Thomas Jungas

Ephrin-B1 reverse signaling controls a posttranscriptional feedback mechanism via miR-124.

ABSTRACT Eph receptors and ephrins exhibit complex and highly dynamic expression patterns during embryonic development. In addition, changes in their expression levels are often associated with pathological situations in adults. Yet, little is known about the mechanisms regulating their expression. Here we report that the expression of ephrin-B1 is controlled by a feedback loop involving posttranscriptional regulatory mechanisms. We observed that the EfnB1 3′ untranslated region (3′-UTR) confers instability to mRNA transcripts, and we identified miR-124 as a posttranscriptional repressor of EfnB1 expression. Furthermore, we showed that miR-124 is itself regulated by ephrin-B1 reverse signaling, thus revealing the existence of a mutually repressive interaction between ephrin-B1 and this microRNA (miRNA). Lastly, we demonstrated the relevance of this mutual inhibition for neuronal differentiation. Our results suggest that miRNAs could be important effectors of Eph/ephrin signaling to refine domains of expression and to regulate function.

Eph:ephrin-B1 forward signaling controls fasciculation of sensory and motor axons

Axon fasciculation is one of the processes controlling topographic innervation during embryonic development. While axon guidance steers extending axons in the accurate direction, axon fasciculation allows sets of co-extending axons to grow in tight bundles. The Eph:ephrin family has been involved both in axon guidance and fasciculation, yet it remains unclear how these two distinct types of responses are elicited. Herein we have characterized the role of ephrin-B1, a member of the ephrinB family in sensory and motor innervation of the limb. We show that ephrin-B1 is expressed in sensory axons and in the limb bud mesenchyme while EphB2 is expressed in motor and sensory axons. Loss of ephrin-B1 had no impact on the accurate dorso-ventral innervation of the limb by motor axons, yet EfnB1 mutants exhibited decreased fasciculation of peripheral motor and sensory nerves. Using tissue-specific excision of EfnB1 and in vitro experiments, we demonstrate that ephrin-B1 controls fasciculation of axons via a surround repulsion mechanism involving growth cone collapse of EphB2-expressing axons. Altogether, our results highlight the complex role of Eph:ephrin signaling in the development of the sensory-motor circuit innervating the limb.

Eph-mediated tyrosine phosphorylation of citron kinase controls abscission

Abscission is the last step of cytokinesis, allowing the physical separation of daughter cells at the end of cell division. It has been considered a cell autonomous process, yet Jungas et al. report that Ephrin/Eph signaling controls the completion of abscission.

Generation of transgenic mice overexpressing EfnB2 in endothelial cells

Genetic studies have shown that ephrin‐B2 and its cognate EphB4 receptor are necessary for normal embryonic angiogenesis. Moreover, there is overwhelming evidence that ephrin‐B2 is involved in tumor vascularization, yet its role in adult angiogenesis has been difficult to track genetically. Here, we report the generation of transgenic mice that over‐express EfnB2 specifically in endothelial cells (ECs). We show that exogenous expression of EfnB2 under the control of the Tie2 promoter/enhancer regions in ECs does not affect viability or growth of the transgenic animals. We further show that targeted expression of EfnB2 in ECs is not sufficient to rescue severe cardiovascular defects at mid‐gestation stages but rescues early embryonic lethality associated with loss‐of‐function mutation in EfnB2. This mouse model will be useful to study the role of ephrin‐B2 in physiological and pathological angiogenesis.genesis 49:811–820, 2011.

Ephrin-B1 reverse signaling controls a post-transcriptional feedback mechanism in neural progenitors

Habituation is a form of non-associative conditioning in which there is a reduction in response to a specific stimulus presented repetitively over time. Components of the cAMP mediated signaling pathway have been previously shown to be important for normal habituation. A kinase anchoring proteins (AKAPS) are a large family of proteins originally identified in mammals which modulate the specificity of protein kinase A (PKA) function by targeting and compartmentalizing PKA to various sub-cellular structures. Rugose (rg) encodes a Drosophila A kinase anchor protein (DAKAP550) which has been previously shown to be required normal pattern formation in the developing eye (Shamloula et al 2002). We present data which show mutations in rugose(rg), which encodes a (DAKAP550) alter habituation and synaptic properties. Data from behavioral, electrophysiological and cell biological studies on the adult as well as the larval neuromuscular junction are presented here.

EPH-ective control of cytokinesis

Experimental evidence in the unicellular choanoflagellate Salpingoeca rosetta, the closest living relative of animals, proposes that emergence of multicellularity – one of the founding step of animal life – is caused by incomplete separation of daughter cells at the last step of cell division, cytokinesis. Incomplete cytokinesis also occurs in animals, giving rise to polyploid cells (hepatocytes) or syncitia (germ cells). Until recently cytokinesis was viewed as a mechanism orchestrated only by cell intrinsic factors, yet in Salpingoeca Rosetta the switch from unicellular to multicellular state can be driven by extrinsic factors such as predators, changes in ocean chemistry or emergence of new ecological niches. These observations indicate that completion of cytokinesis may be controlled by factors present in the environment of dividing cells. In our recent study, we asked whether cytokinesis is similarly controlled by extracellular cues in mammals, focusing on the role of local communication via Eph/ephrin signaling. Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, are best known for their role in modulating cell adhesion and migration, mostly through the regulation of the actin cytoskeleton. Using live and fixed cell microscopy, we observed in various cell lines that activation of the EphB2 receptor induced cytokinesis delays and/or failures, which correlated with an increased number of polyploid cells. We showed that EphB2 tyrosine kinase activity was necessary for cytokinesis defects and we identified Citron kinase (CitK) as a downstream target of EphB2 signaling. CitK is a serine/threonine kinase that plays a well characterized function in cytokinesis. We found that CitK interacts with EphB2; that it is phosphorylated on tyrosine upon activation of Eph signaling and that this phosphorylation is mediated by Src. Using Mass Spectrometry we identified 2 tyrosine residues located in the Rho Binding domain of CitK that are phosphorylated by Src. Functional assays with engineered phosphomimetic and unphosphorylatable mutants of CitK confirmed that phosphorylation of these residues modulates CitK interaction with RhoA and alters cytokinesis. To demonstrate the physiological relevance of these in vitro observations, we focused on dividing neural progenitors (NPs) of the neocortex since Eph receptors and ephrins are expressed in these cells and CitK has been shown in mouse, rat and humans to play a critical role in NP cytokinesis. We showed that CitK and EphB2 partially co-localize in dividing NPs in vivo and that CitK is phosphorylated on tyrosine in these cells. More importantly, we found that pups lacking EphB2 signaling exhibit a decrease in the fraction of polyploid cortical neurons which we used as a read-out of NP cytokinesis failure. Thus, our study reports for the first time a complete molecular cascade downstream of Eph signaling controling cytokinesis. Interestingly, all the actors of this cascade are present in the genome of Salpingoeca rosetta: Eph receptors, Src kinase and a citron kinase-like protein. In addition, our study confirms that cytokinesis can be controlled by cues present in the environment of dividing cells in mammals and suggests that CitK plays a key role in the completion of cytokinesis as an integrator of both intrinsic and extrinsic information. Our findings raise a number of questions (Fig. 1), first with respect to the role of Eph/ephrin signaling in cancer. Indeed, Eph signaling deregulation is observed in many cancers, but until now its role was restricted to metastatic diffusion and tumor aggressiveness. Our data showing that Eph-induced cytokinesis failure leads to aneuploidy, suggests that Eph/ephrin signaling may play a role in cancer initiation by promoting genetic instability. More puzzling questions raised by our study concern the observation that Eph/ephrin signaling controls neuronal polyploidy. In mammals, the majority of cells are diploid except for a handful of cell types (gametes, hepatocytes and cardiomyocytes). The existence of polyploid neurons in mammals was reported several years ago, yet little is known about these neurons. Is there an advantage for neurons in being polyploid? Is polyploidy an additional source of neuronal diversity and does it participate in the complexity of the mammalian brain? What is the significance of its regulation by local signaling via Eph/ephrin? In conclusion, modulation of cytokinesis by environmental information appears as an important mechanism driving evolution, from emergence of multicellularity to generation of neuronal diversity.

Developmental Upregulation of Ephrin-B1 Silences Sema3C/Neuropilin-1 Signaling during Post-crossing Navigation of Corpus Callosum Axons

The corpus callosum is the largest commissure in thexa0brain, whose main function is to ensure communication between homotopic regions of the cerebral cortex. During fetal development, corpus callosum axons (CCAs) grow toward and across the brain midline and then away on the contralateral hemisphere to their targets. A particular feature of this circuit, which raises a key developmental question, is that the outgoing trajectory of post-crossing CCAs is mirror-symmetric with the incoming trajectory of pre-crossing axons. Here, we show that post-crossing CCAs switch off their response to axon guidance cues, among which the secreted Semaphorin-3C (Sema3C), that act as attractants for pre-crossing axons on their way to the midline. This change is concomitant with an upregulation of the surface protein Ephrin-B1, which acts in CCAs to inhibit Sema3C signaling via interaction with the Neuropilin-1 (Nrp1) receptor. This silencing activity is independent of Eph receptors and involves axa0N-glycosylation site (N-139) in the extracellular domain of Ephrin-B1. Together, our results reveal a molecular mechanism, involving interaction between the two unrelated guidance receptors Ephrin-B1 and Nrp1, that is used to control the navigation of post-crossing axons in the corpus callosum.

Cross Talk between One-Carbon Metabolism, Eph Signaling, and Histone Methylation Promotes Neural Stem Cell Differentiation

Metabolic pathways, once seen as a mere consequence of cell states, have emerged as active players in dictating different cellular events such as proliferation, self-renewal, and differentiation. Several studies have reported a role for folate-dependent one-carbon (1C) metabolism in stem cells; however, its exact mode of action and how it interacts with other cues are largely unknown. Here, we report a link between the Eph:ephrin cell-cell communication pathway and 1C metabolism in controlling neural stem cell differentiation. Transcriptional and functional analyses following ephrin stimulation revealed alterations in folate metabolism-related genes and enzymatic activity. Inxa0vitro and inxa0vivo data indicate that Eph-B forward signaling alters the methylation state of H3K4 by regulating 1C metabolism and locksxa0neural stem cell in a differentiation-ready state. Ourxa0study highlights a functional link between cell-cell communication, metabolism, and epigenomic remodeling in the control of stem cell self-renewal.

Crosstalk between one carbon metabolism and eph signaling promotes neural stem cells differentiation through epigenetic remodeling

Metabolic pathways, once seen as a mere consequence of cell states, have emerged as active players in dictating different cellular events such as proliferation, self-renewal and differentiation. Several studies have reported a role for folate-dependent 1-carbon (1C) metabolism in stem cells, however, its exact mode of action and how it interacts with other cues is largely unknown. Here, we report a link between the Eph:ephrin cell-cell communication pathway and 1C metabolism in controlling differentiation of neural stem cells. Transcriptional and functional analyses following ephrin stimulation revealed alterations in folate metabolism-related genes and enzymatic activity. In vitro and in vivo data indicate that Eph-B forward signaling alters the methylation state of H3K4 by regulating 1C metabolism, and locks neural stem cells in a differentiation-ready state. The functional link between cell-cell communication, metabolism and epigenetic remodeling identifies a novel triad in the control of stem cell self-renewal vs. differentiation. HIGHLIGHTS 1C folate metabolism is regulated by local cell-to-cell communication Description of Eph-B transcriptional response in NSC Eph activation decreases the expression and activity of DHFR Inhibition of DHFR modifies epigenetic marks and impairs self-renewal of neural stem cells Decreased H3K4 methylation locks neural stem cells in a pro-differentiation state eTOC BLURB Fawal et al. present evidence that Eph-B forward signaling inhibits 1C folate metabolism in neural stem cells leading to alteration of H3K4 methylation on key progenitor genes. In addition, they show that these epigenetic changes are inherited and maintained in the long term, thus locking NSC into a differentiation ready state.

13-P004 A positive feedback loop involving miR-124 regulates ephrin-B1 expression in neural progenitors

The way in which organisms generate patterns of differentiated tissues is of fundamental interest to developmental biology. One way to do this involves the formation of pattern without positional information and is fascinating because it raises the question of how cells can adopt different fates within a uniform environment. We are using the social amoeba, D. discoideum to show that it is the relative sensitivity of cells to inducing signals within the developing system that is important in making initial cell fate choice decisions in uniform environments. We show that relative levels of Ras activation are important in determining the sensitivity of cells to inducing signals during development and that this sensitivity is further modulated by differences in glucose metabolism between cells. Understanding the mechanisms by which Ras regulation and glucose metabolism interact to affect sensitivity to signals will provide valuable insight into the mechanisms that allow organisms to pattern without positional information