Ephrin Expression Research Articles

Phosphorylation of Ephrin-B1 Regulates Dissemination of Gastric Scirrhous Carcinoma

Interaction of the Eph family of receptor protein tyrosine kinase and its ligand ephrin family induces bidirectional signaling via cell-cell contacts. High expression of B-type ephrin is frequently found in various cancer cells, and their expression levels are associated with high invasion of tumors and poor prognosis. However, whether ephrin-B1 actually promotes invasion of cancer cells in vivo has not been shown. We investigated the involvement of ephrin-B1 in regulating the invasiveness of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Reduction of ephrin-B1 expression by short inter-fering RNA or overexpression of phosphorylation-defective mutant suppressed migration and invasion of scirrhous gastric cancer cells in vitro without affecting tumor cell proliferation and apoptosis. Blocking of tyrosine phosphorylation of ephrin-B1 attenuates not only dissemination of cancer cells injected intraperitoneally but also local invasion and dissemination of orthotopically implanted cancer cells in the gastric wall of nude mice. Furthermore, blocking of ephrin-B1 phosphorylation attenuated the activation of Rac1 GTPase in these invasive gastric cancer cells. Our results suggest that tyrosine phosphorylation of ephrin-B1 promotes invasion of cancer cells in vivo and is a potential therapeutic target in some types of gastrointestinal cancers.

The C-terminus of ephrin-B1 regulates metalloproteinase secretion and invasion of cancer cells

Interaction of the Eph family of receptor protein tyrosine kinases and their ligands, ephrin family members, induces bi-directional signaling via cell-cell contacts. High expression of B-type ephrin is associated with high invasion potential of tumors, however, the mechanism by which ephrin-B promotes cancer cell invasion is poorly understood. We show that interaction of ephrin-B1 with the Eph receptor B2 (EphB2) significantly enhances processing of the extracellular domain of ephrin-B1, which is regulated by the C-terminus. Matrix metalloproteinase-8 (MMP-8) is the key protease that cleaves ephrin-B1, and the C-terminus of ephrin-B1 regulates activation of the extracellular release of MMP-8 without requirement of de novo protein synthesis. One possible mechanism by which ephrin-B1 regulates the exocytosis of MMP-8 is the activation of Arf1 GTPase, a critical regulator of membrane trafficking. In support of this hypothesis, activation of ephrin-B1 increased GTP-bound Arf1, and the secretion of MMP-8 was reduced by expression of a dominant-negative mutant of Arf1. Expression of ephrin-B1 promoted the invasion of cancer cells in vivo, which required the C-terminus of ephrin-B1. Our results suggest a novel function of the C-terminus of ephrin-B1 in activating MMP-8 secretion, which promotes the invasion of cancer cells.

EphA5 and ephrin‐A2 expression during optic nerve regeneration: a ‘two‐edged sword’

During development, gradients of EphA receptors (nasal(low)-temporal(high)) and their ligands ephrin-As (rostral(low)-caudal(high)) are involved in establishing topography between retinal ganglion cells (RGCs) and the superior colliculus (SC). EphA5-expressing RGC axons are repulsed by ephrin-A2-expressing SC neurones. In adult rats RGCs maintain graded EphA5 expression but ephrin-A2 expression is down-regulated in the SC to a weak gradient. At 1 month after optic nerve transection, EphA5 expression is reduced in the few remaining RGCs and is no longer graded; by contrast, SC ephrin-A2 is up-regulated to a rostral(low)-caudal(high) gradient. Here we examined expression in adult rat 1 month after bridging the retina and SC with a peripheral nerve graft, a procedure that enhances RGC survival and permits RGC axon regeneration. Double labelling with cell markers revealed preservation of a nasal(low)-temporal(high) EphA5 gradient in RGCs and establishment of a rostral(low)-caudal(high) ephrin-A2 gradient within neurones of the SC. The results suggest a potential for guidance cues to restore the topography of RGC axons in the SC. However, high ephrin-A2 levels were also found in astrocytes surrounding the peripheral nerve graft insertion site. The repulsive ephrin-A2 environment offers at least a partial explanation for the observation that only a limited number of RGC axons can exit the graft to enter target central nervous system tissue.

Involvement of BIG-2 in convergence of olfactory axons to target glomeruli

Entorhinal and commissural/associational projections show a non-overlapping distribution in the hippocampus proper and the dentate gyrus. The expression of Ephrins and their Eph receptors in the developing hippocampus indicates that this family of axonal guidance molecules may modulate the formation of these connections. Here we focused on the role of the ephrin-A5 ligand in the development of the main hippocampal afferents. In situ hybridization showed that ephrin-A5 mRNA was detected mainly in the principal cells of the hippocampus proper and in the dentate gyrus throughout postnatal development. Immunocytochemical analyses revealed prominent expression of the EphA3 receptor, a putative receptor for ephrin-A5, in the main cells and the neuropil of the developing hippocampus. Tracing experiments in ephrin-A5(−/−) mice showed that commissural projections were transiently altered in the hippocampus proper at P5, but they were mistargeted throughout the postnatal development in the dentate gyrus.Immunocytochemistry with anti-calbindin antibodies revealed that the dentate mossy fiber projection was not altered in ephrin-A5(−/−) mice. Electron microscopy studies showed alterations in the density of synapses and spines in commissural/associational layers, but not in entorhinal layers, and in the mossy fibers in these animals. Taken together, these findings indicate that ephrin-A5 signaling is involved in the formation and maturation of synapses in the hippocampus.

Expression of Eph Receptor Tyrosine Kinases and their Ligands in Human Granulosa Lutein Cells and Human Umbilical Vein Endothelial Cells

Corpus luteum development is regulated by gonadotropins and accompanied by extremely rapid vascularization of the avascular granulosa cell compartiment by endothelial cells (EC). The proliferation of Granulosa cells (GC) and EC is a complex interplay and takes place in a spatially and temporarily coordinated manner. The erythropoietin-producing hepatoma amplified sequence (Eph) receptors and their ligands-the ephrins- are a recently detected family of membrane located protein tyrosine kinases which play a crucial role in the growth and development of nerve and blood vessel network. We report about the mRNA expression pattern of Ephs and their ligands in human GC, in human EC, and in carcinoma cell lines OvCar-3 and Hela. The mRNA of EphA4, EphA7, ephrinA4, ephrinB1 and ephrinB2 was detected in GC and EC, while EphA2 was expressed only in GC. The expression of various Ephs and ephrins did not change in GC after stimulation with human chorion gonadotropin. Our study analyzes for the first time the expression of the complete human Eph/ephriny-system in GC and in EC. The remarkable similarity between these two cell types supports the theory of a functional relationship of EC and GC. In addition, it was shown that hCG is not a major determinant of Eph/ephrin regulation in GC.

Pax7 and superior collicular polarity: insights from Pax6 (Sey) mutant mice

Pax genes are important modulators of CNS development. Pax7 and Pax6 polarise the neural tube and regionalise the brain. Pax7 is pivotal in specifying the superior colliculus/tectum, an important centre for integration of visuomotor responses and a target for Pax6+ retinal ganglion cell axons during retinocollicular mapping. Whilst initial Pax7-specification of the mesencephalon is well-established, a role in regulating polarity within the maturing mouse superior colliculus is yet to be defined, although already detailed for the chick tectum. We therefore quantified Pax7 cellular distribution and expression levels at three functionally distinct stages of superior collicular development, and analysed Pax7 expression in response to aberrant axonal input and altered forebrain/midbrain boundary placement in Pax6 mutant mice. Comparative expression profiles of ephrin-A2 and its co-localisation with Pax7 were determined in wildtype and Pax6 mutant mice. Results indicate that graded Pax7 expression in wildtype mice is perturbed in Pax6 mutant mice; changes manifest as a shift in polarity, loss of graded expression and dramatically reduced protein levels during RGC synaptogenesis. Ephrin-A2 expression is similarly altered. These results implicate Pax7 as an important determinant of polarity within the mouse superior colliculus, and suggest a role in retinotopic mapping.

Ephrin A5 expression promotes invasion and transformation of murine fibroblasts

Emerging evidence suggests involvement of the ephrin/Eph receptor system in tumourigenesis. Research on this new role has centred on the contribution of Eph receptors. In contrast, we focused on the elucidation of the role of ephrins, specifically ephrin A5. Results indicated an increase in invasive potential of ephrin A5-expressing murine fibroblasts, which was abolished by addition of a Src family kinase inhibitor. Furthermore, anchorage-independent growth was increased in ephrin A5-expressing cells. Stimulation with EphA5-Fc receptor increased colony size, but not colony number in ephrin A5 transfectants. Moreover, we observed morphogenetic transformation of ephrin A5-expressing 3T3 cells into a branching network when plated onto Matrigel. This behaviour was specific to ephrin A5 transfectants, as 3T3 cells expressing ephrin B1 displayed a phenotype similar to control 3T3 cells. We conclude that ectopic expression of ephrin A5 in murine fibroblasts elevates oncogenic potential, including increased invasive behaviour, anchorage-independent growth, and morphological transformation.

Expression of Ephrin in Retinal Neovascularization and Iris Rubeosis

We investigated expression of ephrin-B2 and Eph-B4 in the retinal tissues of six primate eyes with neovascularization and iris rubeosis secondary to laser-induced central retinal vein occlusion and in tissue from 10 human eyes with proliferative diabetic retinopathy. Two primate eyes with rubeosis and retinal neovascularization were enucleated 1, 2 and 4 weeks after the creation of central retinal vein occlusion. Antibodies were localized using the avidin-biotin reaction. In the primate eyes, ephrin-B2 was negative at I week and positive at 2 and 4 weeks in the rubeotic tissue, but was positive only at 2 weeks in the retinal neovascular membrane. Eph-B4 was negative in all the primate eye specimens. In the human tissue, ephrin-B2 was detected in two of the five eyes with rubeosis and three of the five eyes with retinal neovascularization. These data suggest that ephrin-B2 is a key regulator of neovascularization.

Graded ephrin-A2 expression in the developing hamster superior colliculus

During development, ephrin gradients guide retinal ganglion cell axons to their appropriate topographic locations in the superior colliculus (SC). Expression of ephrin-A2, assessed immunohistochemically in the developing hamster SC, revealed a rostral(low) to caudal (high) gradient that is most prominent at postnatal days P4 and P7 when topography is established. Double-labelling immunohistochemistry for ephrin-A2 and cell specific markers revealed that ephrin-A2 is expressed exclusively by a subset of neurons. The expression pattern has implications for mechanisms underlying establishment of topography during development and following injury.

Over-expression of Eph and ephrin genes in advanced ovarian cancer: ephrin gene expression correlates with shortened survival

BackgroundIncreased expression of Eph receptor tyrosine kinases and their ephrin ligands has been implicated in tumor progression in a number of malignancies. This report describes aberrant expression of these genes in ovarian cancer, the commonest cause of death amongst gynaecological malignancies.MethodsEph and ephrin expression was determined using quantitative real time RT-PCR. Correlation of gene expression was measured using Spearman's rho statistic. Survival was analysed using log-rank analysis and (was visualised by) Kaplan-Meier survival curves.ResultsGreater than 10 fold over-expression of EphA1 and a more modest over-expression of EphA2 were observed in partially overlapping subsets of tumors. Over-expression of EphA1 strongly correlated (r = 0.801; p < 0.01) with the high affinity ligand ephrin A1. A similar trend was observed between EphA2 and ephrin A1 (r = 0.387; p = 0.06). A striking correlation of both ephrin A1 and ephrin A5 expression with poor survival (r = -0.470; p = 0.02 and r = -0.562; p < 0.01) was observed. Intriguingly, there was no correlation between survival and other clinical parameters or Eph expression.ConclusionThese data imply that increased levels of ephrins A1 and A5 in the presence of high expression of Ephs A1 and A2 lead to a more aggressive tumor phenotype. The known functions of Eph/ephrin signalling in cell de-adhesion and movement may explain the observed correlation of ephrin expression with poor prognosis.

Conjunctival Cytokine Expression in Symptomatic Moderate Dry Eye Subjects

To compare ocular surface cytokine expression in healthy controls and subjects with moderate dry eye and to study the ability of interleukin (IL)-1beta to modulate cytokine expression in cultured human conjunctival epithelial cells (CECs). Subjective (symptom questionnaire) and objective (tear osmolality, fluorescein tear break-up time [TBUT]) measures of dry eye were determined in five healthy controls and five subjects with moderate dry eye. Tear clearance rates were measured with a fluorophotometer. Enzyme immunoassay and a cytokine bead assay were used to quantify IL-1beta in tear fluid. RT-PCR was performed to detect expression of IL-1beta, IL-6, IL-8, growth-related oncogene (GRO)-beta, intercellular adhesion molecule (ICAM)-1, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and ephrin A5 in conjunctival impression cytology (CIC) samples and in CECs (IOBA-NHC cell line, n=3; primary cultured CEC, n=3) exposed to 10 ng/mL IL-1beta for 6 hours. Subjects with moderate dry eye had significantly higher symptom scores, higher tear osmolality, and shorter TBUT than healthy controls. Subjects with dry eye demonstrated slightly slower tear clearance (13.1% per minute) than healthy controls (15.4% per minute). Very low levels of IL-1beta protein were detected in the tear fluid of both groups. TRAIL was constitutively expressed in CIC samples, whereas IL-1beta, IL-6, and GRO-beta were absent. Weak expression of IL-8 (two healthy, four dry eye), ICAM-1 (four healthy, four dry eye), and ephrin A5 (one healthy, two dry eye) was observed. IL-1beta upregulated its own expression and that of IL-6, IL-8, GRO-beta, and ICAM-1 in cultured CECs but not that of ephrin A5 or TRAIL. The lack of major differences in ocular surface cytokine expression between the two groups of subjects implies other inflammatory pathways or etiologies are involved in moderate dry eye. Although IL-1beta modulated the expression of various cytokines in cultured CECs, its absence in tear fluid and CIC samples suggests that IL-1beta does not play a modulatory role in moderate dry eye.

EphB4 controls blood vascular morphogenesis during postnatal angiogenesis

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin-1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non-neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non-neoplastic angiogenesis in general. This implies that both neoplastic and non-neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.

Expression of EphA2 and Ephrin A-1 in Carcinoma of the Urinary Bladder

The EphA2 receptor tyrosine kinase is believed to play a role in tumor growth and metastasis. The clinical significance of the expression of EphA2 was observed in breast, prostate, colon, skin, cervical, ovarian, and lung cancers. The purpose of this work was to determine the expression of EphA2 and its ligand, Ephrin A-1, and E-cadherin in carcinoma of the urinary bladder, and determine EphA2 as a new target for therapy in bladder cancer. EphA2 mRNA and protein expression was investigated by reverse transcription-PCR and Western blot, respectively, in bladder cancer cell lines. In addition, the expression of EphA2, Ephrin A-1, and E-cadherin in tissues from patients with different stages of urinary bladder cancer was determined by immunohistochemistry. Furthermore, the ability of Ephrin A-1 to inhibit growth of bladder cancer cells was also investigated using an adenoviral delivery system. Western blot analysis showed high EphA2 expression in TCCSUP, T24, and UMUC-3 cell lines. In tissues, the staining intensity of EphA2 was less in normal urothelium but increased greatly in advancing stages of urothelial carcinoma (P < 0.05). Similarly, the staining intensity of Ephrin A-1 was low in normal tissues and high in cancerous tissues, but it was similar across the various stages of urothelial carcinoma (T(a)-T(4)). E-cadherin immunoreactivity decreased in urothelial cancer. Association of EphA2 and Ephrin A-1 expression was found to be significant between T(a) stage and T(1)-T(2) (P < 0.04) and T(a) and T(3)-T(4) stages (P < 0.0001). Adenovirus delivery of Ephrin A-1 inhibited proliferation of TCCSUP cells. EphA2 may serve as a novel target for bladder cancer therapy.

Ephrin-A5 modulates the topographic mapping and connectivity of commissural axons in murine hippocampus

Entorhinal and commissural/associational projections show a non-overlapping distribution in the hippocampus proper and the dentate gyrus. The expression of Ephrins and their Eph receptors in the developing hippocampus indicates that this family of axonal guidance molecules may modulate the formation of these connections. Here we focused on the role of the ephrin-A5 ligand in the development of the main hippocampal afferents. In situ hybridization showed that ephrin-A5 mRNA was detected mainly in the principal cells of the hippocampus proper and in the dentate gyrus throughout postnatal development. Immunocytochemical analyses revealed prominent expression of the EphA3 receptor, a putative receptor for ephrin-A5, in the main cells and the neuropil of the developing hippocampus. Tracing experiments in ephrin-A5(−/−) mice showed that commissural projections were transiently altered in the hippocampus proper at P5, but they were mistargeted throughout the postnatal development in the dentate gyrus.Immunocytochemistry with anti-calbindin antibodies revealed that the dentate mossy fiber projection was not altered in ephrin-A5(−/−) mice. Electron microscopy studies showed alterations in the density of synapses and spines in commissural/associational layers, but not in entorhinal layers, and in the mossy fibers in these animals. Taken together, these findings indicate that ephrin-A5 signaling is involved in the formation and maturation of synapses in the hippocampus.

Dissociation of Corticothalamic and Thalamocortical Axon Targeting by an EphA7-Mediated Mechanism

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, we show that EphA7-mediated signaling on neocortical axons controls the within-nucleus topography of CT projections in the thalamus. Notably, CT axons that mis-express 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, mis-expression of cortical EphA7 results in disrupted topography of CT projections, but unchanged inter- and intra-areal topography of TC projections. Our 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.

Eph/ephrin expression in the adult rat visual system following localized retinal lesions: localized and transneuronal up‐regulation in the retina and superior colliculus

Following unilateral optic nerve section in adult PVG hooded rat, the axon guidance cue ephrin-A2 is up-regulated in caudal but not rostral superior colliculus (SC) and the EphA5 receptor is down-regulated in axotomised retinal ganglion cells (RGCs). Changes occur bilaterally despite the retino-collicular projection being mostly crossed. Here we investigate the dynamics of Eph/ephrin expression using in situ hybridization and semi-quantitative immunohistochemistry after localized retinal lesions. Unilateral krypton laser lesions to dorso-nasal retina ablated contralaterally projecting RGCs (DN group); ventro-temporal lesions ablated contralaterally and ipsilaterally projecting RGCs (VT group). Lesions of the entire retina served as controls (Total group). Results are compared to normal animals in which tectal ephrin-A2 and retinal EphA5 are expressed, respectively, as shallow ascending rostro-caudal and naso-temporal gradients. In both SCs of DN and Total groups, tectal ephrin-A2 was up-regulated caudally; in the VT group, expression remained normal bilaterally. Unilateral collicular ablation indicated that bilateral changes in ephrin-A2 expression are mediated via intercollicular pathways. EphA5 expression in the VT group was elevated in the intact nasal region of experimental retinae. For each experimental group, EphA5 expression was also elevated in nasal retina of the opposite eye, resulting in uniform expression across the naso-temporal axis. Up-regulation of ephrin-A2 in caudal, but not rostral, SC suggests the enhancement of developmental positional information as a result of injury. Bilateral increases in retinal EphA5 expression demonstrate that signals for up-regulation operate interocularly. The study demonstrates that signals regulating guidance cue expression are both localized and relayed transneuronally.

Hypoxia up‐regulates expression of Eph receptors and ephrins in mouse skin

Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.

Opposing Gradients of Ephrin-As and EphA7 in the Superior Colliculus Are Essential for Topographic Mapping in the Mammalian Visual System

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. Here we investigate the role of an EphA7 gradient in the SC, which has the reverse orientation to the ephrin-A gradient. We find that 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. Our 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.

Methylmercury Alters Eph and Ephrin Expression During Neuronal Differentiation of P19 Embryonal Carcinoma Cells

Developmental exposure to methylmercury (MeHg) induces a spectrum of neurological impairment characterized by cognitive disturbance, sensory/motor deficit, and diffuse structural abnormalities of the brain. These alterations may arise from neural path-finding errors during brain development, resulting from disturbances in the function of morphoregulatory guidance molecules. The Eph family of tyrosine kinase receptors and their ligands, the ephrins, guide neuronal migration and neurite pathfinding mainly via repulsive intercellular interactions. The present study examined the effects of MeHg on mRNA and protein expression profiles of Ephs and ephrins in the P19 embryonal carcinoma (EC) cell line and its neuronal derivatives. Undifferentiated control P19 cells displayed low- to undetectable levels of mRNA for ephrins or Ephs, with the sole exception of EphA2 which was highly expressed. Upon differentiation into neurons, the ephrin expression increased progressively through day 10. Similarly, expression of the Ephs, including EphsA3, -A4, -A8, -B2, -B3, -B4, and -B6, increased significantly. In contrast, EphA2 expression decreased in day 2, 6 and 10 control neurons. Treatment with MeHg did not affect the expression of mRNA for ephrins or Ephs in undifferentiated P19 cells. However, treatment of differentiating neurons with MeHg for 24 h caused consistent increases in ligand mRNA expression, particularly ephrin-A5, -A6, -B1, and -B2. Similarly, MeHg induced variable increases in mRNA expression of receptors EphA2, -A3, -B3, and -B6. A trend toward a concentration–response relationship was observed for the alterations in Eph receptor mRNA expression although increases at the low and mid concentrations did not reach statistical significance. Immunoblots for ligand and receptor proteins mirrored the increases in the mRNA levels at the 0.5 and 1.5 μM MeHg concentrations but showed decreased protein levels compared to controls at the 3.0 μM concentration. Alterations in the Eph/ephrin family of repulsion molecules may represent an important mechanism in developmental MeHg neurotoxicity.

Ephrin-A5 inhibits growth of embryonic sensory neurons

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