Ephrin Genes Research Articles

Spatial-temporal comparison of Eph/Ephrin gene expression in ocular lenses from aging and knockout mice.

Cataracts, defined as any opacity in the transparent ocular lens, remain the leading cause of blindness and visual impairment in the world; however, the etiology of this pathology is not fully understood. Studies in mice and humans have found that the EphA2 receptor and the ephrin-A5 ligand play important roles in maintaining lens homeostasis and transparency. However, due to the diversity of the family of Eph receptors and ephrin ligands and their promiscuous binding, identifying functional interacting partners remains a challenge. Previously, 12 of the 14 Ephs and 8 of 8 ephrins in mice were characterized to be expressed in the mouse lens. To further narrow down possible genes of interest in life-long lens homeostasis, we collected and separated the lens epithelium from the fiber cell mass and isolated RNA from each compartment in samples from young adult and middle-aged mice that were either wild-type, EphA2-/- (knockout), or ephrin-A5 -/- . Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was implemented to compare transcript levels of 33 Eph and ephrin gene variants in each tissue compartment. Our results show that, of the Eph and ephrin variants screened, 5 of 33 showed age-related changes, and 2 of 33 showed genotype-related changes in lens epithelium. In the isolated fibers, more dynamic gene expression changes were observed, in which 12 of 33 variants showed age-related changes, and 6 of 33 showed genotype-related changes. These data allow for a more informed decision in determining mechanistic leads in Eph-ephrin-mediated signaling in the lens.

Transcriptome and methylome of the supraoptic nucleus provides insights into the age-dependent loss of neuronal plasticity.

The age-dependent loss of neuronal plasticity is a well-known phenomenon that is poorly understood. The loss of this capacity for axonal regeneration is emphasized following traumatic brain injury, which is a major cause of disability and death among adults in the US. We have previously shown the intrinsic capacity of magnocellular neurons within the supraoptic nucleus to undergo axonal regeneration following unilateral axotomization in an age-dependent manner. The aim of this research was to determine the age-dependent molecular mechanisms that may underlie this phenomenon. As such, we characterized the transcriptome and DNA methylome of the supraoptic nucleus in uninjured 35-day old rats and 125-day old rats. Our data indicates the downregulation of a large number of axonogenesis related transcripts in 125-day old rats compared to 35-day old rats. Specifically, several semaphorin and ephrin genes were downregulated, as well as growth factors including FGF's, insulin-like growth factors (IGFs), and brain-derived neurotrophic factor (BDNF). Differential methylation analysis indicates enrichment of biological processes involved in axonogenesis and axon guidance. Conversely, we observed a robust and specific upregulation of MHCI related transcripts. This may involve the activator protein 1 (AP-1) transcription factor complex as motif analysis of differentially methylated regions indicate enrichment of AP-1 binding sites in hypomethylated regions. Together, our data suggests a loss of pro-regenerative capabilities with age which would prevent axonal growth and appropriate innervation following injury.

A comprehensive prognostic and immunological analysis of ephrin family genes in hepatocellular carcinoma.

Background: Ephrins, a series of Eph-associated receptor tyrosine kinase ligands, play an important role in the tumorigenesis and progression of various cancers. However, their contributions to hepatocellular carcinoma (HCC) remain unclear. Thus, we aimed to explore their prognostic value and immune implications in HCC. Methods: Multiple public databases, such as TCGA, GTEx, and UCSC XENA, were used to analyze the expression of ephrin genes across cancers. Kaplan-Meier analysis and Cox regression were used to explore the prognostic role of ephrin genes in HCC. A logistic regression model was utilized to evaluate the association between ephrin gene expression and clinical characteristics. Gene set enrichment analysis (GSEA) was conducted to elucidate their potential biological mechanisms. Various immune algorithms were utilized to investigate the correlation between ephrin genes and tumor immunity. We also analyzed their association with drug sensitivity, and gene mutations. Finally, RT–qPCR was performed to validate the expression of ephrin family genes in HCC cells and clinical tissues. Results: The expression of EFNA1, EFNA2, EFNA3, EFNA4, EFNB1, and EFNB2 was upregulated in most cancer types, while EFNA5 and EFNB3 was downregulated in most cancers. In HCC, the expression levels of EFNA1, EFNA3, EFNA4, EFNB1, and EFNB2 were significantly higher in tumor tissues than in normal tissues. High expression of EFNA3, EFNA4, and EFNB1 was associated with tumor progression and worse prognosis in HCC patients. The expression of EFNA3 and EFNA4 was negatively associated with the stromal/ESTIMATE scores, while EFNB1 was positively correlated with the immune/stromal/ESTIMATE scores. Moreover, these ephrin genes were closely relevant to the infiltration of immune cells, such as B cells, CD4+ T cells, CD8+ T cells, neutrophil cells, macrophage cells, and dendritic cells. EFNB1 expression was positively associated with most immune-related genes, while EFNA3/EFNA4 was positively related to TMB and MSI. In addition, EFNA3, EFNA4, and EFNB1 were related to drug sensitivity and affected the mutation frequency of some genes in HCC. Conclusion: EFNA3, EFNA4, and EFNB1 are independent prognostic factors for HCC patients and are closely correlated with tumor immunity, which may provide a new direction for exploring novel therapeutic targets and biomarkers for immunotherapy.

Aggressive and recurrent ovarian cancers upregulate ephrinA5, a non-canonical effector of EphA2 signaling duality

Erythropoietin producing hepatocellular (Eph) receptors and their membrane-bound ligands ephrins are variably expressed in epithelial cancers, with context-dependent implications to both tumor-promoting and -suppressive processes in ways that remain incompletely understood. Using ovarian cancer tissue microarrays and longitudinally collected patient cells, we show here that ephrinA5/EFNA5 is specifically overexpressed in the most aggressive high-grade serous carcinoma (HGSC) subtype, and increased in the HGSC cells upon disease progression. Among all the eight ephrin genes, high EFNA5 expression was most strongly associated with poor overall survival in HGSC patients from multiple independent datasets. In contrast, high EFNA3 predicted improved overall and progression-free survival in The Cancer Genome Atlas HGSC dataset, as expected for a canonical inducer of tumor-suppressive Eph receptor tyrosine kinase signaling. While depletion of either EFNA5 or the more extensively studied, canonically acting EFNA1 in HGSC cells increased the oncogenic EphA2-S897 phosphorylation, EFNA5 depletion left unaltered, or even increased the ligand-dependent EphA2-Y588 phosphorylation. Moreover, treatment with recombinant ephrinA5 led to limited EphA2 tyrosine phosphorylation, internalization and degradation compared to ephrinA1. Altogether, our results suggest a unique function for ephrinA5 in Eph-ephrin signaling and highlight the clinical potential of ephrinA5 as a cell surface biomarker in the most aggressive HGSCs.

Changes in ephrin gene expression during bone healing identify a restricted repertoire of ephrinsmediating fracture repair.

To identify the repertoire of ephrin genes that might regulate endochondral bone fracture repair, we examined changes in ephrin ligand and receptor (Eph) gene expression in fracture callus tissues during bone fracture healing. Ephrin and Eph proteins were then localized in the fracture callus tissues present when changes in gene expression were observed. Ephrin gene expression was widespread in fracture tissues, but the repertoire of ephrin genes with significant changes in expression that might suggest a regulatory role in fracture callus development was restricted to the ephrin A family members Epha4, Epha5 and the ephrin B family member Efnb1. After 3 weeks of healing, Epha4 fracture expression was downregulated from 1.3- to 0.8-fold and Epha5 fracture expression was upregulated from 1.2- to 1.5-fold of intact contralateral femur expression, respectively. Efnb1 expression was downregulated from 1.5- to 1.2-fold after 2 weeks post-fracture. These ephrin proteins were localized to fracture callus prehypertrophic chondrocytes and osteoblasts, as well as to the periosteum and fibrous tissues. The observed positive correlation between mRNA levels of EfnB1 with Col10 and Epha5 with Bglap, together with colocalized expression with their respective proteins, suggest that EfnB1 is a positive mediator of prehypertrophic chondrocyte development and that Epha5 contributes to osteoblast-mediated mineralization of fracture callus. In contrast, mRNA levels of Epha4 and Efnb1 correlated negatively with Bglap, thus suggesting a negative role for these two ephrin family members in mature osteoblast functions. Given the number of family members and widespread expression of the ephrins, a characterization of changes in ephrin gene expression provides a basis for identifying ephrin family members that might regulate the molecular pathways of bone fracture repair. This approach suggests that a highly restricted repertoire of ephrins, EfnB1 and EphA5, are the major mediators of fracture callus cartilage hypertrophy and ossification, respectively, and proposes candidates for additional functional study and eventual therapeutic application.

Oxycodone Self-Administration Induces Alterations in Expression of Integrin, Semaphorin and Ephrin Genes in the Mouse Striatum.

Oxycodone is one a commonly used medication for pain, and is also a widely abused prescription opioid, like other short-acting MOPr agonists. Neurochemical and structural adaptations in brain following chronic MOPr-agonist administration are thought to underlie pathogenesis and persistence of opiate addiction. Many axon guidance molecules, such as integrins, semaphorins, and ephrins may contribute to oxycodone-induced neuroadaptations through alterations in axon-target connections and synaptogenesis, that may be implicated in the behaviors associated with opiate addiction. However, little is known about this important area. The aim of this study is to investigate alterations in expression of selected integrin, semaphorin, ephrins, netrin, and slit genes in the nucleus accumbens (NAc) and caudate putamen (CPu) of mice following extended 14-day oxycodone self-administration (SA), using RNAseq.Methods: Total RNA from the NAc and CPu were isolated from adult male C57BL/6J mice within 1 h after the last session of oxycodone in a 14-day self-administration paradigm (4h/day, 0.25 mg/kg/infusion, FR1) or from yoked saline controls. Gene expressions were examined using RNA sequencing (RNA-Seq) technology. RNA-Seq libraries were prepared using Illumina's TruSeq® Stranded Total RNA LT kit. The reads were aligned to the mouse reference genome (version mm10) using STAR. DESeq2 was applied to the counts of protein coding genes to estimate the fold change between the treatment groups. False Discovery Rate (FDR) q < 0.1 were used to select genes that have a significant expression change. For selection of a subset of genes related to axon guidance pathway, REACTOME was used.Results: Among 38 known genes of the integrin, semaphorin, and ephrin gene families, RNA-seq data revealed up-regulation of six genes in the NAc: heterodimer receptor, integrins Itgal, Itgb2, and Itgam, and its ligand semaphorin Sema7a, two semaphorin receptors, plexins Plxnd1 and Plxdc1. There was down-regulation of eight genes in this region: two integrin genes Itga3 and Itgb8, semaphorins Sema3c, Sema4g, Sema6a, Sema6d, semaphorin receptor neuropilin Nrp2, and ephrin receptor Epha3. In the CPu, there were five differentially expressed axon guidance genes: up-regulation of three integrin genes, Itgal, Itgb2, Itga1, and down-regulation of Itga9 and ephrin Efna3 were thus observed. No significant alterations in expression of Netrin-1 or Slit were observed.Conclusion: We provide evidence for alterations in the expression of selective axon guidance genes in adult mouse brain following chronic self-administration of oxycodone. Further examination of oxycodone-induced changes in the expression of these specific axon guidance molecules and integrin genes in relation to behavior may provide new insights into development of addiction to oxycodone.

Abstract 127: Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia

Abstract Guidance molecules from the Netrin, Slit, Ephrin, and Semaphorin gene families were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to have critical roles in tumor invasion. The SLIT2/mir-218-1 signaling axis has properties of a tumor suppressor pathway via the inhibition of directional migration and is epigenetically silenced in many cancers. We propose that mir-218-1 expression and function is independent of SLIT2 signaling in pancreatic ductal adenocarcinoma and inhibits tumor dissemination following the conversion of precursor lesions to invasive carcinoma. We determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 mRNA while KRAS-independent lines show silenced SLIT2 expression. Using chromatin immunoprecipitation (ChIP) and Sequenom, we confirmed that loss of SLIT2 in KRAS-independent lines was due to enrichment of silencing histone mark H3K27me3 and DNA hypermethylation at the SLIT2 promoter, respectively. Activating marks H3K4me2 and H4ac are found at the SLIT2 promoter in KRAS-dependent lines suggesting that SLIT2 is lost during pancreatic cancer progression due to epigenetic silencing. mir-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. Many intronic microRNAs are transcribed from the host gene promoter; however, mir-218-1 expression in pancreatic cancer cell lines does not correlate with SLIT2 gene expression. Therefore, we examined whether mir-218-1 had an alternative promoter independent of the SLIT2 promoter. Using ChIP for H3K4me3, a marker for promoter regions, and H4ac, a marker of permissive chromatin, we discovered a putative alternative promoter of mir-218-1 in all pancreatic cancer cell lines regardless of KRAS dependence. The putative alternative mir-218-1 promoter was also found to have basal transcription via enhanced luciferase reporter activity. This suggests that mir-218-1 expression is important and may contain its own tumor suppressor properties independent of SLIT2 signaling. Since pancreatic cancer is an invasive disease we examined the role of miR-218 target genes in specialized cellular structures, termed invadopodia. These structures have been found to facilitate the invasive phenotype of many cancer types and are dynamic actin-rich cellular projections that localize proteases to degrade extracellular matrix. Transfection of a miR-218 antagomir enhanced invasion in a modified Boyden chamber while addition of mir-218 reduced cell invasion. Our results further demonstrate that mir-218 functions in the suppression of invadopodia maturation and metalloproteinase matrix degradation. Overall, our data establishes that the SLIT2/mir-218-1 signaling axis is an important mediator of pancreatic cancer invasion through the extracellular matrix. Citation Format: Brenna A. Rheinheimer, Lukas Vrba, Bernard W. Futscher, Ronald L. Heimark. Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 127. doi:10.1158/1538-7445.AM2014-127

Abstract 4034: An integrated analysis of the eph/ephrin family: implications in endometrial cancer.

Abstract Objective: Members of the Eph/Ephrin family are known to play seminal roles in cancer growth and metastasis in many solid tumors; however, a comprehensive analysis has not been carried out. We analyzed the clinical relevance of all Eph/Ephrin family members in endometrial cancer. Methods: Level 3 data from The Cancer Genome Atlas (TCGA) was utilized to assess the clinical significance of all 14 Eph receptors available in the database (EphA1-8, EphA10, EphB1-4, and EphB6), as well as EphrinA1-A5 and EphrinB1-B3. We examined gene methylation status and copy number changes in these tumors. Clinical information extracted included age, tumor histology, tumor grade, stage, estrogen(ER)/progesterone(PR) receptor status, and overall survival. Results: A total of 320 samples were available for analysis. Increased EphA2, EphA4, EphB1, and EphB2 expression was significantly correlated with poorer overall survival (p=0.03, p=0.04, p=0.01, and p=0.008, respectively) and lack of ER and PR expression (r=-0.34, p=2.25e−6; r-0.24, p=0.0007; r=-0.22, p=0.002; r=-0.46, p=2.38e−11, respectively). Compared to endometrioid histology, serous histology was associated with increased expression of EphA2 (p&amp;lt;0.0001), EphA4 (p&amp;lt;0.0002), EphB1 (p&amp;lt;0.0001), EphB2 (p&amp;lt;0.0001), and EphrinA3 (p=0.04). Grade 3 tumor samples had increased expression of EphA2 (p&amp;lt;0.03), EphA4 (p&amp;lt;0.006), EphB1 (p&amp;lt;0.0001), EphB2 (p&amp;lt;0.0001), and EphrinA3 (p=0.04) compared to lower grade samples. EphA2, EphB1, and EphB2 expression was higher in samples from patients with advanced stage (FIGO Stage IIIA-IVB) compared to those with early stage disease (p=0.008, p=0.0183, and p&amp;lt;0.001, respectively). Patients age &amp;gt; 63 years had greater tumoral expression of EphA4 (p&amp;lt;0.0001), EphB2 (p=0.001), and EphrinA3 (p=0.03). Increased copy number correlated with EphB2 (r=0.41, p=0.001) and EphrinA3 (r=0.23, p=0.02) expression, but was not correlated with other Eph or Ephrin gene expression. Gene methylation inversely correlated with EphA2, EphA4, EphB1, EphB2, EphrinA3, and EphrinA5 receptor expression (r&amp;lt;-0.22, p&amp;lt;0.0001; r&amp;lt;-0.26, p&amp;lt;0.0002; r&amp;lt;-0.23, p&amp;lt;0.0006; r&amp;lt;-0.21, p&amp;lt;0.003; r&amp;lt;-0.2, p&amp;lt;0.01; r&amp;lt;-0.2, p&amp;lt;0.01, respectively). In patients with advanced stage expression of EphrinA3 and EphrinA5 was associated with poorer OS (p=0.002 and p=0.012). In a multivariate analysis, stage and Eph expression were the strongest independent predictors of poor survival, of which, EphA4 was the most significant (p=0.0024). In further support of the significance of EphA4, increased expression of its ligands EphrinA3 and EprhinA5 correlated with poorer OS. Conclusions: Concordant with emerging roles for the Eph receptor and Eprhins in driving malignant biology, increased EphA2, EphA4, EphB1, and EphB2 expression, as well as EphrinA3 and EphrinA5 expression, is predictive of poor patient outcome and constitutes attractive targets for biological therapies. Citation Format: Heather J. Dalton, Cristina Ivan, Chad V. Pecot, Rajesha Rupamaiole, Behrouz Zand, Justin Bottsford-Miller, Wei Hu, Alpa M. Nick, Robert L. Coleman, Anil K. Sood. An integrated analysis of the eph/ephrin family: implications in endometrial cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4034. doi:10.1158/1538-7445.AM2013-4034

Expression of Ephrin Receptors and Ligands in Postmortem Brains of HIV-Infected Subjects With and Without Cognitive Impairment

Despite the successes of combination antiretroviral therapy, HIV-associated neurocognitive disorders persist in many infected individuals. Earlier studies showed that neurocognitive impairment was associated with glutamate toxicity and synaptodendritic damage. We examined alterations in expression of four ephrin genes that are involved in synapse formation and recruitment of glutamate receptors to synapses, in the caudate and anterior cingulate in postmortem brain of cognitively characterized HIV-infected subjects, along with expression of neuronal and astroglial/macroglial markers. Postmortem tissues of HIV-infected and control subjects were obtained from the Manhattan HIV Brain Bank. HIV-infected subjects underwent neurocognitive assessment prior to death. Quantification of mRNA of genes of chemokine receptors and chemokines (CCR5, CXCR4, CCL2), astroglial/microglial markers (GFAP, CD163, CD68), the neuronal marker SNAP25, ephrin receptors EPHA4 and EPHB2, and ephrin ligands EFNB1 and EFNB2 was performed using SYBR Green RT-PCR. Proinflammatory chemokine and glial/macrophage mRNA levels in both regions were significantly greater in HIV+ than in HIV- subjects. Levels of EPHA4 and EFNB2 mRNA in the caudate, and EPHB2 mRNA in anterior cingulate were significantly lower in HIV+ subjects (p < 0.002, p < 0.02, p < 0.05, respectively). These transcripts also showed correlations with immune status and cognitive function within the HIV-infected group. Decreased levels of EFNB2 mRNA in the caudate correlated with lower CD4 counts (P < 0.05). Cognitive associations were limited to the cingulate, where decreased levels of EPHB2 mRNA were associated with better global cognitive status. Decreased cingulate expression of EPHB2 may represent a compensatory mechanism minimizing excitotoxic injury in the face of chronic inflammation.

Global Evaluation of Eph Receptors and Ephrins in Lung Adenocarcinomas Identifies EphA4 as an Inhibitor of Cell Migration and Invasion

The Eph family of receptors is the largest family of receptor tyrosine kinases, but it remains poorly studied in lung cancer. We aimed to systematically explore the human Eph receptors and their ligands, the ephrins, in lung adenocarcinoma. The prognostic impact of Eph receptor and ephrin gene expression was analyzed using 2 independent cohorts of lung adenocarcinoma. Gene expression profiles in lung adenocarcinoma compared with normal adjacent lung were studied in 3 independent cohorts and in cell lines. Gene expression profiles were validated with quantitative polymerase chain reaction (qPCR) and Western blotting in cell lines. Functional studies to assess the role of Eph receptor A4 (EphA4) were carried out in vitro. The biological effects of EphA4 in lung cancer cell lines were assayed following overexpression and knockdown. Of the 11 Eph receptors and 8 ephrins analyzed, only EphA4 and ephrin A1 gene expression were consistently associated with an improved outcome in patients with lung adenocarcinoma. Expression levels of EphA4 by microarray correlated well with expression levels measured by qPCR and Western blotting. EphA4 overexpression reduced cell migration and invasion but did not affect cell cycle, apoptosis, or drug sensitivity. Surprisingly, EphA4 was expressed at higher levels in cancer compared with non-cancer tissues and cell lines. EphA4 gene expression is associated with an improved outcome in patients with resected lung adenocarcinoma, possibly by affecting cancer cell migration and invasion.

Overexpression of the B-type Eph and ephrin genes correlates with progression and pain in human pancreatic cancer.

B-type erythropoietin-producing hepatocellular (EphB) receptors and their ephrin ligands are associated with aggressive behavior and poor prognosis in a number of malignancies. The aim of this study was to determine the association between the aberrant expression of these genes and prognosis in human pancreatic cancer. The expression of EphB and the ephrin ligands was determined using real-time quantitative reverse transcription PCR in 46 human primary pancreatic cancers. Overexpression of EphB2 and a more modest overexpression of ephrin-B2 mRNA were observed in more than 44% (20/46) of the pancreatic cancer specimens examined. Overexpression (>upper quartile) of EphB2 and ephrin-B2 was markedly associated with abdominal and/or back pain. Multivariate analysis identified the overexpression of EphB2 as an independent prognostic factor for disease-free and overall survival (both P<0.05). The results of the present study suggest that an increased level of ephrin-B2, in the presence of a high expression of EphB2, leads to a more aggressive tumor phenotype and that EphB2 may be used as a prognostic factor in human pancreatic cancer.

Complex expression patterns of Eph receptor tyrosine kinases and their ephrin ligands in colorectal carcinogenesis

Aberrant expression of Eph and ephrin proteins in human cancers is extensively documented. However, data are frequently limited to one gene and therefore incomplete and in some instances conflicting. We analysed expression of all Eph and ephrin genes in colorectal cancer (CRC) cell lines and 153 clinical specimens, providing for the first time a comprehensive analysis of this system in CRC. Eph/ephrin mRNA expression was assessed by quantitative real-time PCR and correlated with protein expression (flow cytometry, Western blotting and immunocytochemistry). These data show that EphA1, EphA2, EphB2 and EphB4 were significantly over expressed in CRC. In all cases, at least one Eph gene was found in normal colon (EphA1, EphA2, EphB2, EphB4), where expression was observed at high levels in most CRCs. However, other Eph gene expression was lost in individual CRCs compared to the corresponding normal, EphA7 being a striking example. Loss of expression was more common in advanced disease and thus correlated with poor survival. This is consistent with the redundant functionality of Eph receptors, such that expression of a single Eph gene is sufficient for effector function. Overall, the data suggest a progressive loss of expression of individual Eph genes suggesting that individual CRCs need to be phenotyped to determine which Eph genes are highly expressed. Targeted therapies could then be selected from a group of specific antibodies, such as those developed for EphA1.

Meis1 specifies positional information in the retina and tectum to organize the zebrafish visual system

BackgroundDuring visual system development, multiple signalling pathways cooperate to specify axial polarity within the retina and optic tectum. This information is required for the topographic mapping of retinal ganglion cell axons on the tectum. Meis1 is a TALE-class homeodomain transcription factor known to specify anterior-posterior identity in the hindbrain, but its role in visual system patterning has not been investigated.Resultsmeis1 is expressed in both the presumptive retina and tectum. An analysis of retinal patterning reveals that Meis1 is required to correctly specify both dorsal-ventral and nasal-temporal identity in the zebrafish retina. Meis1-knockdown results in a loss of smad1 expression and an upregulation in follistatin expression, thereby causing lower levels of Bmp signalling and a partial ventralization of the retina. Additionally, Meis1-deficient embryos exhibit ectopic Fgf signalling in the developing retina and a corresponding loss of temporal identity. Meis1 also positively regulates ephrin gene expression in the tectum. Consistent with these patterning phenotypes, a knockdown of Meis1 ultimately results in retinotectal mapping defects.ConclusionsIn this work we describe a novel role for Meis1 in regulating Bmp signalling and in specifying temporal identity in the retina. By patterning both the retina and tectum, Meis1 plays an important role in establishing the retinotectal map and organizing the visual system.

Aberrant DNA methylation and epigenetic inactivation of Eph receptor tyrosine kinases and ephrin ligands in acute lymphoblastic leukemia

Eph receptors and their ephrin ligands are involved in normal hematopoietic development and tumorigenesis. Using methylated CpG island amplification/DNA promoter microarray, we identified several EPH receptor and EPHRIN genes as potential hypermethylation targets in acute lymphoblastic leukemia (ALL). We subsequently studied the DNA methylation status of the Eph/ephrin family by bisulfite pyrosequencing. Hypermethylation of EPHA2, -A4, -A5, -A6, -A7, -A10, EPHB1, -B2, -B3, -B4, EFNA1, -A3, -A5, and EFNB1 and -B2 genes was detected in leukemia cell lines and primary ALL bone marrow samples. Expression analysis of EPHB4, EFNB2, and EFNA5 genes demonstrated that DNA methylation was associated with gene silencing. We cloned the promoter region of EPHB4 and demonstrated that promoter hypermethylation can result in EPHB4 transcriptional silencing. Restoration of EPHB4 expression by lentiviral transduction resulted in reduced proliferation and apoptotic cell death in Raji cells in which EPHB4 is methylated and silenced. Finally, we demonstrated that phosphorylated Akt is down-regulated in Raji cells transduced with EPHB4. These results suggest that epigenetic silencing by hypermethylation of EPH/EPHRIN family genes contributes to ALL pathogenesis and that EPHB4 can function as a tumor suppressor in ALL.

The role of Eph receptors and ephrin ligands in colorectal cancer

Eph receptors and their ephrin ligands constitute the largest subfamily of receptor tyrosine kinases and are components of the cell signaling pathways involved during development. Eph and ephrin overexpression have been documented in a variety of human cancers including gastrointestinal malignancies and in particular colorectal malignancies. EphB and ephrin B proteins have been implicated in the homeostasis of the gastrointestinal tract where EphB2- and EphB3-ephrin B signaling regulates cell sorting in the mature epithelium. These proteins are also reported to be upregulated in colon carcinomas. The EphA/ephrin A system has also been implicated in epithelial tissue structure and function. More recently, EphA receptors and their corresponding ligands have been implicated in numerous malignancies. Of these, EphA2 in particular has been intensively investigated and has been proposed as a therapeutic target. An interesting observation emerging from these studies is the role for Ephs and ephrins in critical aspects of cell adhesion, migration and positioning, and a crucial role in tumor progression and metastasis. However, the underlying role of Ephs and ephrins in these processes has generally been studied on individual Eph or ephrin genes. Given the multiplicity of Eph expression on gut epithelial cells, a more global approach is needed to define the precise role of Eph-ephrin interaction in malignant transformation. Here, we will review the recent advances on the role of Eph-ephrin signaling in colorectal malignancies.

A novel 1.4 Mb de novo microdeletion of chromosome 1q21.3 in a child with microcephaly, dysmorphic features and mental retardation

A 2.5 years old girl presented with moderate mental retardation, microcephaly, arching eyebrows, low set ears, long eyelashes, persistent fetal pads and clinodactyly. About 1 Mb deletion in the chromosomal region 1q21.3 was identified using BAC array CGH analysis. The parental follow up FISH analysis was normal. Further study of the deletion using a 244K oligo-array of Agilent Technologies Inc., Santa Clara, CA, USA defined the deleted region to span about 1.4 Mb with approximate genomic location chr1:152,511,593-153,993,103 (NCBI genome build 36). This is a novel deletion, not reported to-date. Larger proximal 1q deletions that were previously reported typically included microcephaly, mental retardation and multiple congenital anomalies. The deleted region reported here includes at least 30 coding genes. Among them of interest is a three-gene cluster of the ephrin gene family ( EFNA1, EFNA3 and EFNA4). This is a group of receptor protein-tyrosine kinase type genes with presumed, but not completely characterized function in nervous system development.

Disrupted Intercellular Communication Causes a Disfiguring Birth Defect

Before a fertilized egg begins the repeated rounds of cell division that turn the single cell into a proliferating, streaming, differentiating mass of cells, its fate may already be sealed. Inherited mutations in genes involved in segregating and sorting embryonic cells can result in serious abnormalities in body patterning and appear to underlie an inherited X-linked disorder (so-called because the mutated genes lie on the X chromosome) called craniofrontonasal syndrome (CFNS). X-linked disorders tend to affect males more severely than females, because boys inherit just one X chromosome while girls inherit two: if one gene is defective, the other can fill in. CFNS is a rare departure from this pattern, with females exhibiting the most severe symptoms. This disfiguring disorder is characterized by a range of skull aberrations, including facial asymmetry, widely spaced eyes, and abnormal head shape, as well as polydactyly and fused digits. A class of receptor protein-tyrosine kinases called Ephs and their ephrin binding partners (called ligands) regulate tissue patterning by restricting cell interactions, ensuring proper cell sorting, and establishing developmental compartment boundaries. Mutations in one ephrin gene, ephrin-B1, have been identified in patients with CFNS and have been associated with aberrant skeletal patterning in mutant “heterozygous” female mice, which carry one normal and one nonfunctional copy of the ephrin-B1 gene. Mutations in connexins, structural proteins that form gap junction pores, also lead to cranial and skeletal defects in both mice and humans. In a new study, Alice Davy, Jeffrey Bush, and Philippe Soriano elucidate the mechanisms of ephrin-mediated cell sorting, and show how the breakdown of the process causes physical abnormalities. The researchers worked with ephrin-B1 heterozygous female mice, polydactyl mutants with abnormally developed frontal bones in the skull (called the calvarial phenotype, after the name of the bones). They show that Eph/ephrin signaling regulates gap junction communication, which in turn controls cell sorting. Their results indicate that flawed cell sorting, resulting from dysregulated communication at gap junctions—intracellular membrane channels with pores that allow coupled cells to exchange small molecules—underlies the skeletal abnormalities observed in the mice. Previous studies established that ephrin-B1 heterozygous females exhibit polydactyly while males lacking their copy of ephrin-B1 and females lacking both copies do not. Polydactyly accompanied a random inactivation of X chromosomes in female cells (in which one X chromosome is silenced in some cells and the second is silenced in others) that created a mosaic pattern of ephrin-B1 expression, with ephrin-B1-expressing cells segregated from cells that didn’t express the gene. Ephrin-B1 mutants also develop multiple defects in tissue derived from neural crest cells—which give rise to cartilage, bone, connective tissue, and other specialized tissues. In this study, Davy et al. show that the mosaic loss of ephrin-B1 blocked the differentiation of neural crest cells by disrupting the distribution of a connexin (Cx43) that regulates bone cell differentiation and forms gap junctional pores. Cx43 aggregated between wild-type (nonmutant) cells and between cells that lack ephrin-B1, but was rarely seen at the border between ephrin-B1-positive and -negative cells, suggesting that the mosaic cells restricted the number of junctional pores. Expression of the ephrin-B1 receptor, EphB2, is elevated in ephrin-B1-negative regions in ephrin-B1 heterozygous embryos, so the researchers suspected that interactions between the receptor and ligand reduced Cx43 levels and disrupted gap junction formation—which they confirmed by tracking gap junction communication in cell cultures. This defect might be mediated by a physical interaction between ephrin-B1 and Cx43. The researchers propose that gap junction communication is inhibited by interactions between Eph-positive and ephrin-positive cells that cause Cx43 to be sequestered inside cells, where they can’t form gap junction pores and establish cell-to-cell communication—leading to skeletal abnormalities. This explains why the CFNS phenotype is more prevalent in females (who exhibit mosaic expression of ephrin-B1 through X inactivation). By contributing a mouse model with skull and digit defects that mimic those seen in humans, the researchers have provided a valuable platform for future investigations into the role of ephrins and gap junction communication in disfiguring skeletal disorders.

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.

Eph receptors and their ephrin ligands are expressed in developing mouse pancreas

Pancreas development involves branching morphogenesis concomitantly to differentiation of endocrine, exocrine and ductal cell types from a single population of pancreatic precursors. These processes depend on many signals and factors that also control development of the central nervous system. In the latter, Eph receptors and their class-A (GPI-anchored) and class-B (transmembrane) ephrin ligands control cell migration and axon-pathfinding, help establish regional patterns and act as labels for cell positioning. This raised the question as to whether and where Ephs and ephrins are expressed during pancreas development. Here we have identified the Eph and ephrin genes that are expressed in mouse embryonic pancreas, as detected by RT-PCR analysis. In situ hybridization experiments showed that Ephs and ephrins are mainly expressed in the burgeoning structures of the epithelium which differentiate into exocrine acini. Binding experiments on whole pancreas demonstrated the presence of functional Eph receptors. They showed that EphBs are expressed by the pancreatic epithelium at embryonic day (e) 12.5 and that, from e14.5 on, Ephs of both classes are expressed by the pancreatic epithelium and then become restricted to developing acini. We conclude that specific members of the Eph/ephrin family are expressed in embryonic pancreas according to a dynamic temporal and regional pattern.

Vessels and Nerves: Marching to the Same Tune

The patterning of the nervous system is achieved through coordinated action of a variety of repulsive or attractive neuronal guidance factors that direct the growth of growing axons to specific pathways. Recent studies suggest that the same or similar sets of factors also guide migration of endothelial cells, helping to direct the stereotypical embryonic patterning of the developing vertebrate vascular system.