Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos

Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. Here, we have uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. We first demonstrate that the segregation of notochord and neural fates is an autonomous property of the mother cell and that the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. We show that these cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. Finally, we demonstrate that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. We propose a model whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification.     

Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation

The Eph receptors and their ephrin ligands play crucial roles in a large number of cell–cell interaction events, including those associated with axon pathfinding, neuronal cell migration and vasculogenesis. They are also involved in the patterning of most tissues and overall cell positioning in the development of the vertebrate body plan. The Eph/ephrin signaling system manifests several unique features that differentiate it from other receptor tyrosine kinases, including initiation of bi-directional signaling cascades and the existence of ligand and receptor subclasses displaying promiscuous intra-subclass interactions, but very rare inter-subclass interactions. In this review we briefly discuss these features and focus on recent studies of the unique and expansive high-affinity Eph/ephrin assemblies that form at the sites of cell–cell contact and are required for Eph signaling initiation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Establishment of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

In an attempt to estimate the number of pigment precursor cells in sea urchin embryos, DNA synthesis and cell divisions were blocked with aphidicolin from various stages of development. Interestingly, pigment cells differentiated on a normal time schedule, even if the embryos were treated from late cleavage stages on. In most of the embryos treated from 10 h on, 10-15 pigment cells differentiated. Thereafter, the number of pigment cells in the aphidicolin-treated embryos further increased, as the initiation of the treatment was delayed. On the other hand, total cell volumes in the pigment lineage, calculated from the averaged number and diameter of differentiated pigment cells, were almost the same irrespective of the time of the initiation of aphidicolin treatment. This indicated that the increase in the number was caused by divisions of the pre-existing cells in the pigment lineage. Thus, the founder cells that exclusively produce pigment cells could be identified. They are nine times-cleaved blastomeres and specified by 10 h post-fertilization. The obtained results also clarified the division schedule in the pigment lineage; the founder cells divide once (10th) until hatching, and divide once more (11th) by the end of gastrulation.     

Regulation of cell differentiation by Eph receptor and ephrin signaling

There is increasing evidence that in addition to having major roles in morphogenesis, in some tissues Eph receptor and ephrin signaling regulates the differentiation of cells. In one mode of deployment, cell contact dependent Eph-ephrin activation induces a distinct fate of cells at the interface of their expression domains, for example in early ascidian embryos and in the vertebrate hindbrain. In another mode, overlapping Eph receptor and ephrin expression underlies activation within a cell population, which promotes or inhibits cell differentiation in bone remodelling, neural progenitors and keratinocytes. Eph-ephrin activation also contributes to formation of the appropriate number of progenitor cells by increasing or decreasing cell proliferation. These multiple roles of Eph receptor and ephrin signaling may enable a coupling between morphogenesis and the differentiation and proliferation of cells.     

Regulation of cell differentiation by Eph receptor and ephrin signaling

There is increasing evidence that in addition to having major roles in morphogenesis, in some tissues Eph receptor and ephrin signaling regulates the differentiation of cells. In one mode of deployment, cell contact dependent Eph-ephrin activation induces a distinct fate of cells at the interface of their expression domains, for example in early ascidian embryos and in the vertebrate hindbrain. In another mode, overlapping Eph receptor and ephrin expression underlies activation within a cell population, which promotes or inhibits cell differentiation in bone remodelling, neural progenitors and keratinocytes. Eph-ephrin activation also contributes to formation of the appropriate number of progenitor cells by increasing or decreasing cell proliferation. These multiple roles of Eph receptor and ephrin signaling may enable a coupling between morphogenesis and the differentiation and proliferation of cells.     

The role of the Eph-ephrin signalling system in the regulation of developmental patterning

The Eph and ephrin system, consisting of fourteen Eph receptor tyrosine kinase proteins and nine ephrin membrane proteins in vertebrates, has been implicated in the regulation of many critical events during development. Binding of cell surface Eph and ephrin proteins results in bi-directional signals, which regulate the cytoskeletal, adhesive and motile properties of the interacting cells. Through these signals Eph and ephrin proteins are involved in early embryonic cell movements, which establish the germ layers, cell movements involved in formation of tissue boundaries and the pathfinding of axons. This review focuses on two vertebrate models, the zebrafish and mouse, in which experimental perturbation of Eph and/or ephrin expression in vivo have provided important insights into the role and functioning of the Eph/ephrin system.     

Role of cell adhesion in the specification of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

To clarify the role of cell adhesion in the specification of pigment cell lineage in sea urchin embryos, cell contacts were inhibited by Ca²⁺-free artificial seawater (ASW) treatment, and the number of differentiated pigment cells was examined by the method devised for the present study. Obtained results showed that inhibition of cell contacts during mid-to-late blastula stage greatly affects the number of pigment cells. Treatment with Ca²⁺-free ASW during 7.5–10.5h of development drastically decreased the number of pigment cells, indicating that cell adhesion during this period is indispensable for the specification of pigment cell lineage. On the other hand, the number of pigment cells were increased by the treatment during 9.5–12.5 h of development. It was suggested that this increase was caused by excess divisions of the precursor cells, that is, the division schedule of the precursor cells was altered by inhibition of cell contacts at this period. Interestingly, the number of pigment cells was a multiple of four in a majority of embryos in which pigment cells were drastically decreased in number. These findings suggest that the founder blastomeres of the pigment cell lineage are specified during 7–10 h of development, and that these blastomeres divide twice before they differentiate into pigment cells.     

The cleavage program in the 2d cell lineage of Tubifex embryos

Early development in clitellate annelids is characterized by a highly stereotyped sequence of unequal, spiral cleavages. Cell 2d (i.e., the second micromere of the D quadrant) in the oligochaete Tubifex tubifex also undergoes an evolutionarily conserved sequence of cell division to produce four bilateral pairs of ectodermal teloblasts that act as embryonic stem cells. This study was conducted to characterize each of the 15 rounds of cell division that occur in the 2d cell lineage in this clitellate. After its occurrence, cell 2d undergoes three rounds of highly unequal divisions, giving off the first smaller daughter cell toward the posterior right of the larger daughter cell, the second cell toward the posterior left, and the third cell toward the anterior side of the cell; the larger daughter cell that results from the third division (i.e., the great-granddaughter cell of 2d) then divides equally into a bilateral pair of NOPQ proteloblasts. Cell NOPQ on either side of the embryo undergoes 11 rounds of cell division, during which ectoteloblasts N, Q, and O/P are produced in this order. After its appearance, NOPQ undergoes highly unequal divisions twice cutting off the smaller cells toward the anterior end of the embryo and then divides almost equally into ectoteloblast N and proteloblast OPQ. After its appearance, OPQ undergoes highly unequal divisions twice giving off the first smaller cell toward the anterior and the second smaller cell toward the posterior of the embryo and then divides almost equally into ectoteloblast Q and proteloblast OP. Finally, OP undergoes highly unequal division four times after its birth budding off the smaller cells toward the anterior and then cleaves equally into ectoteloblasts O and P. In the unequally dividing cells of the 2d cell lineage, the mitotic apparatus (MA), which forms at the cell's center, moves eccentrically toward the cortical site where the smaller cell will be given off. The moving MA is oriented perpendicular to the surface it approaches, and its peripheral pole becomes closely associated with the cell cortex. In contrast, the MA involved in the equal divisions remains in the cell center throughout mitosis. The key features of the cleavage program in the 2d cell lineage are discussed in light of the present observations. The mechanical aspects of unequal cleavage in the 2d cell lineage and the modes of specification of MA orientation are discussed. A comparison of the cleavage mode in the 2d cell lineage is also performed among six selected clitellate annelid species.     

Morphogenesis of prechordal plate and notochord requires intact Eph/ephrin B signaling

Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migration processes during development. We report the molecular cloning and tissue distribution of zebrafish transmembrane ephrins that represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrin B sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously to several Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands are confined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins are expressed as early as 30% epiboly and during gastrula stages: in the germ ring, shield, prechordal plate, and notochord. Ectopic overexpression of dominant-negative soluble ephrin B constructs yields reproducible defects in the morphology of the notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does not completely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesis of the prechordal plate and the notochord is likely a consequence of a cell movement defect. Our observations suggest Eph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation.     

Role of ephrin B2 in human retinal endothelial cell proliferation and migration

This study was designed to determine the presence of Eph B4 or ephrin B2 in human retinal endothelial cells (REC) and their signal transduction. Human retinal endothelial cells were stimulated with an Eph B4/Fc chimera and probed for phosphorylation of phosphatidylinositol-3-kinase (PI3K), Src, and mitogen-activated protein kinase (MAPK) pathways. Proliferation and migration were investigated after Eph B4/Fc stimulation in the presence of various pathway inhibitors. Human retinal endothelial cells express ephrin B2, with little expression of Eph B4. Treatment with EphB4/Fc chimera resulted in activation of PI3K, Src, and MAPK pathways. Eph B4-stimulated endothelial cell proliferation was mediated via PI3K, nitric oxide synthase, and extracellular signal-regulated kinase 1/2 (ERK1/2). Blockade of Src-PI3K pathways produced significant attenuation of Eph B4/Fc-stimulated migration. These results demonstrate for the first time that ephrin B2 is present in human retinal endothelial cells. Additionally, it appears that vascular growth may be modulated in the retina through activation of the PI3K pathway and its downstream components.     

EphrinA/EphA signal facilitates insulin-like growth factor-I-induced myogenic differentiation through suppression of the Ras/extracellular signal-regulated kinase 1/2 cascade in myoblast cell lines

Insulin-like growth factor-I (IGF-I) activates not only the phosphatidylinositol 3-kinase (PI3K)-AKT cascade that is essential for myogenic differentiation but also the extracellular signal-regulated kinase (ERK) 1/2 cascade that inhibits myogenesis. We hypothesized that there must be a signal that inhibits ERK1/2 upon cell-cell contact required for skeletal myogenesis. Cell-cell contact-induced engagement of ephrin ligands and Eph receptors leads to downregulation of the Ras-ERK1/2 pathway through p120 Ras GTPase-activating protein (p120RasGAP). We therefore investigated the significance of the ephrin/Eph signal in IGF-I-induced myogenesis. EphrinA1-Fc suppressed IGF-I-induced activation of Ras and ERK1/2, but not that of AKT, in C2C12 myoblasts, whereas ephrinB1-Fc affected neither ERK1/2 nor AKT activated by IGF-I. IGF-I-dependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc failed to suppress the Ras-ERK1/2 cascade by IGF-I and to promote IGF-I-mediated myogenesis. EphrinA1-Fc did not promote IGF-I-dependent myogenesis when the ERK1/2 was constitutively activated. Furthermore, a dominant-negative EphA receptor blunted IGF-I-induced myogenesis in C2C12 and L6 myoblasts. However, the inhibition of IGF-I-mediated myogenesis by down-regulation of ephrinA/EphA signal was canceled by inactivation of the ERK1/2 pathway. Collectively, these findings demonstrate that the ephrinA/EphA signal facilitates IGF-I-induced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines.     

Using an adherent cell culture of the mouse subependymal zone to study the behavior of adult neural stem cells on a single-cell level

A comprehensive understanding of the cell biology of adult neural stem cells (aNSCs) requires direct observation of aNSC division and lineage progression in the absence of niche-dependent signals. Here we describe a culture preparation of the adult mouse subependymal zone (SEZ), which allows for continuous single-cell tracking of aNSC behavior. The protocol involves the isolation (approximately 3 h) and culture of cells from the adult SEZ at low density in the absence of mitogenic growth factors in chemically defined medium and subsequent live imaging using time-lapse video microscopy (5-7 d); these steps are followed by postimaging immunocytochemistry to identify progeny (approximately 7 h). This protocol enables the observation of the progression from slow-dividing aNSCs of radial/astroglial identity up to the neuroblast stage, involving asymmetric and symmetric cell divisions of distinct fast-dividing precursors. This culture provides an experimental system for studying instructive or permissive effects of signal molecules on aNSC modes of cell division and lineage progression.     

Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway

Interactions between Eph receptor tyrosine kinases (RTKs) and membrane-anchored ephrin ligands critically regulate axon pathfinding and development of the cardiovascular system, as well as migration of neural cells. Similar to other RTKs, ligand-activated Eph kinases recruit multiple signalling and adaptor proteins, several of which are involved in growth regulation. However, in contrast to other RTKs, activation of Eph receptors fails to promote cell proliferation or to transform rodent fibroblasts, indicating that Eph kinases may initiate signalling pathways that are distinct from those transmitted by other RTKs. Here we show that stimulation of endogenous EphA kinases with ephrin-A1 potently inhibits the Ras/MAPK cascade in a range of cell types, and attenuates activation of mitogen-activated protein kinase (MAPK) by receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). In prostatic epithelial cells and endothelial cells, but not fibroblasts, treatment with ephrin-A1 inhibits cell proliferation. Our results identify EphA kinases as negative regulators of the Ras/MAPK pathway that exert anti-mitogenic functions in a cell-type-specific manner.     

The role of ephrins and Eph receptors in cancer

Eph receptors are the largest receptor tyrosine kinase family of transmembrane proteins with an extracellular domain capable of recognizing signals from the cells’ environment and influencing cell–cell interaction and cell migration. Ephrins are the ligands to Eph receptors and stimulate bi-directional signaling of the Eph/ephrin axis. Eph receptor and ephrin overexpression can result in tumorigenesis as related to tumor growth and survival and is associated with angiogenesis and metastasis in many types of human cancer. Recent data suggest that Eph/ephrin signaling could play an important role in the development of novel inhibition strategies and cancer treatments to potentially target this receptor tyrosine kinase and/or its ligand. A deeper understanding of the molecular basis for normal versus defective cell–cell interaction through the Eph/ephrin axis will enable the potential development of novel cancer treatments. This review emphasizes the biology of Eph/ephrin as well as the potential for novel targeted therapy through this pathway.     

Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. We genetically created such neuroblasts, and found that in embryos, they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. Our results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus.     

Decreased maternal behavior and anxiety in ephrin‐A5−/− mice

During development of the nervous system, molecular signals mediating cell–cell interactions play critical roles in the guidance of axonal growth and establishment of synaptic functions. The Eph family of tyrosine kinase receptors and their ephrin ligands has been shown to mediate neuronal interactions in the development of topographic axon projection maps in several brain regions, and the loss of Eph activities result in defects in select axonal pathways. However, effects of deficiencies of the Eph signals on animal behavior have not been well documented. In this study, we showed that inactivation of a ligand of the Eph receptors, ephrin‐A5, resulted in defects in maternal behavior and alterations in anxiety. Female ephrin‐A5 ^?/? mice show significant defects in nest building and pup retrieval. In addition, lower levels of anxiety were observed in both male and female null mice. These changes were not due to deficiencies in estradiol, progesterone or corticosterone levels. Our observations suggest that ephrin‐A5 plays a key role in the development and/or function of neural pathways mediating mouse maternal care and anxiety.     

Promiscuous and specific recognition among ephrins and Eph receptors

Eph–ephrin interactions control the signal transduction between cells and play an important role in carcinogenesis and other diseases. The interactions between Eph receptors and ephrins of the same subclass are promiscuous; there are cross-interactions between some subclasses, but not all. To understand how Eph–ephrin interactions can be both promiscuous and specific, we investigated sixteen energy landscapes of four Eph receptors (A2, A4, B2, and B4) interacting with four ephrin ligands (A1, A2, A5, and B2). We generated conformational ensembles and recognition energy landscapes starting from separated Eph and ephrin molecules and proceeding up to the formation of Eph–ephrin complexes. Analysis of the Eph–ephrin recognition trajectories and the co-evolution entropy of 400 ligand binding domains of Eph receptor and 241 ephrin ligands identified conserved residues during the recognition process. Our study correctly predicted the promiscuity and specificity of the interactions and provided insights into their recognition. The dynamic conformational changes during Eph–ephrin recognition can be described by progressive conformational selection and population shift events, with two dynamic salt bridges between EphB4 and ephrin-B2 contributing to the specific recognition. EphA3 cancer-related mutations lowered the binding energies. The specificity is not only controlled by the final stage of the interaction across the protein–protein interface, but also has large contributions from binding kinetics with the help of dynamic intermediates along the pathway from the separated Eph and ephrin to the Eph–ephrin complex.     

Eph/ephrin对树突棘的调控与中枢神经系统疾病

树突棘是神经元树突上的功能性突起结构,通常作为突触后成份与投射来的轴突共同构成完整的突触连接。树突棘的形态与结构具有明显的可塑性,其变化通常会引起突触功能的改变。Eph受体酪氨酸激酶家族分子与其配体ephrin都是重要的神经导向因子,同时对树突棘结构也有直接的调控作用。Eph受体的活化可以促进树突棘的发生并影响树突棘的形态及内部结构;而Eph受体的异常也往往会损害正常的突触功能,甚至导致许多与树突棘结构异常相关的神经系统病变的发生。