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PubMed2005

Differential expression of Eph receptors and ephrins in the cochlear ganglion and eighth cranial nerve of the chick embryo.

The cochleovestibular ganglion of the chick differentiates at early embryonic stages as VIIIth nerve axons enter the brainstem. The tonotopic organization of the auditory portion of the VIIIth nerve can be discerned at the time axons initially reach their brainstem targets. The mechanisms underlying this early organization are not known. Eph receptor tyrosine kinases and their ligands, the ephrins, have a demonstrated role in guiding axons to topographically appropriate locations in other areas of the nervous system. In order to begin to test whether Eph proteins have a similar role in the auditory system, we investigated the tonotopic expression of several Eph receptors and ephrins in the VIIIth nerve during embryonic ages corresponding to the initial innervation of the auditory brainstem. Expression patterns of EphA4, EphB2, EphB5, ephrin-A2, and ephrin-B1 were evaluated immunohistochemically at embryonic days 4 through 10. Protein expression was observed in the cochlear ganglion and VIIIth nerve axons at these ages. EphB5, ephrin-A2, and ephrin-B1 were expressed throughout the nerve. EphA4 and EphB2 had complementary expression patterns within the nerve, with EphA4 expression higher in the dorsolateral part of the nerve and EphB2 expression higher in the ventromedial part of the nerve. These regions may correspond to auditory and vestibular components, respectively. Moreover, EphA4 expression was higher toward the low-frequency region of both the centrally and peripherally projecting branches of cochlear ganglion cells. Regional variation of Eph protein expression may influence the target selection and topography of developing VIIIth nerve projections.

Animals↗

PubMed2005

Retinoic acid regulates the expression of dorsoventral topographic guidance molecules in the chick retina.

Asymmetric expression of several genes in the early eye anlagen is required for the dorsoventral (DV) and anteroposterior (AP) patterning of the retina. Some of these early patterning genes play a role in determining the graded expression of molecules that are needed to form the retinotectal map. The polarized expression of retinoic acid synthesizing and degrading enzymes along the DV axis in the retina leads to several zones of varied retinoic acid (RA) activity. This is suggestive of RA playing a role in DV patterning of the retina. A dominant-negative form of the retinoic acid receptor alpha (DNhRARalpha) was expressed in the chick retina to block RA activity. RA signaling was found to play a role in regulating the expression of EphB2, EphB3 and ephrin B2, three molecules whose graded expression in the retina along the DV axis is important for establishing the correct retinotectal map. Blocking RA signaling by misexpression of a RA degrading enzyme, Cyp26A1 recapitulated some but not all the effects of DNhRARalpha. It also was found that Vax, a ventrally expressed transcription factor that regulates the expression of the EphB and ephrin B molecules, functions upstream of, or in parallel to, RA. Expression of DNhRARalpha led to increased levels of RA-synthesizing enzymes and loss of RA-degrading enzymes. Activation of such compensatory mechanisms when RA activity is blocked suggests that RA homeostasis is very strictly regulated in the retina.

Animals↗

PubMed2004

Expression of Ephb2 and Ephb4 in breast carcinoma.

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, play key roles in diverse biological processes. Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, EphB receptors and ephrinB ligands are later proved to be crucial regulators of vasculogenesis and embryogenesis. More studies indicate that Eph receptors are involved in angiogenesis and tumorigenesis. This study aimed to investigate the expression of EphB2 and EphB4 in breast carcinomas. Semiquantitative RT-PCR and immunohistochemistry were used to examine the expression patterns of EphB2 and EphB4. Clinicopathological and survival correlations were statistically analyzed in a series of 94 breast carcinomas, 9 normal specimens and 4 breast carcinoma cell lines. 1(1%), 16(17%), 29(31%), 48(51%) of the 94 tumors were negative, weak, moderate and strong EphB2 protein expression, respectively. 6(6%), 27(29%), 28(30%), 33(35%) of the tumors were negative, weak, moderate and strong EphB4 expression, respectively. Both EphB2 and EphB4 RTPCR products could be detected in all specimens. Increased EphB2 protein expression was negatively associated with overall survival, and there was a trend that increased EphB2 protein expression was correlated with shorter disease free survival, while EphB4 protein expression was associated with histological grade and stage. EphB4 membrane staining was increased with S phase fraction and associated with DNA aneuploidy. These findings indicate that both EphB2 and EphB4 are involved in the development of breast cancer and that both molecules could be potential predictive markers.

Aneuploidy↗

PubMed2001

Association among EPHB2, TrkA, and MYCN expression in low-stage neuroblastomas.

BACKGROUND: The EPH family is the largest subfamily of receptor protein-tyrosine kinases, consisting of EPHA and EPHB subgroups. Ligands of EPH family receptors are called ephrins, which include ephrin-A and ephrin-B subgroups. We recently found that transcripts encoding the EPHB subgroup (EPHB) and the ephrin-B subgroup (EFNB) were expressed together in neuroblastoma (NB) cell lines. PROCEDURE: In this study, we examined the expression of EPHB and EFNB transcripts in 24 NB specimens representing all clinical stages. We found that several EPHB and EFNB transcripts were expressed together in all NBs examined. RESULTS: Among the transcripts examined, EPHB6 expression was most significantly associated with low stage tumors (stages 1, 2, and 4S; P = 0.0048). TrkA expression was significantly correlated with EPHB6, EFNB2, and EFNB3 expression (P < 0.01 in each case). Taken together, these data indicate that the expression of EPHB6, EFNB2, and EFNB3 may serve as prognostic indicators of favorable NBs. In the low-stage NBs without MYCN amplification, EPHB2 expression was correlated both with MYCN expression and with TrkA expression (P < 0.01 in each case). Moreover, MYCN expression was correlated with TrkA expression (P < 0.01) in the low-stage NBs. CONCLUSIONS: This observation points to the possibility that MYCN expression might contribute to favorable outcome of low-stage NBs.

Gene Expression Regulation, Neoplastic↗

PubMed2006

High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability.

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

DNA Mutational Analysis↗

PubMed2005

EphB2/R-Ras signaling regulates glioma cell adhesion, growth, and invasion.

Eph receptor tyrosine kinases mediate neurodevelopmental processes such as boundary formation, vasculogenesis, and cell migration. Recently, we found that overexpression of EphB2 in glioma cells results in reduced cell adhesion and increased cell invasion. Since R-Ras has been shown to play a critical role in EphB2 regulation of integrin activity, we explored whether the biological role of EphB2 in glioma invasion is mediated by downstream R-Ras activation. On EphB2 activation, R-Ras associated with the receptor and became highly phosphorylated. Depletion of endogenous R-Ras expression by siRNA abrogated EphB2 effects on glioma cell adhesion, proliferation, and invasion in ex vivo rat brain slices. Anti-proliferative responses to EphB2 activation were consistent with suppressed mitogen-activated protein kinase activity. Moreover, R-Ras was highly phosphorylated in the invading glioma cells. In human brain tumor specimens, R-Ras expression and phosphorylation correlated with increasing grade of gliomas. Laser capture microdissection of invading glioblastoma cells revealed elevated R-Ras mRNA (1.5- to 26-fold) in 100% (eight of eight) of biopsy specimens, and immunohistochemistry revealed high R-Ras localization primarily in glioblastoma cells. The phosphorylation ratio of R-Ras positively correlated with the phosphorylation ratio of EphB2 in glioblastoma tissues. These results demonstrate that R-Ras plays an important role in glioma pathology, further suggesting the EphB2/R-Ras signaling pathway as a potential therapeutic target.

Animals↗

PubMed2005

Altered expression patterns of EphrinB2 and EphB2 in human umbilical vessels and congenital venous malformations.

Vascular malformations cause discomfort and pain in children and are often associated with skeletal hypertrophy. Their molecular basis is poorly understood. Ephrin ligands and Eph receptor tyrosine kinases are involved in embryonic vascular development. In mice, some ephrin/Eph family members show a complementary expression pattern in blood vessels, with ephrinB2 being expressed on arterial and EphB4 on venous endothelium. Targeted deletions of the genes reveal their essential roles for conduit vessel development in mice, suggesting similar functions during human vascular development and deregulation in vascular malformations. Here, we have defined the expression patterns of human ephrinB2, EphB4, and EphB2 in normal vessels of neonates (i.e. umbilici) and adults and compared them with those in congenital venous malformations. In adults, normal vessels of the skin, muscle, and legs express ephrinB2 and EphB2 on arterial endothelial cells (ECs), whereas EphB4 is found in arteries and veins. In the umbilicus, EphB2 is a specific marker of arterial ECs, whereas ephrinB2 is additionally expressed in venous ECs, suggesting an arterial function of the veins. In venous malformations, the expression of EphB4 is not altered, but both ephrinB2 and EphB2 are ectopically expressed in venous ECs. This may reflect a nonphysiologic arterialization of malformed veins. Our study shows that the arterial markers ephrin B2 and EphB2 are expressed in a subset of veins, and it remains to be studied whether this is cause or consequence of an altered vascular identity.

Adult↗

PubMed2006

Distribution of EphB receptors and ephrin-B1 in the developing vertebrate spinal cord.

Contact-dependent interactions between EphB receptors and ephrin-B ligands mediate a variety of cell-cell communication events in the developing and mature central nervous system (CNS). These predominantly repulsive interactions occur at the interface between what are considered to be mutually exclusive EphB and ephrin-B expression domains. We previously used receptor and ligand affinity probes to show that ephrin-B ligands are expressed in the floor plate and within a dorsal region of the embryonic mouse spinal cord, while EphB receptors are present on decussated segments of commissural axons that navigate between these ephrin-B domains. Here we present the generation and characterization of two new monoclonal antibodies, mAb EfB1-3, which recognizes EphB1, EphB2, and EphB3, and mAb efrnB1, which is specific for ephrin-B1. We use these reagents and polyclonal antibodies specific for EphB1, EphB2, EphB3, or ephrin-B1 to describe the spatiotemporal expression patterns of EphB receptors and ephrin-B1 in the vertebrate spinal cord. Consistent with affinity probe binding, we show that EphB1, EphB2, and EphB3 are each preferentially expressed on decussated segments of commissural axons in vivo and in vitro, and that ephrin-B1 is expressed in a dorsal domain of the spinal cord that includes the roof plate. In contrast to affinity probe binding profiles, we show here that EphB1, EphB2, and EphB3 are present on the ventral commissure, and that EphB1 and EphB3 are expressed on axons that compose the dorsal funiculus. In addition, we unexpectedly find that mesenchymal cells, which surround the spinal cord and dorsal root ganglion, express ephrin-B1.

Animals↗

PubMed2005

Intramolecular regulatory switch in ZAP-70: analogy with receptor tyrosine kinases.

ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.

Amino Acid Sequence↗

PubMedDate not supplied

Crystal structure of an Eph receptor-ephrin complex.

The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are important in regulating cell-cell interactions as they initiate a unique bidirectional signal transduction cascade whereby information is communicated into both the Eph-expressing and the ephrin-expressing cells. Initially identified as regulators of axon pathfinding and neuronal cell migration, Ephs and ephrins are now known to have roles in many other cell-cell interactions, including those of vascular endothelial cells and specialized epithelia. Here we report the crystal structure of the complex formed between EphB2 and ephrin-B2, determined at 2.7 A resolution. Each Eph receptor binds an ephrin ligand through an expansive dimerization interface dominated by the insertion of an extended ephrin loop into a channel at the surface of the receptor. Two Eph-Ephrin dimers then join to form a tetramer, in which each ligand interacts with two receptors and each receptor interacts with two ligands. The Eph and ephrin molecules are precisely positioned and orientated in these complexes, promoting higher-order clustering and the initiation of bidirectional signalling.

Amino Acid Sequence↗

PubMed2003

Dihydrotestesterone induction of EPHB2 expression in the female genital tubercle mimics male pattern of expression during embryogenesis.

PURPOSE: The Eph family constitutes the largest class of membrane bound tyrosine kinase receptors in higher vertebrates, being involved in complex cell-to-cell recognition events via their membrane anchored ligands, the ephrins. To uncover evidence for possible molecular involvement in genitourinary development, we evaluated the pattern of EphB2 expression in male and female genital tubercles (GTs) after culture with and without dihydrotestosterone (DHT). MATERIALS AND METHODS: GTs of embryos from time pregnant CD1 mice heterozygous for the EphB2lacZ mutation were explanted at 12.5 to 14.5 days after conception. In these mutant mice the exons encoding the EphB2 tyrosine kinase and C-terminal domains were replaced with bacterial lacZ sequence encoding beta-galactosidase to demonstrate EphB2 expression. The GTs were microdissected, cultured for 24 to 48 hours with or without addition of 10 nM DHT and then stained with X-gal to visualize the beta-galactosidase reporter of EphB2 expression. RESULTS: LacZ staining was detected along the borders of the urethral plate (UP) in males. Females expressed EphB2 at the GT base but DHT exposure induced EphB2 expression along the UP. Under culture conditions, progressive approximation of the UP borders was noted in male fetuses and androgen exposed females. UP approximation was not seen in the nonexposed female tubercles. CONCLUSIONS: EphB2 expression appears to be temporally and spatially regulated in the developing mouse GT and differs between sexes. Male pattern of expression can be induced in the female GT by DHT exposure. Given DHT exposure alters EphB2 expression, EphB2 is a candidate androgen regulated gene. These findings may give insight into UP closure in normal and pathological conditions.

Animals↗

PubMed2002

Manipulation of EphB2 regulatory motifs and SH2 binding sites switches MAPK signaling and biological activity.

Signaling by the Eph family of receptor tyrosine kinases (RTKs) is complex, because they can interact with a variety of intracellular targets, and can potentially induce distinct responses in different cell types. In NG108 neuronal cells, activated EphB2 recruits p120RasGAP, in a fashion that is associated with down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway and neurite retraction. To pursue the role of the Ras-MAPK pathway in EphB2-mediated growth cone collapse, and to explore the biochemical and biological functions of Eph receptors, we sought to re-engineer the signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM) RasGAP binding sites but incorporated a Grb2 binding motif at an alternate RasGAP binding site within the kinase domain had little effect on basal Erk MAPK activation. In contrast, elimination of all RasGAP binding sites, accompanied by the addition of a Grb2 binding site within the kinase domain, led to an increase in phospho-Erk levels in NG108 cells following ephrin-B1 stimulation. Functional assays indicated a correlation between neurite retraction and the ability of the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data suggest that EphB2 can be designed to repress, stabilize, or activate the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2 domain binding sites and support the notion that Erk MAPK regulation plays a significant role in axon guidance. The behavior of EphB2 variants with mutations in the JM region and kinase domains suggests an intricate pattern of regulation and target recognition by Eph receptors.

Amino Acid Sequence↗

PubMed1999

Up-regulation of Eph tyrosine kinase receptors after excitotoxic injury in adult hippocampus.

The molecular mechanisms underlying the response to injury in the central nervous system are incompletely understood. Many cell activation systems may be involved. Tyrosine kinase receptors and their ligands play key roles in cell activation throughout life. The Eph family of tyrosine kinase receptors/ ligands are developmentally regulated and have been implicated in neural pathfinding. However, nothing is known about their role in the adult brain. We have used a model of central nervous system lesion in the rat, in which intraventricular injection of kainate was performed. This produced neuronal death in the CA3-CA4 fields and glial activation in the hippocampus. Highly degenerate primers, corresponding to the catalytic domain of the tyrosine kinase family, were used for reverse transcription-polymerase chain reaction of pooled RNA extracted from injured hippocampi. The amplified products were cloned and 100 clones (arbitrarily named TK1-TK100) were examined and inserts sequenced. We obtained four clones containing inserts which belong to the Eph receptor family. Two of these inserts (TK17 and TK63) were EphA4 and the other were EphB2 (TK25) and EphA5 (TK23). We performed in situ hybridization, and we found our clones to be present in all fields of the hippocampus, their expression being mainly neuronal. Three days after lesion, prominent expression appeared in CA1 as compared to the same field in the non-treated contralateral hippocampus. We performed northern blot analysis for quantification, and found that, three days after injury, the values decreased to 33 +/- 4%, 33 +/- 1% and 46 +/- 1% of control values for TK63 (EphA4), TK25 (EphB2) and TK23 (EphA5), respectively. Neuronal death in CA3-CA4 might account for this fact. Later, five days post-injury, the expression increased to 63 +/- 3%, 71 +/- 1% and 111 +/- 5% of control values, respectively. This increase was due to an up-regulation of these genes in the hippocampal neurons that survive after the injury, as indicated by in situ hybridization.

Amino Acid Sequence↗

PubMed2001

Eph receptors tingle the spine.

During the development of excitatory synapses, molecules cluster on dendrites to form postsynaptic densities, and specialized structures known as spines appear. EphB2 is demonstrated to control this process by associating with and phosphorylating a key postsynaptic molecule, syndecan-2, thereby initiating the maturation of dendritic spines.

Dendrites↗

PubMed1999

Multiple in vivo tyrosine phosphorylation sites in EphB receptors.

Autophosphorylation regulates the function of receptor tyrosine kinases. To dissect the mechanism by which Eph receptors transmit signals, we have developed an approach using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to map systematically their in vivo tyrosine phosphorylation sites. With this approach, phosphorylated peptides from receptors digested with various endoproteinases were selectively isolated on immobilized anti-phosphotyrosine antibodies and analyzed directly by MALDI mass spectrometry. Multiple in vivo tyrosine phosphorylation sites were identified in the juxtamembrane region, kinase domain, and carboxy-terminal tail of EphB2 and EphB5, and found to be remarkably conserved between these EphB receptors. A number of these sites were also identified as in vitro autophosphorylation sites of EphB5 by phosphopeptide mapping using two-dimensional chromatography. Only two in vitro tyrosine phosphorylation sites had previously been directly identified for Eph receptors. Our data further indicate that in vivo EphB2 and EphB5 are also extensively phosphorylated on serine and threonine residues. Because phosphorylation at each site can affect receptor signaling properties, the multiple phosphorylation sites identified here for the EphB receptors suggest a complex regulation of their functions, presumably achieved by autophosphorylation as well as phosphorylation by other kinases. In addition, we show that MALDI mass spectrometry can be used to determine the binding sites for Src homology 2 (SH2) domains by identifying the EphB2 phosphopeptides that bind to the SH2 domain of the Src kinase.

Amino Acid Sequence↗

PubMed2000

Eph receptors and ephrins in the developing chick cerebellum: relationship to sagittal patterning and granule cell migration.

Spatiotemporal expression patterns of six members of the Eph gene family (EphA4, EphA3, EphB2, ephrin-B1, ephrin-A2, and ephrin-A5) were characterized immunocytochemically at various stages of chick cerebellar development. EphA4 expression is observed in the cerebellar anlage as early as embryonic day 5 (E5) and continues in the posthatch cerebellum. During the early period of cerebellar development (E3-E8), complementarity is observed between EphA4 and ephrin-A5 expression within the cerebellar-isthmal region. By E8, differential expression of EphA4 in parasagittal Purkinje cell bands is evident, and the expression remains banded in the posthatch cerebellum. Banded expression of the ephrin-A5 ligand complements EphA4 expression during the middle period (E9-E15). During this period, ephrin-A2 and EphA3 are coexpressed in a banded pattern and with variable correlation to EphA4. Variability in the banding expression is observed for EphA4, EphA3, ephrin-A5, and ephrin-A2 across different lobes, and graded complementarity in the expression pattern of EphA3 and ephrin-A5 is observed in the external granular layer between the posterior and anterior lobes. Analysis of Purkinje cell birth date in correlation with Eph-ephrin expression during the middle period reveals that early-born cells express EphA4, whereas late-born cells express ephrin-A5. Finally, EphA4 expression domains are respected by migrating granule cell ribbons, which express both ephrin-B1 and EphB2. These expression patterns suggest multiple roles for the Eph-ephrin system in cerebellar development, including demarcation/enforcement of boundaries of the cerebellar anlage, formation/maintenance of Purkinje cell compartments, and restriction of the early phase of granule cell migration to ribbons.

Animals↗

PubMed2006

EphB2 and ephrin-B2 regulate the ionic homeostasis of vestibular endolymph.

The ability to transport cations and anions across epithelia is critical for the regulation of pH, ionic homeostasis, and volume of extracellular fluids. Although the transporters and channels that facilitate ion and water movement across cell membranes are well known, the molecular mechanisms and signal transduction events that regulate these activities remain poorly understood. The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are well known to transduce bidirectional signals that control axon guidance and other cell migration/adhesion events during development. However, these molecules are also expressed in non-motile epithelial cells, including EphB2 in K(+)-secreting vestibular dark cells and ephrin-B2 in the adjacent transitional cells of the inner ear. Consistent with these expression patterns, mice with cytoplasmic domain mutations that interfere with EphB2 forward signaling or ephrin-B2 reverse signaling exhibit a hyperactive circling (waltzing) locomotion associated with a decreased amount of endolymph fluid that normally fills the vestibular labyrinth. Endolymph is unusual as an extracellular fluid in that it is normally high in K(+) and low in Na(+). Direct measurement of this fluid in live animals revealed significant decreases in K(+) concentration and endolymphatic potential in both EphB2 and ephrin-B2 mutant mice. Our findings provide evidence that bidirectional signaling mediated by B-subclass Ephs and ephrins controls the production and ionic homeostasis of endolymph fluid and thereby provide the first evidence that these molecules can control the activities of mature epithelial cells.

Animals↗

PubMed1997

Graded and lamina-specific distributions of ligands of EphB receptor tyrosine kinases in the developing retinotectal system.

Molecular gradients have been postulated to control the topographic mapping of retinal axons in their central targets. Based initially on their expression patterns, and more recently on functional studies, members of the EphA subfamily of receptor tyrosine kinases and their ephrin-A ligands have been implicated in the guidance of retinal axons along the anterior-posterior axis of the chick optic tectum. The report that a receptor of the EphB subfamily, EphB2/Cek5/Nuk/Sek3, is expressed in a high ventral to low dorsal gradient in the developing chick retina and is present on ganglion cell axons suggests that it may be involved in the mapping of retinal axons along the corresponding dorsal-ventral axis of the tectum. To address this issue, we have determined the expression and distribution of ephrin-B1/LERK-2/Cek5-L and ephrin-B2/LERK-5/Htk-L/ELF-2, ligands for EphB2, in the developing chick retinotectal system using riboprobes, immunocytochemistry, and receptor affinity probes. Both ephrin-B1 and ephrin-B2 transcripts are expressed in a high dorsal to low ventral gradient in the developing retina, complementary to the distribution of EphB2. Ephrin-B1 and ephrin-B2 proteins are predominantly found in the developing plexiform layers, suggesting a role in the development of intraretinal connections. Neither protein is detected on ganglion cell axons. In tectum, ephrin-B1 transcripts are expressed in a high dorsal to low ventral gradient in the neuroepithelium and the protein is present along the processes of radial glia and is concentrated at their endfeet in the stratum opticum, at the time retinal axons are growing through it. This distribution of ephrin-B1 suggests that it influences retinal axon mapping along the dorsal-ventral tectal axis and may also be involved in intratectal development. In contrast, ephrin-B2 transcripts and protein are localized to the deeper retinorecipient laminae in the tectum at the time retinal axons begin to arborize in them, suggesting that this ligand may influence the laminar patterning of retinal axon terminations.

Animals↗