Reanalysis of EphA3 Knock-In Double Maps in Mouse Suggests That Stochasticity in Topographic Map Formation Acts at the Retina Rather than between Competing Mechanisms at the Colliculus.

It has been suggested that stochasticity acts in the formation of topographically ordered maps in the visual system through the opposing chemoaffinity and neural activity forces acting on the innervating nerve fibers being held in an unstable equilibrium. Evidence comes from the Islet2-EphA3 knock-in mouse, in which ∼50% of the retinal ganglion cells, distributed across the retina, acquire the EphA3 receptor, thus having an enhanced density of EphA which specifies retinotopic order along the rostrocaudal (RC) axis of the colliculus. Sampling EphA3 knock-in maps in heterozygotes at different positions along the mediolateral (ML) extent of the colliculus had found single 1D maps [as in wild types (WTs)], double maps (as in homozygous knock-ins) or both single and double maps. We constructed full 2D maps from the same mouse dataset. We found either single maps or maps where the visual field projects rostrally, with a part-projection more caudally to form a double map, the extent and location of this duplication varying considerably. Contrary to previous analyses, there was no strict demarcation between heterozygous and homozygous maps. These maps were replicated in a computational model where, as the level of EphA3 was increased, there was a smooth transition from single to double maps. Our results suggest that the diversity in these retinotopic maps has its origin in a variability over the retina in the effective amount of EphA3, such as through variability in gene expression or the proportion of EphA3+ retinal ganglion cells, rather than the result of competing mechanisms acting at the colliculus.

The Ephrin tyrosine kinase a3 (EphA3) is a novel mediator of RAGE-prompted motility of breast cancer cells.

BACKGROUND: The receptor for advanced glycation-end products (RAGE) and its ligands have been implicated in obesity and associated inflammatory processes as well as in metabolic alterations like diabetes. In addition, RAGE-mediated signaling has been reported to contribute to the metastatic progression of breast cancer (BC), although mechanistic insights are still required. Here, we provide novel findings regarding the transcriptomic landscape and the molecular events through which RAGE may prompt aggressive features in estrogen receptor (ER)-positive BC. METHODS: MCF7 and T47D BC cells stably overexpressing human RAGE were used as a model system to evaluate important changes like cell protrusions, migration, invasion and colony formation both in vitro through scanning electron microscopy, clonogenic, migration and invasion assays and in vivo through zebrafish xenografts experiments. The whole transcriptome of RAGE-overexpressing BC cells was screened by high-throughput RNA sequencing. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses allowed the prediction of potential functions of differentially expressed genes (DEGs). Flow cytometry, real time-PCR, chromatin immunoprecipitation, immunofluorescence and western blot assays were performed to investigate the molecular network involved in the regulation of a novel RAGE target gene namely EphA3. The clinical significance of EphA3 was explored in the TCGA cohort of patients through the survivALL package, whereas the pro-migratory role of EphA3 signaling was ascertained in both BC cells and cancer-associated fibroblasts (CAFs). Statistical analysis was performed by t-tests. RESULTS: RNA-seq findings and GSEA analysis revealed that RAGE overexpression leads to a motility-related gene signature in ER-positive BC cells. Accordingly, we found that RAGE-overexpressing BC cells exhibit long filopodia-like membrane protrusions as well as an enhanced dissemination potential, as determined by the diverse experimental assays. Mechanistically, we established for the first time that EphA3 signaling may act as a physical mediator of BC cells and CAFs motility through both homotypic and heterotypic interactions. CONCLUSIONS: Our data demonstrate that RAGE up-regulation leads to migratory ability in ER-positive BC cells. Noteworthy, our findings suggest that EphA3 may be considered as a novel RAGE target gene facilitating BC invasion and scattering from the primary tumor mass. Overall, the current results may provide useful insights for more comprehensive therapeutic approaches in BC, particularly in obese and diabetic patients that are characterized by high RAGE levels.

The Hippo effector YAP1/TEAD1 regulates EPHA3 expression to control cell contact and motility.

The EPHA3 protein tyrosine kinase, a member of the ephrin receptor family, regulates cell fate, cell motility, and cell-cell interaction. These cellular events are critical for tissue development, immunological responses, and the processes of tumorigenesis. Earlier studies revealed that signaling via the STK4-encoded MST1 serine-threonine protein kinase, a core component of the Hippo pathway, attenuated EPHA3 expression. Here, we investigated the mechanism by which MST1 regulates EPHA3. Our findings have revealed that the transcriptional regulators YAP1 and TEAD1 are crucial activators of EPHA3 transcription. Silencing YAP1 and TEAD1 suppressed the EPHA3 protein and mRNA levels. In addition, we identified putative TEAD enhancers in the distal EPHA3 promoter, where YAP1 and TEAD1 bind and promote EPHA3 expression. Furthermore, EPHA3 knockout by CRISPR/Cas9 technology reduced cell-cell interaction and cell motility. These findings demonstrate that EPHA3 is transcriptionally regulated by YAP1/TEAD1 of the Hippo pathway, suggesting that it is sensitive to cell contact-dependent interactions.

EPHA3 Contributes to Epigenetic Suppression of PTEN in Radioresistant Head and Neck Cancer.

EPHA3, a member of the EPH family, is overexpressed in various cancers. We demonstrated previously that EPHA3 is associated with radiation resistance in head and neck cancer via the PTEN/Akt/EMT pathway; the inhibition of EPHA3 significantly enhances the efficacy of radiotherapy in vitro and in vivo. In this study, we investigated the mechanisms of PTEN regulation through EPHA3-related signaling. Increased DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2) levels, along with increased histone H3 lysine 27 trimethylation (H3K27me3) levels, correlated with decreased levels of PTEN in radioresistant head and neck cancer cells. Furthermore, PTEN is regulated in two ways: DNMT1-mediated DNA methylation, and EZH2-mediated histone methylation through EPHA3/C-myc signaling. Our results suggest that EPHA3 could display a novel regulatory mechanism for the epigenetic regulation of PTEN in radioresistant head and neck cancer cells.

Critical role of EphA3 in cancer and current state of EphA3 drug therapeutics.

The erythropoietin-producing human hepatocellular (Eph) receptors are transmembrane glycoprotein members of the tyrosine kinase receptors family. The Ephs may bind to various ephrin ligands resulting in the phosphorylation of their tyrosine kinase domain and the activation of the Eph receptor. In this review we focus on EphA3, one receptor of the 14 different Ephs, as it carries out both redundant and restricted functions in the germline development of mammals and in the maintenance of various adult tissues. The loss of EphA3 regulation is correlated with various human malignancies, the most notable being cancer. This receptor is overexpressed and/or mutated in multiple tumors, and is also associated with poor prognosis and decreased survival in patients. Here we highlight the role of EphA3 in normal and malignant tissues that are specific to cancer; these include hematologic disorders, gastric cancer, glioblastoma multiforme, colorectal cancer, lung cancer, renal cell carcinoma, and prostate cancer. Moreover, various anticancer agents against EphA3 have been developed to either inhibit its kinase domain activity or to function as agonists. Thus, we examine the most potent small molecule drugs and mAb-based therapeutics against EphA3 that are currently in pre-clinical or clinical stages.

Sam68 Promotes the Progression of Human Breast Cancer through inducing Activation of EphA3.

BACKGROUND: Src associated with mitosis of 68 kDa (Sam68), is often highly expressed in human cancers. Overexpression of Sam68 has been shown to be correlated with poor survival prognosis in some cancer patients. However, little is known whether Sam68 plays a role in promoting metastasis in breast cancer. MATERIALS AND METHODS: The expression of Sam68 protein in breast cancer tissue was detected by immunohistochemistry. Trans-well assay, wound-healing, real-time PCR and Western blotting analysis were used to detect the effect of Sam68 on promoting EMT or metastasis of breast cancer. Next-generation RNA sequencing was used to analyze genes that may be regulated by Sam68. RESULTS: Sam68 plays a positive role in promoting breast cancer metastasis. Sam68 was found to be overexpressed in breast cancer along with lymph node metastasis. MMP-9 was also found to be overexpressed in breast cancer tissue and was correlated to the expression of Sam68 (P<0.01). Xenograft in NOD/SCID mice and in vitro experiments confirmed that the invasion and metastatic ability of breast cancer cells were regulated by Sam68. And EPHA3 could be up-regulated by Sam68 in breast cancer. CONCLUSION: High expression of Sam68 participates in breast cancer metastasis by up-regulating the EPHA3 gene.

Glucose homeostasis is regulated by pancreatic ß-cell cilia via endosomal EphA-processing.

Diabetes mellitus affects one in eleven adults worldwide. Most suffer from Type 2 Diabetes which features elevated blood glucose levels and an inability to adequately secrete or respond to insulin. Insulin producing ß-cells have primary cilia which are implicated in the regulation of glucose metabolism, insulin signaling and secretion. To better understand how ß-cell cilia affect glucose handling, we ablate cilia from mature ß-cells by deleting key cilia component Ift88. Here we report that glucose homeostasis and insulin secretion deteriorate over 12 weeks post-induction. Cilia/basal body components are required to suppress spontaneous auto-activation of EphA3 and hyper-phosphorylation of EphA receptors inhibits insulin secretion. In ß-cells, loss of cilia/basal body function leads to polarity defects and epithelial-to-mesenchymal transition. Defective insulin secretion from IFT88-depleted human islets and elevated pEPHA3 in islets from diabetic donors both point to a role for cilia/basal body proteins in human glucose homeostasis.

EPHA3 enhances macrophage autophagy and apoptosis by disrupting the mTOR signaling pathway in mice with endometriosis.

Background: Endometriosis is a chronic fibrotic disease characterized by agonizing pelvic pain and low fertility, mainly affecting middle-aged women. The aim of the present study is to assess the potential effects of erythropoietin-producing hepatocellular carcinoma A3 (EPHA3) on endometriosis, with emphasis on the autophagy and apoptosis of macrophages via inhibition of the mammalian target of rapamycin (mTOR) signaling pathway.Methods: The mouse models of endometriosis were established followed by culturing the macrophages and macrophage transfection via the EPHA3 vector, siRNA EPHA3, and RAPA (an inhibitor of the mTOR signaling pathway). The expression of EPHA3, related factors in the mTOR signaling pathway, macrophage autophagy (autophagy-related gene 3 (Atg3), light chain 3-I (LC3-I), light chain 3-II (LC3-II) and Beclin1) and apoptosis (B-cell lymphoma-2 (bcl-2), bax and fas) were all detected and documented, respectively. The changes of autophagic lysosomes and the apoptosis of macrophages in each group following transfection were also inspected and detected.Results: The results of the in silico analysis ascertained EPHA3 to be a candidate gene of endometriosis. After successful modeling, the uterine tissues of endometriosis mice presented with a low expression of EPHA3 and activated mTOR signaling pathway. Overexpression of EPHA3 inhibited the activation of the mTOR signaling pathway, down-regulated bcl-2 expression, up-regulated the expression of Atg3, LC3-II/LC3-I, Beclin1, bax and fas, and also promoted the autophagy and apoptosis of macrophages in endometriosis mice.Conclusion: Altogether, EPHA3 could potentially promote the autophagy and apoptosis of macrophages in endometriosis via inhibition of the mTOR signaling pathway, highlighting the potential of EPHA3 as the target to treat endometriosis.

Prognostic Significance of High EphA1-4 Expression Levels in Locally Advanced Gastric Cancer.

BACKGROUND/AIM: Erythropoietin-producing hepatocellular carcinoma receptor A (EphA) is associated with angiogenesis and invasive tumor progression. In this study, we evaluated the EphA1-4 expression levels in advanced gastric cancer. PATIENTS AND METHODS: Tumor tissues obtained from 114 patients with advanced gastric adenocarcinoma who underwent gastrectomy were analyzed. In addition, the impact of EPHA 1-4 mRNA expression on survival was analyzed using the Kaplan-Meier plotter database on the website. RESULTS: High EphA 1, 2, and 4 expression levels were significantly related to recurrence (p<0.01, p=0.04, and p<0.01). Both high EphA 1 and 4 expression levels were independent predictors of relapse-free interval (hazard ratio [HR]=2.0, p=0.03; HR=2.4, p=0.03) and disease-specific survival (HR=2.0, 95% p=0.03; HR=2.5, p=0.02) on multivariate analysis. In the Kaplan-Meier plotter database, high EPHA2 mRNA expression was significantly associated with poor survival in patients with gastric cancer (p=0.0098), and high expression levels of EPHA1 and 4 tended to be associated with poor survival (p=0.050, p=0.052). CONCLUSION: EphA 1, 2, and 4 may play key roles in recurrence and survival in patients with advanced gastric cancer.

Visual Neurons in the Superior Colliculus Innervated by Islet2+ or Islet2- Retinal Ganglion Cells Display Distinct Tuning Properties.

Throughout the visual system, different subtypes of neurons are tuned to distinct aspects of the visual scene, establishing parallel circuits. Defining the mechanisms by which such tuning arises has been a long-standing challenge for neuroscience. To investigate this, we have focused on the retina's projection to the superior colliculus (SC), where multiple visual neuron subtypes have been described. The SC receives inputs from a variety of retinal ganglion cell (RGC) subtypes; however, which RGCs drive the tuning of different SC neurons remains unclear. Here, we pursued a genetic approach that allowed us to determine the tuning properties of neurons innervated by molecularly defined subpopulations of RGCs. In homozygous Islet2-EphA3 knock-in (Isl2EA3/EA3) mice, Isl2+ and Isl2- RGCs project to non-overlapping sub-regions of the SC. Based on molecular and anatomic data, we show that significantly more Isl2- RGCs are direction-selective (DS) in comparison with Isl2+ RGCs. Targeted recordings of visual responses from each SC sub-region in Isl2EA3/EA3 mice revealed that Isl2- RGC-innervated neurons were significantly more DS than those innervated by Isl2+ RGCs. Axis-selective (AS) neurons were found in both sub-regions, though AS neurons innervated by Isl2+ RGCs were more tightly tuned. Despite this segregation, DS and AS neurons innervated by Isl2+ or Isl2- RGCs did not differ in their spatial summation or spatial frequency (SF) tuning. Further, we did not observe alterations in receptive field (RF) size or structure of SC neurons innervated by Isl2+ or Isl2- RGCs. Together, these data show that innervation by Isl2+ and Isl2- RGCs results in distinct tuning in the SC and set the stage for future studies investigating the mechanisms by which these circuits are built.

EphA3 targeting reduces in vitro adhesion and invasion and in vivo growth and angiogenesis of multiple myeloma cells.

PURPOSE: Multiple myeloma (MM) is a hematologic malignancy characterized by a clonal expansion of plasma cells (PCs) in the bone marrow (BM). Since MM has so far remained incurable, further insights into its pathogenesis and the concomitant identification of new therapeutic targets are urgently needed. The tyrosine kinase receptor EphA3 is known to be involved in various cellular processes including cell viability, cell movement and cell-cell interactions. Recently, EphA3 has emerged as a potential therapeutic target in several hematologic and solid tumors. Here, we aimed to uncover the role of EphA3 in MM. METHODS: EphA3 mRNA and protein expression in primary MM bone marrow plasma cells (BMPCs), in MM-derived cell lines and in healthy controls (HCs) was assessed using qRT-PCR, Western blotting and flow cytometry. The effects of siRNA-mediated EphA3 silencing and anti EphA3 antibody (EphA3mAb) treatment on MM PC trafficking and viability were evaluated using in vitro assays. The effects of EphA3mAb treatment were also assessed in two MM-derived mouse xenograft models. RESULTS: We found that EphA3 was overexpressed in primary MM BMPCs and MM-derived cell lines compared to HCs. We also found that siRNA-mediated EphA3 silencing and EphA3mAb treatment significantly inhibited the ability of MM PCs to adhere to fibronectin and stromal cells and to invade in vitro, without affecting cell proliferation and viability. Gene expression profiling showed that EphA3 silencing resulted in expression modulation of several molecules that regulate adhesion, migration and invasion processes. Importantly, we found that EphA3mAb treatment significantly inhibited in vivo tumor growth and angiogenesis in two MM-derived mouse xenograft models. CONCLUSIONS: Our findings suggest that EphA3 plays an important role in the pathogenesis of MM and provide support for the notion that its targeting may represent a novel therapeutic opportunity for MM.

The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis.

Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wild-type lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis.

Defective response inhibition and collicular noradrenaline enrichment in mice with duplicated retinotopic map in the superior colliculus.

The superior colliculus is a hub for multisensory integration necessary for visuo-spatial orientation, control of gaze movements and attention. The multiple functions of the superior colliculus have prompted hypotheses about its involvement in neuropsychiatric conditions, but to date, this topic has not been addressed experimentally. We describe experiments on genetically modified mice, the Isl2-EphA3 knock-in line, that show a well-characterized duplication of the retino-collicular and cortico-collicular axonal projections leading to hyperstimulation of the superior colliculus. To explore the functional impact of collicular hyperstimulation, we compared the performance of homozygous knock-in, heterozygous knock-in and wild-type mice in several behavioral tasks requiring collicular activity. The light/dark box test and Go/No-Go conditioning task revealed that homozygous mutant mice exhibit defective response inhibition, a form of impulsivity. This defect was specific to attention as other tests showed no differences in visually driven behavior, motivation, visuo-spatial learning and sensorimotor abilities among the different groups of mice. Monoamine quantification and gene expression profiling demonstrated a specific enrichment of noradrenaline only in the superficial layers of the superior colliculus of Isl2-EphA3 knock-in mice, where the retinotopy is duplicated, whereas transcript levels of receptors, transporters and metabolic enzymes of the monoaminergic pathway were not affected. We demonstrate that the defect in response inhibition is a consequence of noradrenaline imbalance in the superficial layers of the superior colliculus caused by retinotopic map duplication. Our results suggest that structural abnormalities in the superior colliculus can cause defective response inhibition, a key feature of attention-deficit disorders.

Hypoxia-controlled EphA3 marks a human endometrium-derived multipotent mesenchymal stromal cell that supports vascular growth.

Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF)-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs), but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.

The Ephrin receptor EphA4 restricts axonal sprouting and enhances branching in the injured mouse optic nerve.

The lack of axonal regeneration in the adult central nervous system is in part attributable to the presence of inhibitory molecules present in the environment of injured axons such as the myelin-associated proteins Nogo-A and MAG and the repulsive guidance molecules Ephrins, Netrins and Semaphorins. In the present study, we hypothesized that EphA4 and one of its potential binding partners EphrinA3 may participate in the inhibition of adult axon regeneration in the model of adult mouse optic nerve injury. Axonal regeneration was analysed in three dimensions after tissue clearing of EphA4 knockout (KO), EphrinA3 KO and wild-type (WT) optic nerves. By immunohistochemistry, EphA4 was highly expressed in Müller glia endfeet in the retina and in astrocytes in the retina and the optic nerve, while EphrinA3 was present in retinal ganglion cells and oligodendrocytes. Optic nerve crush did not cause expression changes. Significantly more axons grew in the crushed optic nerve of EphA4 KO mice than in WT or EphrinA3 KO animals. Single axon analysis revealed that EphA4 KO axons were less prone to form aberrant branching than axons in the other mouse groups. The expression of growth-associated proteins Sprr1a and Gap-43 did not vary between EphA4 KO and WT retinae. However, glial fibrillary acidic protein-expressing astrocytes were withdrawn from the perilesional area in EphA4 KO, suggesting that gliosis down-regulation may locally contribute to improve axonal growth at the injury site. In summary, our three-dimensional analysis of injured mouse optic nerves reveals beneficial effects of EphA4 ablation on the intensity and the pattern of optic nerve axon regeneration.

Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands.

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.

On the Importance of Countergradients for the Development of Retinotopy: Insights from a Generalised Gierer Model.

During the development of the topographic map from vertebrate retina to superior colliculus (SC), EphA receptors are expressed in a gradient along the nasotemporal retinal axis. Their ligands, ephrin-As, are expressed in a gradient along the rostrocaudal axis of the SC. Countergradients of ephrin-As in the retina and EphAs in the SC are also expressed. Disruption of any of these gradients leads to mapping errors. Gierer's (1981) model, which uses well-matched pairs of gradients and countergradients to establish the mapping, can account for the formation of wild type maps, but not the double maps found in EphA knock-in experiments. I show that these maps can be explained by models, such as Gierer's (1983), which have gradients and no countergradients, together with a powerful compensatory mechanism that helps to distribute connections evenly over the target region. However, this type of model cannot explain mapping errors found when the countergradients are knocked out partially. I examine the relative importance of countergradients as against compensatory mechanisms by generalising Gierer's (1983) model so that the strength of compensation is adjustable. Either matching gradients and countergradients alone or poorly matching gradients and countergradients together with a strong compensatory mechanism are sufficient to establish an ordered mapping. With a weaker compensatory mechanism, gradients without countergradients lead to a poorer map, but the addition of countergradients improves the mapping. This model produces the double maps in simulated EphA knock-in experiments and a map consistent with the Math5 knock-out phenotype. Simulations of a set of phenotypes from the literature substantiate the finding that countergradients and compensation can be traded off against each other to give similar maps. I conclude that a successful model of retinotopy should contain countergradients and some form of compensation mechanism, but not in the strong form put forward by Gierer.

EPHA3 as a novel therapeutic target in the hematological malignancies.

The Eph receptors are the largest family of tyrosine kinases and are of increasing interest in developmental therapeutics. Their unique method of interaction with their ligands, the ephrins, via bidirectional signaling, and their variable expression in different tissues are well documented. Ephs are upregulated in, and critical to, embryological processes, most notably development of the neurological system. They are central in many processes involving cell motility and adhesion. Recent findings on elevated expression of Eph receptors in human malignancies as well as in stem cell environments are of particular interest. With increasing focus on molecularly targeted anticancer therapies, exploration of the potential of Eph receptors as therapeutic targets in both solid and hematologic malignancies has begun. The most promising of the Eph receptors in this regard is EPHA3, which is overexpressed in many hematologic malignancies. Preclinical data support the value of pursuing this target for further development, and lead compounds are now entering the clinic.

EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

Copy number variations of EphA3 are associated with multiple types of hematologic malignancies.

BACKGROUND: EphA3 is a component of the Eph receptor family, the largest subgroup of the receptor tyrosine kinase (RTK) family. A recent array-based study implicated the presence of copy-number variations (CNVs) of EphA3 in the genomes of acute myelogenous leukemia. CNVs are present in the general population at varying degrees, and have been found to associate with various types of diseases including hematologic malignancies. However, most of the current studies focused on the genome-wide screening of CNVs, and the functional impact of such regions needs to be extensively investigated in large number of clinical samples. PATIENTS AND METHODS: In our study, we collected 617 bone marrow samples from multiple types of hematologic malignancies as well as healthy controls. DNA copy numbers and mRNA levels of EphA3 in these samples were examined. RESULTS: We found significant association between the CNVs of EphA3 and these hematologic malignancies including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), and myelodysplastic syndrome (MDS). We also observed a positive correlation between the relative mRNA level and gene dosage of EphA3. CONCLUSION: The CNVs of EphA3 were associated with multiple types of hematologic malignancies including ALL, AML, CLL, CML, MM, and MDS.