The molecular phylogeny of eph receptors and ephrin ligands

Background  

The tissue distributions and functions of Eph receptors and their ephrin ligands have been well studied, however less is known about their evolutionary history. We have undertaken a phylogenetic analysis of Eph receptors and ephrins from a number of invertebrate and vertebrate species.     

Eph receptors and ephrins

The Eph receptors are the largest known family of receptor tyrosine kinases. The Eph receptors and their membrane-attached ligands, ephrins, show diverse expression patterns during development. Recent studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes, including neuronal network formation, the patterning of the neural tube and the paraxial mesoderm, the guidance of cell migration, and vascular formation. In the nervous system, Eph receptors and ephrins have been shown to act as positional labels to establish topographic projections. They also play a key role in pathway finding by axons and neural crest cells. The crucial roles of Eph receptors and ephrins during development suggest involvement of these genes in congenital disorders affecting the nervous system and other tissues. It has also been suggested that Eph receptors and ephrins may be involved in carcinogenesis. It is therefore of clinical importance to further analyse the function of these molecules, as manipulation of their function may have therapeutic applications.     

Eph/ephrins and N-cadherin coordinate to control the pattern of sympathetic ganglia

Previous studies have suggested that the segmental pattern of neural-crest-derived sympathetic ganglia arises as a direct result of signals that restrict neural crest cell migratory streams through rostral somite halves. We recently showed that the spatiotemporal pattern of chick sympathetic ganglia formation is a two-phase process. Neural crest cells migrate laterally to the dorsal aorta, then surprisingly spread out in the longitudinal direction, before sorting into discrete ganglia. Here, we investigate the function of two families of molecules that are thought to regulate cell sorting and aggregation. By blocking Eph/ephrins or N-cadherin function, we measure changes in neural crest cell migratory behaviors that lead to alterations in sympathetic ganglia formation using a recently developed sagittal slice explant culture and 3D confocal time-lapse imaging. Our results demonstrate that local inhibitory interactions within inter-ganglionic regions, mediated by Eph/ephrins, and adhesive cell-cell contacts at ganglia sites, mediated by N-cadherin, coordinate to sculpt discrete sympathetic ganglia.     

EphA5 and EphA6: regulation of neuronal and spine morphology

Background

The Eph family of receptor tyrosine kinases plays important roles in neural development. Previous studies have implicated Eph receptors and their ligands, the ephrins, in neuronal migration, axon bundling and guidance to specific targets, dendritic spine formation and neural plasticity. However, specific contributions of EphA5 and EphA6 receptors to the regulation of neuronal cell morphology have not been well studied.

Results

Here we show that deletion of EphA5 and EphA6 results in abnormal Golgi staining patterns of cells in the brain, and abnormal spine morphology.

Conclusion

These observations suggest novel functions of these Eph receptors in the regulation of neuronal and spine structure in brain development and function.

     

Roles of Eph receptors and ephrins in segmental patterning

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have key roles in patterning and morphogenesis. Interactions between these molecules are promiscuous, but largely fall into two groups: EphA receptors bind to glycosylphosphatidyl inositol-anchored ephrin-A ligands, and EphB receptors bind to transmembrane ephrin-B proteins. Ephrin-B proteins transduce signals, such that bidirectional signalling can occur upon interaction with the Eph receptor. In many tissues, there are complementary and overlapping expression domains of interacting Eph receptors and ephrins. An important role of Eph receptors and ephrins is to mediate cell contact-dependent repulsion, and this has been implicated in the pathfinding of axons and neural crest cells, and the restriction of cell intermingling between hindbrain segments. Studies in an in vitro system show that bidirectional activation is required to prevent intermingling between cell populations, whereas unidirectional activation can restrict cell communication via gap junctions. Recent work indicates that Eph receptors can also upregulate cell adhesion, but the biochemical basis of repulsion versus adhesion responses is unclear. Eph receptors and ephrins have thus emerged as key regulators that, in parallel with cell adhesion molecules, underlie the establishment and maintenance of patterns of cellular organization.     

Ephrin-B ligands play a dual role in the control of neural crest cell migration

Little is known about the mechanisms that direct neural crest cells to the appropriate migratory pathways. Our aim was to determine how neural crest cells that are specified as neurons and glial cells only migrate ventrally and are prevented from migrating dorsolaterally into the skin, whereas neural crest cells specified as melanoblasts are directed into the dorsolateral pathway. Eph receptors and their ephrin ligands have been shown to be essential for migration of many cell types during embryonic development. Consequently, we asked if ephrin-B proteins participate in the guidance of melanoblasts along the dorsolateral pathway, and prevent early migratory neural crest cells from invading the dorsolateral pathway. Using Fc fusion proteins, we detected the expression of ephrin-B ligands in the dorsolateral pathway at the stage when neural crest cells are migrating ventrally. Furthermore, we show that ephrins block dorsolateral migration of early-migrating neural crest cells because when we disrupt the Eph-ephrin interactions by addition of soluble ephrin-B ligand to trunk explants, early neural crest cells migrate inappropriately into the dorsolateral pathway. Surprisingly, we discovered the ephrin-B ligands continue to be expressed along the dorsolateral pathway during melanoblast migration. RT-PCR analysis, in situ hybridisation, and cell surface-labelling of neural crest cell cultures demonstrate that melanoblasts express several EphB receptors. In adhesion assays, engagement of ephrin-B ligands to EphB receptors increases melanoblast attachment to fibronectin. Cell migration assays demonstrate that ephrin-B ligands stimulate the migration of melanoblasts. Furthermore, when Eph signalling is disrupted in vivo, melanoblasts are prevented from migrating dorsolaterally, suggesting ephrin-B ligands promote the dorsolateral migration of melanoblasts. Thus, transmembrane ephrins act as bifunctional guidance cues: they first repel early migratory neural crest cells from the dorsolateral path, and then later stimulate the migration of melanoblasts into this pathway. The mechanisms by which ephrins regulate repulsion or attraction in neural crest cells are unknown. One possibility is that the cellular response involves signalling to the actin cytoskeleton, potentially involving the activation of Cdc42/Rac family of GTPases. In support of this hypothesis, we show that adhesion of early migratory cells to an ephrin-B-derivatized substratum results in cell rounding and disruption of the actin cytoskeleton, whereas plating of melanoblasts on an ephrin-B substratum induces the formation of microspikes filled with F-actin.     

Eph/ephrin interactions modulate muscle satellite cell motility and patterning

During development and regeneration, directed migration of cells, including neural crest cells, endothelial cells, axonal growth cones and many types of adult stem cells, to specific areas distant from their origin is necessary for their function. We have recently shown that adult skeletal muscle stem cells (satellite cells), once activated by isolation or injury, are a highly motile population with the potential to respond to multiple guidance cues, based on their expression of classical guidance receptors. We show here that, in vivo, differentiated and regenerating myofibers dynamically express a subset of ephrin guidance ligands, as well as Eph receptors. This expression has previously only been examined in the context of muscle-nerve interactions; however, we propose that it might also play a role in satellite cell-mediated muscle repair. Therefore, we investigated whether Eph-ephrin signaling would produce changes in satellite cell directional motility. Using a classical ephrin 'stripe' assay, we found that satellite cells respond to a subset of ephrins with repulsive behavior in vitro; patterning of differentiating myotubes is also parallel to ephrin stripes. This behavior can be replicated in a heterologous in vivo system, the hindbrain of the developing quail, in which neural crest cells are directed in streams to the branchial arches and to the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite. We hypothesize that guidance signaling might impact multiple steps in muscle regeneration, including escape from the niche, directed migration to sites of injury, cell-cell interactions among satellite cell progeny, and differentiation and patterning of regenerated muscle.     

Tissue transglutaminase clusters soluble A-type ephrins into functionally active high molecular weight oligomers

The Eph receptors and their ligands, the ephrins, are thought to act at points of close cell-cell contact to elicit bi-directional signaling in receptor and ligand expressing cells. However, when cultured in vitro, some A-type ephrins are released from the cell surface and it is unclear if these soluble ephrins participate in Eph receptor activation. We show that soluble ephrin A5 is subject to oligomerization. Ephrins A1 and A5 are substrates for a cross-linking enzyme, tissue transglutaminase, which mediates the formation of oligomeric ephrin. Transglutaminase-cross-linked ephrin binds to A-type Eph receptors, stimulates Eph kinase activity, and promotes invasion and migration of HeLa cells. Transglutaminase-mediated oligomerization of soluble ephrin potentially represents a novel mechanism of forward signaling through Eph receptors and may extend the influence of A-type ephrins beyond cell contact mediated signaling.     

Eph受体家族及其配体的信号转导途径及功能

Eph受体是已知最大的酪氨酸蛋白激酶受体家族,Eph受体和其膜附着型配体（ephrin）在发育过程中呈现不同的表达模式,近来研究证明,Eph受体和其配体在包括神经网络形成,神经管和轴旁中胚层的成型（patterning）,细胞迁移导向和轴突路径导引,血管形成等许多的发育过程中起重要作用.Eph受体及其配体也与肿瘤发生有关,因此深入分析这些分子尤其在肿瘤细胞生长中的功能而应用于治疗具有重要的临床意义.     

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.     

Diverse roles of eph receptors and ephrins in the regulation of cell migration and tissue assembly

Eph receptor tyrosine kinases and ephrins have key roles in regulation of the migration and adhesion of cells required to form and stabilize patterns of cell organization during development. Activation of Eph receptors or ephrins can lead either to cell repulsion or to cell adhesion and invasion, and recent work has found that cells can switch between these distinct responses. This review will discuss biochemical mechanisms and developmental roles of the diverse cell responses controlled by Eph receptors and ephrins.     

Angiopoietin 2 signaling plays a critical role in neural crest cell migration

Background

Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood.

Results

Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells.

Conclusions

Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.

     

Zebrafish arl6ip1 is required for neural crest development during embryogenesis

Background

Although the embryonic expression pattern of ADP ribosylation factor-like 6 interacting protein 1 (Arl6ip1) has been reported, its function in neural crest development is unclear.

Methods/Principal Findings

We found that knockdown of Arl6ip1 caused defective embryonic neural crest derivatives that were particularly severe in craniofacial cartilages. Expressions of the ectodermal patterning factors msxb, dlx3b, and pax3 were normal, but the expressions of the neural crest specifier genes foxd3, snai1b, and sox10 were greatly reduced. These findings suggest that arl6ip1 is essential for specification of neural crest derivatives, but not neural crest induction. Furthermore, we revealed that the streams of crestin- and sox10-expressing neural crest cells, which migrate ventrally from neural tube into trunk, were disrupted in arl6ip1 morphants. This migration defect was not only in the trunk neural crest, but also in the enteric tract where the vagal-derived neural crest cells failed to populate the enteric nervous system. We found that this migration defect was induced by dampened Shh signaling, which may have resulted from defective cilia. These data further suggested that arl6ip1 is required for neural crest migration. Finally, by double-staining of TUNEL and crestin, we confirmed that the loss of neural crest cells could not be attributed to apoptosis.

Conclusions/Significance

Therefore, we concluded that arl6ip1 is required for neural crest migration and sublineage specification.     

Emergence and migration of trunk neural crest cells in a snake,the California Kingsnake (<Emphasis Type="Italic">Lampropeltis getula californiae</Emphasis>)

Background  

The neural crest is a group of multipotent cells that emerges after an epithelial-to-mesenchymal transition from the dorsal neural tube early during development. These cells then migrate throughout the embryo, giving rise to a wide variety derivatives including the peripheral nervous system, craniofacial skeleton, pigment cells, and endocrine organs. While much is known about neural crest cells in mammals, birds, amphibians and fish, relatively little is known about their development in non-avian reptiles like snakes and lizards.     

Cell migration, pattern formation and cell fate during head development in lungfishes and amphibians

Summary In the evolution of land-living vertebrates, the transition from spending the entire life cycle in the water to first a biphasic (adult on land, eggs and larvae in water) and later a terrestrial life-history mode was achieved by changes in developmental processes and regulatory mechanisms. Lungfishes, salamanders and frogs are studied as examples of species which span this transition. The migration and fate of the embryonic cells that form the head is studied, using experimental embryology (extirpation and transplantation of cells), molecular markers and novel microscopy techniques — such as confocal microscopy. Knowing the migratory routes and fates of the cells that form head structures is important for an elucidation of the changes that took place e.g. when gill arches transformed into head cartilages, and when the specialised larval mouth structures present in today’s frogs and toads arose as an evolutionary innovation. Results so far indicate that the early migration and pattern formation of neural crest cells in the head region is surprisingly conserved. Both the amphibians investigated and the Australian lungfish have the same number of migrating neural crest streams, and the identity of the streams is preserved. The major difference lies in the timing of migration, where there has been a heterochronic shift such that cell migration starts much later in the Australian lungfish than in the amphibians. The molecular mechanisms regulating the formation of streams of cranial neural crest cells seem, at least in part, to be differential expression of ephrins and ephrin receptors, which mediate cell sorting. Our understanding of the behaviour of migrating cells (primarily the more well characterised neural crest cells) could be enhanced by a modelling approach. I present preliminary ideas on how this could be achieved, inspired by recent work on Dictyostelium development and our own previous work on pigment cells and their pattern formation during salamander embryogenesis.     

Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: correlation to function in a growth cone collapse assay

Eph receptors and ephrins play important roles in regulating cell migration and positioning during both normal and oncogenic tissue development. Using a surface plasma resonance (SPR) biosensor, we examined the binding kinetics of representative monomeric and dimeric ephrins to their corresponding Eph receptors and correlated the apparent binding affinity with their functional activity in a neuronal growth cone collapse assay. Our results indicate that the Eph receptor binding of dimeric ephrins, formed through fusion with disulfide-linked Fc fragments, is best described using a bivalent analyte model as a two-step process involving an initial monovalent 2:1 binding followed by a second bivalent 2:2 binding. The bivalent binding dramatically decreases the apparent dissociation rate constants with little effect on the initial association rate constants, resulting in a 30- to 6000-fold decrease in apparent equilibrium dissociation constants for the binding of dimeric ephrins to Eph receptors relative to their monomeric counterparts. Interestingly, the change was more prominent in the A-class ephrin/Eph interactions than in the B-class of ephrins to Eph receptors. The increase in apparent binding affinities correlated well with increased activation of Eph receptors and the resulting growth cone collapse. Our kinetic analysis and correlation of binding affinity with function helped us better understand the interactions between ephrins and Eph receptors and should be useful in the design of inhibitors that interfere with the interactions.     

Eph receptor-ephrin bidirectional signals that target Ras and Rho proteins

The ability of cells to respond to their surrounding environment and relay signals to the cell interior is essential for numerous processes during the development and maintenance of tissues. Eph receptors and their membrane-bound ligands, the ephrins, are unique in the receptor tyrosine kinase family in that their signaling is bidirectional, through both the receptor and the ligand. Eph receptors and ephrins are essential for a variety of biological processes, and play a particularly important role in regulating cell shape and cell movement. Recent data have linked Eph receptor-ephrin signaling complexes to the Ras and Rho families of small molecular weight GTPases and also to heterotrimeric G proteins. Understanding the signaling networks involved is an important step to understand the molecular basis for normal and defective cell-cell communication through Eph receptors and ephrins.     

Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.     

A Novel Role for Lh3 Dependent ECM Modifications during Neural Crest Cell Migration in Zebrafish

During vertebrate development, trunk neural crest cells delaminate along the entire length of the dorsal neural tube and initially migrate as a non-segmented sheet. As they enter the somites, neural crest cells rearrange into spatially restricted segmental streams. Extracellular matrix components are likely to play critical roles in this transition from a sheet-like to a stream-like mode of migration, yet the extracellular matrix components and their modifying enzymes critical for this transition are largely unknown. Here, we identified the glycosyltransferase Lh3, known to modify extracellular matrix components, and its presumptive substrate Collagen18A1, to provide extrinsic signals critical for neural crest cells to transition from a sheet-like migration behavior to migrating as a segmental stream. Using live cell imaging we show that in lh3 null mutants, neural crest cells fail to transition from a sheet to a stream, and that they consequently enter the somites as multiple streams, or stall shortly after entering the somites. Moreover, we demonstrate that transgenic expression of lh3 in a small subset of somitic cells adjacent to where neural crest cells switch from sheet to stream migration restores segmental neural crest cell migration. Finally, we show that knockdown of the presumptive Lh3 substrate Collagen18A1 recapitulates the neural crest cell migration defects observed in lh3 mutants, consistent with the notion that Lh3 exerts its effect on neural crest cell migration by regulating post-translational modifications of Collagen18A1. Together these data suggest that Lh3–Collagen18A1 dependent ECM modifications regulate the transition of trunk neural crest cells from a non-segmental sheet like migration mode to a segmental stream migration mode.     

Conditional expression of Spry1 in neural crest causes craniofacial and cardiac defects

Background  

Growth factors and their receptors are mediators of organogenesis and must be tightly regulated in a temporal and spatial manner for proper tissue morphogenesis. Intracellular regulators of growth factor signaling pathways provide an additional level of control. Members of the Sprouty family negatively regulate receptor tyrosine kinase pathways in several developmental contexts. To gain insight into the role of Spry1 in neural crest development, we analyzed the developmental effects of conditional expression of Spry1 in neural crest-derived tissues.