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, 1999).
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).
Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. EphB1 regulates cell attachment by discriminating the density at which ephrin-B1 is displayed on a reconstituted surface. EphB1-ephrin-B1 engagement does not promote cell attachment through mechanical tethering, but does activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen is mediated through alphavbeta3 integrin. EphB1 transfection confers ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants fail to stimulate ephrin-B1 dependent attachment. These findings led to the proposal that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment (Huynh-Do, 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).
Contact-mediated axon repulsion by ephrins raises an unresolved question: these cell surface ligands form a high-affinity multivalent complex with their receptors present on axons, yet rather than being bound, axons can be rapidly repelled. Ephrin-A2 forms a stable complex with the metalloprotease Kuzbanian, involving interactions outside the cleavage region and the protease domain. Eph receptor binding triggers ephrin-A2 cleavage in a localized reaction specific to the cognate ligand. A cleavage-inhibiting mutation in ephrin-A2 delays axon withdrawal. These studies reveal mechanisms for protease recognition and control of cell surface proteins, and, for ephrin-A2, they may provide a means for efficient axon detachment and termination of signaling (Hattori, 2000).
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).
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).
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 andneuronal 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 behavior 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).
Bidirectional signals mediated by membrane-anchored ephrins and Eph receptor tyrosine kinases have important functions in cell-cell recognition events, including those that occur during axon pathfinding and hindbrain segmentation. The reverse signal that is transduced into B-ephrin-expressing cells is thought to involve tyrosine phosphorylation of the signal's short, conserved carboxy-terminal cytoplasmic domain. The Src-homology-2 (SH2) domain proteins that associate with activated tyrosine-phosphorylated B-subclass ephrins have not been identified, nor has a defined cellular response to reverse signals been described. The SH2/SH3 domain adaptor protein Grb4 binds to the cytoplasmic domain of B ephrins in a phosphotyrosine-dependent manner. In response to B-ephrin reverse signaling, cells increase FAK catalytic activity, redistribute paxillin, lose focal adhesions, round up, and disassemble F-actin-containing stress fibers. These cellular responses can be blocked in a dominant-negative fashion by expression of the isolated Grb4 SH2 domain. The Grb4 SH3 domains bind a unique set of other proteins that are implicated in cytoskeletal regulation, including the Cbl-associated protein (CAP/ponsin), the Abl-interacting protein-1 (Abi-1), dynamin, PAK1, hnRNPK and axin. These data provide a biochemical pathway whereby cytoskeletal regulators are recruited to Eph-ephrin bidirectional signaling complexes (Cowan, 2001).
EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, three tyrosines of ephrin-B1 have been identified as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 identified as phosphorylation sites in 293 cells are also phosphorylated in retinal tissue. These data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex (Kalo, 2001).
Ephrins are cell surface-associated ligands for Eph receptors and are important regulators of morphogenic processes such as axon guidance and angiogenesis. Transmembrane ephrinB ligands act as 'receptor-like' signaling molecules, in part mediated by tyrosine phosphorylation and by engagement with PDZ domain proteins. However, the underlying cell biology and signaling mechanisms are poorly understood. Src family kinases (SFKs) are positive regulators of ephrinB phosphorylation and phosphotyrosine-mediated reverse signaling. EphB receptor engagement of ephrinB causes rapid recruitment of SFKs to ephrinB expression domains and transient SFK activation. With delayed kinetics, ephrinB ligands recruit the cytoplasmic PDZ domain containing protein tyrosine phosphatase PTP-BL and are dephosphorylated. These data suggest the presence of a switch mechanism that allows a shift from phosphotyrosine/SFK-dependent signaling to PDZ-dependent signaling (Palmer, 2002).
A first example of ephrinB signaling via PDZ domain proteins has been shown for cerebellar granule cells, which require the PDZ-RGS3 protein to respond to chemoattractants signaling through heterotrimeric G proteins. PTP-BL contains five PDZ domains, which interact with other cytoplasmic proteins including a GTPase-activating protein (GAP) with specificity for the Ras-like GTPase Rho. Rho GTPases are important regulators of actin cytoskeleton dynamics in response to external stimuli, and ephrinB-mediated axon guidance is thought to involve rapid changes in actin filament organization. Moreover, SFKs are known to regulate cell morphology, adhesion, and migration by association with and phosphorylation of focal adhesion kinase (FAK) and p190 RhoGAP. FAK is a downstream phosphorylation target of ephrinB reverse signaling. Mice deficient in p190 RhoGAP exhibit a lack of the anterior commissure, a phenotype associated with a lack of ephrinB reverse signaling in ephB2-/- mice. Thus, it will be interesting to analyze the role of the Src/p190 RhoGAP pathway and how these proteins contribute to both phosphotyrosine-dependent and PDZ domain-dependent signaling linked to rearrangements of the actin cytoskeleton downstream of ephrinB ligands (Palmer, 2002).
Eph receptors and ephrin ligands are key players in many developmental processes including embryo patterning, angiogenesis, and axon guidance. Eph/ephrin interactions lead to the generation of a bidirectional signal, in which both the Eph receptors and the ephrins activate downstream signaling cascades simultaneously. To understand the role of ephrin-B1 and the importance of ephrin-B1-induced reverse signaling during embryonic development, mouse lines carrying mutations in the efnb1 gene were generated. Complete ablation of ephrin-B1 results in perinatal lethality associated with a range of phenotypes, including defects in neural crest cell (NCC)-derived tissues, incomplete body wall closure, and abnormal skeletal patterning. Conditional deletion of ephrin-B1 demonstrated that ephrin-B1 acts autonomously in NCCs, and controls their migration. Last, a mutation in the PDZ binding domain indicates that ephrin-B1-induced reverse signaling is required in NCCs. These results demonstrate that ephrin-B1 acts both as a ligand and as a receptor in a tissue-specific manner during embryogenesis (Davy, 2004).
This work suggests that the role of ephrin-B1 in NCCs is to control directional migration toward target tissues, in agreement with known functions of ephrin/Eph signaling. In the mutant embryos, NCCs exhibited a wandering behavior, which has also been reported for other mutations, in particular for Twist homozygous mutants. The early lethality of the homozygous Twist mutants does not permit an assessment of whether the migration defects would affect similar NCC-derived structures as ephrin-B1 mutation. However, it has been reported that heterozygous Twist mutants present craniofacial defects reminiscent of defects seen in ephrin-B1null mutants. The migration defects observed in the ephrin-B1null animals do not provoke defects in formation and differentiation of cranial ganglia. Mutant embryos presented defects in nerve fasciculation and branching that might contribute to the perinatal lethality. A role in axon fasciculation has been described for EphA/ephrinA members previously (Davy, 2004).
What could be the molecular mechanisms by which ephrin-B1 regulates cell migration? Ephrin-B1 might function in part as a ligand to regulate NCC migration via activation of Eph receptors. Alternatively, ephrin-B1 also controls NCCs migration as a receptor, presumably by activating a signaling cascade involving a PDZ-containing protein. One candidate that might act downstream of ephrin-B1 in NCCs is the PDZ-containing protein PDZ-RGS3 that has been shown to be an effector of ephrin-B1 in regulating the migration of cerebellar granule cells. Preliminary data indicate that PDZ-RGS3 is indeed expressed in branchial arches (Davy, 2004).
In conclusion, this work demonstrates that ephrin-B1 plays an important role in different tissues during embryogenesis and that reverse signaling is an essential component of ephrin-B1 function. At the cellular level ephrin-B1 seems to regulate adhesion/migration processes. Interestingly, the results suggest that a reverse signaling cascade is required downstream of ephrin-B1 in a tissue-specific manner. Further studies will be necessary to clarify the role of ephrin-B1 in each tissue and to identify the specific effectors of ephrin-B1-induced reverse signaling in these tissues (Davy, 2004).
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).
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).
Rhomboid-1 is a serine protease that cleaves the membrane domain of the Drosophila EGF-family protein, Spitz, to release a soluble growth factor. Several vertebrate rhomboid-like proteins have been identified, although their substrates and functions remain unknown. The human rhomboid, RHBDL2, cleaves the membrane domain of Drosophila Spitz when the proteins are co-expressed in mammalian cells. However, the membrane domains of several mammalian EGF-family proteins were not cleaved by RHBDL2, suggesting that the endogenous targets of the human protease are not EGF-related factors. The amino acid sequence at the luminal face of the membrane domain of a substrate protein determines whether it is cleaved by RHBDL2. Based on this finding, B-type ephrins are predicted as potential RHBDL2 substrates. One of these, ephrinB3, was cleaved so efficiently by the protease that little ephrinB3 was detected on the surface of cells co-expressing RHBDL2. These results raise the possibility that RHBDL2-mediated proteolytic processing may regulate intercellular interactions between ephrinB3 and eph receptors (Pascall, 2004).
Although Apc is well characterized as a tumor-suppressor gene in the intestine, the precise mechanism of this suppression remains to be defined. Using a novel inducible Ahcre transgenic line in conjunction with a loxP-flanked Apc allele, loss of Apc is shown to acutely activate Wnt signaling through the nuclear accumulation of ß-catenin. Coincidentally, it perturbs differentiation, migration, proliferation, and apoptosis, such that Apc-deficient cells maintain a 'crypt progenitor-like' phenotype. Critically, a series of Wnt target molecules has been confirmed in an in vivo setting, and a series of new candidate targets has been identified within the same setting (Sansom, 2004).
ß-catenin levels were examined within the Cre+Apcfl/fl tissue at day 5. There was no increase in total ß-catenin in the Cre+Apcfl/fl samples. However, levels of dephosphorylated ß-catenin were moderately elevated and, crucially, ß-catenin relocalized to the nuclei in the Cre+Apcfl/fl tissue. To more precisely define the time scale of nuclear relocalization, immunohistochemical analyses were performed at days 1, 2, 3, and 4 following induction of the cre recombinase. This analysis showed that relocalization occurred at day 3, and this was coincident with the observed onset of changes in morphology, proliferation, and apoptosis (Sansom, 2004).
To test whether nuclear ß-catenin was activating transcription of its known target genes, microarray analysis was performed using the affymetrix U74A chip. RNA samples were derived from sibling Cre+Apcfl/fl and Cre+Apc+/+ mice given four daily injections of ß-napthoflavone and killed at days 4 and 5. Of the 100 most significantly up-regulated genes, 10 have been associated with Wnt signaling (either directly or through arrays that had examined targets of the ß-catenin/TCF4 complex). Of the comparable genes up-regulated at day 5, 45 of 47 showed increases, of which 36 were in excess of twofold. These 36 included c-Myc, CD44, Tiam 1, Sema3c, and EphB3, all of which were confirmed changes at day 5. These data are, therefore, consistent with the notion that these are important early changes following nuclear relocalization of ß-catenin. Use of the larger chip set also revealed up-regulation of other Wnt target genes at day 4, including Sox17 and Axin2 (Sansom, 2004).
To validate the results obtained from the microarray analysis, the expression pattern of a subset of dysregulated genes was examined. Up-regulation of CD44, C-Myc, laminin gamma2, EphB2, and EphB3 was confirmed immunohistochemically in the Apc-deficient tissue. Expression of the EphrinB2 ligand, which is normally restricted to the top of the crypts and villi, was reduced in concordance with the reduction in villus differentiation in the Cre+Apcfl/fl tissue. These data therefore confirm, in an in vivo setting, many of the targets of Wnt signaling that have been implicated from in vitro studies. These include up-regulation of CD44, c-Myc, MMP-7 (matrilysin), gamma-2 laminin, Sema3c (confirmed by RT-PCR) Ets-2, EphB2, EphB3, and GPR49. The array analysis also indicates up-regulation of a series of genes that either interact with CD44 or are targets of CD44. These include MMP-7, TIAM1, FGF4 and its receptor, and TASR-2. The up-regulation of TIAM1 is particularly interesting, since TIAM1 has been shown to mediate Ras signaling. Indeed, mice deficient in TIAM1 are resistant to Ras-induced skin tumors (Sansom, 2004).
Deficiency of EphB3 has been shown to lead to abnormal Paneth cell positioning in the crypt. Wnt-mediated up-regulation of EphB3 yields a similar Paneth cell phenotype, confirming a pivotal role for the EphB/ephrinB mutual repulsion system in defining crypt-villus architecture. These results are also consistent with the notion that Apc mutant cells express the same genetic program as cells at positions 1-2 of the crypt, with notable increases in EphB3, MMP7, and Pla2g2a being characteristic of both Paneth cells and the Apc-deficient cells described in this study (Sansom, 2004).
In summary, Apc has been shown to be a critical determinant of cell fate in the murine small intestinal epithelium. Acute activation of Wnt signaling immediately produces many of the phenotypes associated with early colorectal lesions: failed differentiation, increased proliferation, and aberrant migration. Within a short time scale, multiple processes are affected: interactions with the cellular matrix, interactions with the basement membrane, increased proliferation, and failure of positional cues (EphB/ephrinB) (Sansom, 2004).
Pathfinding of retinal ganglion cell (RGC) axons at the midline optic chiasm determine whether RGCs project to ipsilateral or contralateral brain visual centers, critical for binocular vision. Using Isl2tau-lacZ knockin mice, it has been shown that the LIM-homeodomain transcription factor Isl2 marks only contralaterally projecting RGCs. The transcription factor Zic2 and guidance receptor EphB1, required by RGCs to project ipsilaterally, colocalize in RGCs distinct from Isl2 RGCs in the ventral-temporal crescent (VTC), the source of ipsilateral projections. Isl2 knockout mice have an increased ipsilateral projection originating from significantly more RGCs limited to the VTC. Isl2 knockouts also have increased Zic2 and EphB1 expression and significantly more Zic2 RGCs in the VTC. It is concluded that Isl2 specifies RGC laterality by repressing an ipsilateral pathfinding program unique to VTC RGCs and involving Zic2 and EphB1. This genetic hierarchy controls binocular vision by regulating the magnitude and source of ipsilateral projections and reveals unique retinal domains (Pak, 2004).
These findings indicate that Isl2 normally represses Zic2 expression in RGCs in the VTC and either directly represses EphB1 expression or indirectly through repression of Zic2 and that the increased ipsilateral projection in Isl2-null mice is due to a loss of this repression and upregulation of Zic2 and EphB1. This model is consistent with several pieces of data. (1) Regarding the timing of expression of Isl2 and Zic2 in VTC RGCs, the onset of Isl2 expression in VTC RGCs is similar to that of Zic2: weak Isl2 expression is detected in the VTC as early as E13.5, and moderate levels of Isl2 expression are evident by E14.5, the age when Zic2 expression in VTC RGCs is first detected. (2) Zic2 and EphB1 colocalize in a subset of RGCs distinct from Isl2 RGCs. (3) Increased expression of Zic2 and EphB1 and a significant increase in Zic2-positive RGCs are found in the VTC of Isl2-null retina. (4) The laterality phenotype of Isl2-null mice complements that of Zic2kd/kd and EphB1 mutants (Pak, 2004).
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).
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