB-1 signaling, ephrin-expressing neurons are disorganized. vab-2/efn-1 mutations synergize with vab-1 kinase alleles, suggesting that VAB-2/EFN-1 may partly function in a kinase-independent VAB-1 pathway. These data indicate that ephrin signaling between neurons is required nonautonomously for epidermal morphogenesis in C. elegans (Chin-Sang, 1999).

How might lack of VAB-1 in neurons cause defects in the epidermis? Two models have been proposed for this nonautonomy of VAB-1: the 'steric hindrance' model and the 'reverse signaling' model; these models are not mutually exclusive. In the steric hindrance model, VAB-1 signaling operates between neuroblasts and neurons, and lack of VAB-1 causes neurons to be disorganized; enclosure is defective because the epidermal cells cannot move over the abnormal neuronal substrate. In the reverse signal model, VAB-1 signals directly from neurons to the epidermal cells; in vab-1 mutants such cues are absent and epidermal cells fail to migrate normally. Analysis of vab-2/efn-1 supports a steric hindrance model for the nonautonomous role of ephrin signaling. VAB-2/EFN-1 is mostly expressed in neuroblasts or neurons, and neuron-specific expression of VAB-2/EFN-1 can partly rescue epidermal defects of vab-2/efn-1 mutants, showing that VAB-2/EFN-1 can function in neurons to regulate epidermal development. Furthermore, mutations in the VAB-1 receptor cause disorganization of the VAB-2/EFN-1-expressing neurons. The spreading of VAB-2/EFN-1-expressing cells in vab-1 mutants likely reflects abnormal cell migration or adhesion, since vab-1 mutants do not display cell fate transformations (Chin-Sang, 1999).

This role of VAB-1 in preventing cell spreading is strikingly reminiscent of the cell sorting functions of Eph signaling in vertebrate neurogenesis. It has been concluded that VAB-2/EFN-1 signaling to VAB-1 occurs between neuronal precursors, and regulates cell adhesion or movement. In the absence of VAB-1 or VAB-2/EFN-1, neuroblasts fail to close up the ventral gastrulation cleft and as a result, descendant neurons are disorganized. Disorganized neurons might block epidermal movements directly (true 'steric hindrance') or indirectly: for example, the boundary between VAB-1 and VAB-2/EFN-1-expressing cells might attract migrating epidermal cells; if this boundary is disorganized, epidermal migrations would be disrupted. An analogous situation might occur in vertebrate angiogenesis, where ephrin signaling between endocardial cells is required for the development of overlying myocardial trabeculae. Such models do not rule out signaling from neurons to epidermis, possibly via other ephrins; however, of the three other C. elegans ephrins, at least two are expressed mainly in neurons. The small number of Eph receptors and ephrins in C. elegans suggests that it will be feasible to dissect the complete network of Eph/ephrin signaling in a simple animal (Chin-Sang, 1999).

Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migration processes during development. The molecular cloning and tissue distribution are reported of zebrafish transmembrane ephrins that represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrin B sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously to several Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands are confined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins are expressed as early as 30% epiboly and during gastrula stages: in the germ ring, shield, prechordal plate, and notochord. Ectopic overexpression of dominant-negative soluble ephrin B constructs yields reproducible defects in the morphology of the notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does not completely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesis of the prechordal plate and the notochord is likely a consequence of a cell movement defect. These observations suggest Eph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation (Chan, 2001).

Eph receptor tyrosine kinases (RTK) and their ephrin ligands are involved in the transmission of signals that regulate cytoskeletal organization and cell migration, and are expressed in spatially restricted patterns at discrete phases during embryogenesis. Loss of function mutants of Eph RTK or ephrin genes result in defects in neuronal pathfinding or cell migration. Soluble forms of human EphA3 and ephrin-A5, acting as dominant negative inhibitors, interfere with early events in zebrafish embryogenesis. Exogenous expression of both proteins results in dose-dependent defects in somite development and organization of the midbrain-hindbrain boundary and hindbrain. The nature of the defects as well as the distribution and timing of expression of endogenous ligands/receptors for both proteins suggest that Eph-ephrin interaction is required for the organization of embryonic structures by coordinating the cellular movements of convergence during gastrulation (Oates, 1999).

In vertebrate embryos, neural crest cell migration and motor axon outgrowth are restricted to rostral somite halves by repulsive factors located in the caudal somite compartment. Two Eph family transmembrane ligands, Lerk2 and HtkL, are expressed in caudal somite halves, and crest cells and motor axons express receptors for these ligands. In several independent in vitro assays, preclustered ligand-Fc fusion proteins can repulsively guide both crest migration and motor axon outgrowth. These repulsive activities depend on a graded or discontinuous presentation of the ligands when tested in the context of permissive substrates, such as laminin or fibronectin. These results identify Lerk2 and HtkL as potential determinants of segmental pattern in the peripheral nervous system (Wang, 1997).

Somitogenesis involves the segmentation of the paraxial mesoderm into units along the anteroposterior axis. A role for Eph and ephrin signaling in the patterning of presomitic mesoderm and formation of the somites is shown. Ephrin-A-L1 and ephrin-B2 are expressed in an iterative manner in the developing somites and presomitic mesoderm, as is the Eph receptor EphA4. The role of these proteins was examined by injection of RNA, encoding dominant negative forms of Eph receptors and ephrins. Interruption of Eph signaling leads to abnormal somite boundary formation and reduced or disturbed myoD expression in the myotome. Disruption of Eph family signaling delays the normal down-regulation of her1 and Delta D expression in the anterior presomitic mesoderm and disrupts myogenic differentiation. It is suggested that Eph signaling has a key role in the translation of the patterning of presomitic mesoderm into somites (Durbin, 1998).

The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation suggests that there is a role for long range signaling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. This could be achieved by the expression of the BMP antagonist, Follistatin. Local application of Follistatin leads to the development of ectopic feather buds. A suggestion is made that Follistatin locally antagonizes the action of the BMPs and so permits the cellular changes associated with feather placode formation. Evidence is provided for the role of short range signaling during feather formation. Changes in cellular morphology in feather placodes are correlated with the expression of the gene Eph-A4 that encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. Expression of this gene precedes cellular reorganization required for feather bud formation (Patel, 1999).

Transgenic mice over-expressing the EphB4 receptor tyrosine kinase in the kidney were established. The EphB4 protein localizes to the developing tubular system of both control and transgenic newborn mice. In transgenic adults, transgene expression persists in the proximal tubules and the Bowman's capsules, structures that were not stained in control kidneys. The glomeruli of control animals consist of regular, round vascular baskets with clearly discernable afferent and efferent arterioles. In contrast, approximately 40% of the transgenic glomeruli had an irregular shrivelled appearance and many exhibited fused, horse shoe-like afferent and efferent arterioles bypassing the glomerulus. These abnormal glomerular structures are very reminiscent of aglomerular vascular shunts, a human degenerative glomerulopathy of unknown aetiology (Andres, 2003).

Targeted inactivation of the Eph receptor ligand ephrinB1 in mouse causes perinatal lethality, edema, defective body wall closure, and skeletal abnormalities. In the thorax, sternocostal connections are arranged asymmetrically and sternebrae are fused -- defects that are phenocopied in EphB2/EphB3 receptor mutants. In the wrist, loss of ephrinB1 leads to abnormal cartilage segmentation and the formation of additional skeletal elements. It is concluded that ephrinB1 and B class Eph receptors provide positional cues required for the normal morphogenesis of skeletal elements. Another malformation, preaxial polydactyly, is exclusively seen in heterozygous females in which expression of the X-linked ephrinB1 gene is mosaic, so that ectopic EphB-ephrinB1 interactions led to restricted cell movements and the bifurcation of digital rays. These findings suggest that differential cell adhesion and sorting might be relevant for an unusual class of X-linked human genetic disorders, in which heterozygous females show more severe phenotypes than hemizygous males (Compagni, 2003).

How can ephrinB1-EphB interactions regulate segmentation and patterning processes? One possibility might be that enhanced proliferation of chondrogenic cells leads to a size expansion of cartilaginous condensations and eventually triggers the bifurcation of digits. Arguing against such a passive growth-controlled mechanism, ephrinB1 mutant limbs do not contain enlarged skeletal elements, and numbers of proliferating cells are comparable between mosaic areas. The A class receptor EphA7 facilitates chondrogenic condensation, but ephrinB1 KO, KO/+, and control mesenchyme show very similar chondrogenic capacities. Furthermore, the polydactyly in ephrinB1 KO/+ limbs is independent from changes in the Shh pathway (Compagni, 2003).

Interdigital zones forming between bifurcated digits in ephrinB1 KO/+ mutants are largely devoid of programmed cell death. BMPs and their receptors regulate apoptosis of interdigital cells, and, indeed, transcripts for bmp2, bmpr-1a, and msx-1 were initially absent in ectopic IDZs, which instead showed residual expression of the chondrogenic marker sox9. This indicates that morphological alterations induced by ectopic EphB-ephrinB1 signaling precede and are, to a certain extent, independent from changes in the genetic programs controlling the development of digits and IDZs (Compagni, 2003).

Previous work has shown that the Eph/ephrin system plays important roles in the guidance of neuronal growth cones and migrating cells by providing repulsive cues. Moreover, complementary patterns of Eph and ephrin expression in adjacent hindbrain rhombomeres are critically involved in restricting cell movement and intermingling, as confirmed by studies in zebrafish embryos. In mosaic KO/+ limbs, evidence was found for reduced cell mixing between mosaic areas of mesenchyme expressing EphB receptors and ephrinB1, respectively. It is hypothesized that EphB-ephrinB1 signaling interfaces provide interacting cells with repulsive cues, which, when properly located, lead to diverging cell movements and the splitting of growing digits (Compagni, 2003).

During somitogenesis, segmental patterns of gene activity provide the instructions by which mesenchymal cells epithelialize and form somites. Various members of the Eph family of transmembrane receptor tyrosine kinases and their Ephrin ligands are expressed in a segmental pattern in the rostral presomitic mesoderm. This pattern establishes a receptor/ligand interface at each site of somite furrow formation. This study uses the fused somites (fss) mutant as an in vivo system to study the role of Eph/Ephrin signaling during somite morphogenesis. fss encodes Tbx24, a T box transcription factor involved in maturation of the presomitic mesoderm. fss mutants lack anterior-posterior polarity within presumptive segments of the rostral PSM and fail to form somites. In the fused somites mutant, lack of intersomitic boundaries and epithelial somites is accompanied by a lack of Eph receptor/Ephrin signaling interfaces. These observations suggest a role for Eph/Ephrin signaling in the regulation of somite epithelialization. Restoration of Eph/Ephrin signaling in the paraxial mesoderm of fss mutants rescues most aspects of somite morphogenesis. (1) Restoration of bidirectional or unidirectional EphA4/Ephrin signaling results in the formation and maintenance of morphologically distinct boundaries. (2) Activation of EphA4 leads to the cell-autonomous acquisition of a columnar morphology and apical redistribution of ß-catenin, aspects of epithelialization characteristic of cells at somite boundaries. (3) Activation of EphA4 leads to nonautonomous acquisition of columnar morphology and polarized relocalization of the centrosome and nucleus in cells on the opposite side of the forming boundary. These nonautonomous aspects of epithelialization may involve interplay of EphA4 with other intercellular signaling molecules. These results demonstrate that Eph/Ephrin signaling is an important component of the molecular mechanisms driving somite morphogenesis. A new role is proposed for Eph receptors and Ephrins as intercellular signaling molecules that establish cell polarity during mesenchymal-to-epithelial transition of the paraxial mesoderm (Barrios, 2003).

Eph/ephrin interactions modulate muscle satellite cell motility and patterning.

During development and regeneration, directed migration of cells, including neural crest cells, endothelial cells, axonal growth cones and many types of adult stem cells, to specific areas distant from their origin is necessary for their function. It has recently been shown that adult skeletal muscle stem cells (satellite cells), once activated by isolation or injury, are a highly motile population with the potential to respond to multiple guidance cues, based on their expression of classical guidance receptors. In vivo, differentiated and regenerating myofibers dynamically express a subset of ephrin guidance ligands, as well as Eph receptors. This expression has previously only been examined in the context of muscle-nerve interactions; however, it is proposed that this expression might also play a role in satellite cell-mediated muscle repair. Therefore, whether Eph-ephrin signaling would produce changes in satellite cell directional motility was investigated. Using a classical ephrin 'stripe' assay, it was found that satellite cells respond to a subset of ephrins with repulsive behavior in vitro; patterning of differentiating myotubes is also parallel to ephrin stripes. This behavior can be replicated in a heterologous in vivo system, the hindbrain of the developing quail, in which neural crest cells are directed in streams to the branchial arches and to the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite. It is hypothesized that guidance signaling might impact multiple steps in muscle regeneration, including escape from the niche, directed migration to sites of injury, cell-cell interactions among satellite cell progeny, and differentiation and patterning of regenerated muscle (Stark, 2011).

Eph receptors and transformation

In epithelial tissues, cells expressing oncogenic Ras (hereafter RasV12 cells; see Drosophila Ras oncogene at 85D) are detected by normal neighbors and as a result are often extruded from the tissue. RasV12 cells are eliminated apically, suggesting that extrusion may be a tumor-suppressive process. Extrusion depends on E-cadherin-based cell-cell adhesions and signaling to the actin-myosin cytoskeleton. However, the signals underlying detection of the RasV12 cell and triggering extrusion are poorly understood. This study identified differential EphA2 (see Drosophila Eph) signaling as the mechanism by which RasV12 cells are detected in epithelial cell sheets. Cell-cell interactions between normal cells and RasV12 cells trigger ephrin-A-EphA2 signaling, which induces a cell repulsion response in RasV12 cells. Concomitantly, RasV12 cell contractility increases in an EphA2-dependent manner. Together, these responses drive the separation of RasV12 cells from normal cells. In the absence of ephrin-A-EphA2 signals, RasV12 cells integrate with normal cells and adopt a pro-invasive morphology. Drosophila Eph (DEph) is detected in segregating clones of RasV12 cells and is functionally required to drive segregation of RasV12 cells in vivo, suggesting that the in vitro findings are conserved in evolution. It is proposed that expression of RasV12 in single or small clusters of cells within a healthy epithelium creates ectopic EphA2 boundaries, which drive the segregation and elimination of the transformed cell from the tissue. Thus, deregulation of Eph/ephrin would allow RasV12 cells to go undetected and expand within an epithelium ( Porazinski, 2016).

Protein tyrosine phosphatase receptor type J (PTPRJ) regulates retinal axonal projections by inhibiting Eph and Abl kinases in mice

Eph receptors play pivotal roles in the axon guidance of retinal ganglion cells (RGCs) at the optic chiasm and the establishment of the topographic retinocollicular map. Previous work has demonstrated that protein tyrosine phosphatase receptor type O (PTPRO) is specifically involved in the control of retinotectal projections in chicks through the dephosphorylation of EphA and EphB receptors. It was subsequently revealed that all the mouse R3 subfamily members (PTPRB, PTPRH, PTPRJ, and PTPRO) of the receptor protein tyrosine phosphatase (RPTP) family inhibited Eph receptors as their substrates in cultured mammalian cells. This study investigated the functional roles of R3 RPTPs in the projection of mouse retinal axon of both sexes. Ptpro and Ptprj were expressed in mouse RGCs; however, Ptprj expression levels were markedly higher than those of Ptpro. Consistent with their expression levels, Eph receptor activity was significantly enhanced in Ptprj-knockout (Ptprj-KO) retinas. In Ptprj-KO and Ptprj/Ptpro-double-KO (DKO) mice, the number of retinal axons that projected ipsilaterally or to the contralateral eye was significantly increased. Furthermore, retinal axons in Ptprj-KO and DKO mice formed anteriorly-shifted ectopic terminal zones in the superior colliculus. c-Abl was found to be downstream of ephrin-Eph signaling for the repulsion of retinal axons at the optic chiasm and in the superior colliculus. c-Abl was identified as a novel substrate for PTPRJ and PTPRO, and the phosphorylation of c-Abl was up-regulated in Ptprj-KO and DKO retinas. Thus, PTPRJ regulates retinocollicular projections in mice by controlling the activity of Eph and c-Abl kinases (Yu, 2018).

EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity

Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. This study identified the transmembrane ligand EphrinB2 (see Drosophila Ephrin) and its receptor EphB4 (see Drosophila Eph) as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function (Frye, 2020).


Search PubMed for articles about Drosophila Eph receptor tyrosine kinase

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Chai, A., et al. (2008). hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction. Hum. Mol. Genet. 17: 266-280. PubMed Citation: 17947296

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Ernst, A. F., Jurney, W. M. and McLoon, S. C. (1998). Mechanisms involved in development of retinotectal connections: roles of Eph receptor tyrosine kinases, NMDA receptors and nitric oxide. Prog. Brain Res. 118: 115-31

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Frye, M., Stritt, S., Ortsater, H., Hernandez Vasquez, M., Kaakinen, M., Vicente, A., Wiseman, J., Eklund, L., Martinez-Torrecuadrada, J. L., Vestweber, D. and Makinen, T. (2020). EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity. Elife 9. PubMed ID: 32897857

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Biological Overview

date revised: 2 November 2020

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