During vertebrate head development, neural crest cells migrate from hindbrain segments to specific branchial arches, where they differentiate into distinct patterns of skeletal structures. The rostrocaudal identity of branchial neural crest cells appears to be specified prior to migration, so it is important that they are targeted to the correct destination. In Xenopus embryos, branchial neural crest cells segregate into four streams that are adjacent during early stages of migration. It is not known what restricts the intermingling of these migrating cell populations and targets them to specific branchial arches. Xenopus EphA4 and EphB1 are expressed in migrating neural crest cells and mesoderm of the third arch, and third plus fourth arches, respectively. The ephrin-B2 ligand, which interacts with these receptors, is expressed in the adjacent second arch neural crest and mesoderm. Using truncated receptors, it has been shown that the inhibition of EphA4/EphB1 function leads to abnormal migration of third arch neural crest cells into second and fourth arch territories. Furthermore, ectopic activation of these receptors by overexpression of ephrin-B2 leads to scattering of third arch neural crest cells into adjacent regions. Similar disruptions occur when the expression of ephrin-B2 or truncated receptors is targeted to the neural crest. These data indicate that the complementary expression of EphA4/EphB1 receptors and ephrin-B2 is involved in restricting the intermingling of third and second arch neural crest and in targeting third arch neural crest to the correct destination. Together with previous work showing that Eph receptors and ligands mediate neuronal growth cone repulsion, these findings suggest that similar mechanisms are used for neural crest and axon pathfinding (Smith, 1997).
In the trunk of avian embryos, neural crest migration through the somites is segmental, with neural crest cells entering the rostral half of each somitic sclerotome but avoiding the caudal half. Little is known about the molecular nature of the cues (intrinsic to the somites) that are responsible for this segmental migration of neural crest cells. Eph-related receptor tyrosine kinases and their ligands are essential for the segmental migration of avian trunk neural crest cells through the somites. EphB3 localizes to the rostral half-sclerotome, including the neural crest, and the ligand ephrin-B1 has a complementary pattern of expression in the caudal half-sclerotome. To test the functional significance of this striking asymmetry, soluble ligand ephrin-B1 was added to interfere with receptor function in either whole trunk explants or neural crest cells cultured on alternating stripes of ephrin-B1 versus fibronection. Neural crest cells in vitro avoid migrating on lanes of immobilized ephrin-B1; the addition of soluble ephrin-B1 blocks this inhibition. Similarly, in whole trunk explants, the metameric pattern of neural crest migration is disrupted by the addition of soluble ephrin-B1, allowing entry of neural crest cells into caudal portions of the sclerotome. Thus, both in vivo and in vitro, the addition of soluble ephrin-B1 results in a loss of the metameric migratory pattern and a disorganization of neural crest cell movement. These results demonstrate that Eph-family receptor tyrosine kinases and their transmembrane ligands are involved in interactions between neural crest and sclerotomal cells, mediating an inhibitory activity necessary to constrain neural precursors to specific territories in the developing nervous system (Krull, 1997).
Little is known about the mechanisms that direct neural crest cells to the appropriate migratory pathways. This study set out to determine how neural crest cells that are specified as neurons and glial cells migrate only 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, it was 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, the expression of ephrin-B ligands was detected in the dorsolateral pathway at the stage when neural crest cells are migrating ventrally. Furthermore, ephrins block dorsolateral migration of early-migrating neural crest cells because when the Eph-ephrin interactions are disrupted by addition of soluble ephrin-B ligand to trunk explants, early neural crest cells migrate inappropriately into the dorsolateral pathway. Surprisingly, the ephrin-B ligands continue to be expressed along the dorsolateral pathway during melanoblast migration. RT-PCR analysis, in situ hybridization, 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 signaling 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 signaling to the actin cytoskeleton, potentially involving the activation of the Cdc42/Rac family of GTPases. In support of this hypothesis, the adhesion of early migratory cells to an ephrin-B-derivatized substratum has been shown to result 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 (Santiago, 2002).
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. 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. This study investigated the function of two families of molecules that are thought to regulate cell sorting and aggregation. By blocking Eph/ephrins or N-cadherin function, changes were measured in neural crest cell migratory behaviors that lead to alterations in sympathetic ganglia formation using a sagittal slice explant culture and 3D confocal time-lapse imaging. The 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 (Kasemeier-Kulesa, 2006).
The hippocampus and septum play central roles in one of the most important spheres of brain function: learning and memory. Although their topographic connections have been known for two decades and topography may be critical for cognitive functions, the basis for hippocamposeptal topographic projection is unknown. Elf-1, a membrane-bound eph family ligand, is a candidate molecular tag for the genesis of the hippocamposeptal topographic projection. Elf-1 is expressed in an increasing gradient from dorsal to ventral septum. Furthermore, Elf-1 selectively allows growth of neurites from topographically appropriate lateral hippocampal neurons, while inhibiting neurite outgrowth by medial hippocampal neurons. Complementary to the expression of Elf-1, an eph family receptor, Bsk, is expressed in the hippocampus in a lateral to medial gradient, consistent with a function as a receptor for Elf-1. Further, Elf-1 specifically binds Bsk, eliciting tyrosine kinase activity. It is concluded that the Elf-1/Bsk ligand-receptor pair exhibits traits of a chemoaffinity system for the organization of hippocamposeptal topographic projections (Gao, 1996).
Neuronal connections are arranged topographically such that the spatial organization of neurons is preserved by their termini in the targets. During the development of topographic projections, axons initially explore areas much wider than the final targets, and mistargeted axons are pruned later. The molecules regulating these processes are not known. The ligands of the Eph family tyrosine kinase receptors may regulate both the initial outgrowth and the subsequent pruning of axons. In the presence of ephrins, the outgrowth and branching of the receptor-positive hippocampal axons are enhanced. However, these axons are induced later to degenerate. These observations suggest that the ephrins and their receptors may regulate topographic map formation by stimulating axonal arborization and by pruning mistargeted axons (Gao, 1999).
Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens/olfactory tubercle, respectively, constituting mesostriatal and mesolimbic pathways. The molecular signals that confer target specificity of different dopaminergic neurons are not known. EphB1 and ephrin-B2, a receptor and ligand of the Eph family, are candidate guidance molecules for the development of these distinct pathways. EphB1 and ephrin-B2 are expressed in complementary patterns in the midbrain dopaminergic neurons and their targets, and the ligand specifically inhibits the growth of neurites and induces the cell loss of substantia nigra, but not ventral tegmental, dopaminergic neurons. These studies suggest that the ligand-receptor pair may contribute to the establishment of distinct neural pathways by selectively inhibiting the neurite outgrowth and cell survival of mistargeted neurons. Ephrin-B2 expression is upregulated by cocaine and amphetamine in adult mice, suggesting that ephrin-B2/EphB1 interaction may play a role in drug-induced plasticity in adults as well (Yue, 1999).
Eph tyrosine kinase receptors and their membrane-bound ephrin ligands mediate cell interactions and participate in several developmental processes. Ligand binding to an Eph receptor results in tyrosine phosphorylation of the kinase domain, and repulsion of axonal growth cones and migrating cells. A subpopulation of ephrin-A5 null mice display neural tube defects resembling anencephaly in man. This is caused by the failure of the neural folds to fuse in the dorsal midline, suggesting that ephrin-A5, in addition to its involvement in cell repulsion, can participate in cell adhesion. During neurulation, ephrin-A5 is co-expressed with its cognate receptor EphA7 in cells at the edges of the dorsal neural folds. Three different EphA7 splice variants, a full-length form and two truncated versions lacking kinase domains, are expressed in the neural folds. Co-expression of an endogenously expressed truncated form of EphA7 suppresses tyrosine phosphorylation of the full-length EphA7 receptor and shifts the cellular response from repulsion to adhesion in vitro. It is concluded that alternative usage of different splice forms of a tyrosine kinase receptor can mediate cellular adhesion or repulsion during embryonic development (Holmberg, 2000).
Because both ephrins and Eph receptors are membrane anchored, suppression of repulsive signal transduction could hypothetically turn these proteins into cell-adhesion molecules. Alternatively, the adhesive effect may be indirect where the ligand-receptor interaction may result in kinase-independent signal transduction that could affect other molecules with adhesive properties. Notably, mutations in the gene encoding the C. elegans Eph receptor, VAB-1, result in defective ventral enclosure, a process resembling neurulation. Analysis of different vab-1 mutants reveals that this process is independent of VAB-1 kinase activity, suggesting that the kinase-independent adhesive functions of Eph family receptors may be evolutionarily conserved. Over the past few years it has become clear that several molecules involved in axon guidance and cell migration, including netrins, semaphorins and slits, have both attractive and repulsive actions. The dual function of these molecules is regulated by interaction with different receptors or by modulation of cyclic-nucleotide-dependent signal transduction pathways. The differential response to ephrin-A5 is quite possibly the first example of how the response to a ligand can be modulated between repulsion or adhesion by alternative use of different splice forms of a receptor (Holmberg, 2000 and references therein).
Ephrins are developmentally regulated molecules that may contribute to axonal pathfinding through their binding to Eph receptor tyrosine kinases. In many cases, ephrins act as negative molecules that stimulate growth cone collapse, although some forms may promote axonal growth. The role played by ephrin-B1 during rat postnatal cerebellar development has been addressed. Ephrin-B1 is expressed by both granule and Purkinje neurons whereas EphB is present in granule neurons in early postnatal cerebellum at a time coincident with axonal and dendrite outgrowth. Stably transfected 3T3 cells overexpressing ephrin-B1 enhance survival and neurite growth from cultured cerebellar granule neurons, an effect that is inhibited by the presence of an excess of a soluble EphB protein. Ephrin-B1-induced neuritogenesis is correlated with an increased expression of certain neuronal-specific microtubule-associated proteins (MAPs). Cerebellar granule neurons plated on stably transfected 3T3 cells overexpressing ephrin-B1 show an up-regulation of the expression of axonal MAPs such as Tau and phosphorylated MAP2C compared with neurons cultured on control 3T3 cells. The level of expression of these axonal MAPs is similar to that found in neurons plated on poly-L-lysine. Interestingly, there is a noteworthy up-regulation of somatodendritic MAPs such as high-molecular-weight MAP2 and mode II-phosphorylated MAP1B in neurons cultured on stably transfected 3T3 cells overexpressing ephrin-B1 compared with neurons plated on either control 3T3 cells or poly-L-lysine. In view of these data, it is suggested that ephrin-B1 favors dendritogenesis of granule neurons during cerebellum development (Moreno-Flores, 2002).
The distribution of members of the Eph family were examined during muscle precursor cell development. The EphA4 receptor tyrosine kinase and its ligand, ephrin-A5, are expressed by muscle precursor cells and forelimb mesoderm in unique spatiotemporal patterns during the period when muscle precursors delaminate from the dermomyotome and migrate into the limb. To test the function of EphA4/ephrin-A5 interactions in muscle precursor migration, targeted, in ovo electroporation was used to express ephrin-A5 ectopically, specifically in the presumptive limb mesoderm. In the presence of ectopic ephrin-A5, Pax7-positive muscle precursor cells are significantly reduced in number in the proximal limb, compared with controls, and congregate abnormally near the lateral dermomyotome. In stripe assays, isolated muscle precursor cells avoid substrate-bound ephrin-A5 and this avoidance is abolished by addition of soluble ephrin-A5. These data suggest that ephrin-A5 normally restricts migrating, EphA4-positive muscle precursor cells to their appropriate territories in the forelimb, disallowing entry into abnormal embryonic regions (Swartz, 2001).
The vertebrate circulatory system is composed of arteries and veins. The functional and pathological differences between these vessels have been assumed to reflect physiological differences such as oxygenation and blood pressure. Ephrin-B2, an Eph family transmembrane ligand, marks arterial but not venous endothelial cells from the onset of angiogenesis. Conversely, Eph-B4, a receptor for ephrin-B2, marks veins but not arteries. ephrin-B2 knockout mice display defects in angiogenesis by both arteries and veins in the capillary networks of the head and yolk sac as well as in myocardial trabeculation. These results provide evidence that differences between arteries and veins are in part genetically determined and suggest that reciprocal signaling between these two types of vessels is crucial for morphogenesis of the capillary beds (Wang, 1998).
The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of vascular morphogenesis. EphrinB2 may have an active signaling role, resulting in bi-directional signal transduction downstream of both ephrinB2 and Eph receptors. To separate the ligand and receptor-like functions of ephrinB2 in mice, the endogenous gene was replaced by cDNAs encoding either carboxyterminally truncated (ephrinB2DeltaC) or, as a control, full-length ligand (ephrinB2WT). While homozygous ephrinB2WT/WT animals are viable and fertile, loss of the ephrinB2 cytoplasmic domain results in midgestation lethality similar to ephrinB2 null mutants (ephrinB2KO). The truncated ligand is sufficient to restore guidance of migrating cranial neural crest cells, but ephrinB2DeltaC/DeltaC embryos show defects in vasculogenesis and angiogenesis very similar to those observed in ephrinB2KO/KO animals. These results indicate distinct requirements of functions mediated by the ephrinB carboxyterminus for developmental processes in the vertebrate embryo (Adams, 2001).
The cues and signaling systems that guide the formation of embryonic blood vessels in tissues and organs are poorly understood. Members of the Eph family of receptor tyrosine kinases and their cell membrane-anchored ligands, the ephrins, have been assigned important roles in the control of cell migration during embryogenesis, particularly in axon guidance and neural crest migration. The role of EphB receptors and their ligands during embryonic blood vessel development has been investigated in Xenopus laevis. In a survey of tadpole-stage Xenopus embryos for EphB receptor expression, expression of EphB4 receptors was detected in the posterior cardinal veins and their derivatives, the intersomitic veins. However, vascular expression of other EphB receptors, including EphB1, EphB2 or EphB3, could not be observed, suggesting that EphB4 is the principal EphB receptor of the early embryonic vasculature of Xenopus. Ephrin-B ligands are expressed complementary to EphB4 in the somites adjacent to the migratory pathways taken by intersomitic veins during angiogenic growth. RNA injection experiments were performed to study the function of EphB4 and its ligands in intersomitic vein development. Disruption of EphB4 signaling by dominant negative EphB4 receptors or misexpression of ephrin-B ligands in Xenopus embryos results in intersomitic veins growing abnormally into the adjacent somitic tissue. These findings demonstrate that EphB4 and B-class ephrins act as regulators of angiogenesis, possibly by mediating repulsive guidance cues to migrating endothelial cells (Helbling, 2000).
Angiogenesis can be divided into three phases: initiation (induction of sprouting); invasion (cell proliferation, migration and matrix degradation), and maturation (remodeling, lumen formation and differentiation of endothelial cells). To date, three growth factor systems (VEGF, angiopoietins and ephrins) have been identified as critical players of angiogenesis. As revealed by the vascular phenotypes obtained from gene inactivation and gain-of-function experiments, each signaling system appears to have an essential role during at least one particular phase of angiogenic growth of intersomitic veins. A tentative hierarchy of the signaling systems essential for intersomitic vein development may therefore be deduced. Analysis of heterozygous VEGF mutant embryos, which are less affected than those homozygously deficient for VEGF, has revealed a strong decrease in intersomitic vein sprouts. VEGF is therefore involved in the initiation of intersomitic vein sprouting. Angiopoietin-1 and its receptor Tie-2 appear to be critical later during maturation and stabilization of the intersomitic veins. Indeed, mutant mice initially form intersomitic veins, which in case of Ang-1 mutants undergo subsequent regression in older embryos. Finally, analysis of ephrin-B2- and double EphB2/EphB3-deficient mice as well Xenopus embryos disrupted in EphB4 signaling indicate a requirement for EphB receptors and their ligands during the invasion phase of intersomitic vein development. Therefore, EphB signaling appears to act downstream of VEGF and its receptors, but upstream of the angiopoietin signaling system (Helbling, 2000 and references therein).
EphrinB2, a transmembrane ligand of EphB receptor tyrosine kinases, is specifically expressed in arteries. In ephrinB2 mutant embryos, there is a complete arrest of angiogenesis. However, ephrinB2 expression is not restricted to vascular endothelial cells, and it has been proposed that its essential function may be exerted in adjacent mesenchymal cells. Mice have been generated in which ephrinB2 is specifically deleted in the endothelium and endocardium of the developing vasculature and heart. Such a vascular-specific deletion of ephrinB2 results in angiogenic remodeling defects identical to those seen in the conventional ephrinB2 mutants. These data indicate that ephrinB2 is required specifically in endothelial and endocardial cells for angiogenesis, and that ephrinB2 expression in perivascular mesenchyme is not sufficient to compensate for the loss of ephrinB2 in these vascular cells (Gerety, 2002).
Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between the endocardium and myocardium. Ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1, and BMP10 expression and signaling, and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression, and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro-cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. It is suggested that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease (Grego-Bessa, 2007).
The epidermis of the nematode C. elegans is an epithelium that undergoes epiboly during embryogenesis, where it encloses the embryo ventrally. Ventral enclosure is the result of epidermal cell shape changes and involves two steps: (1) the extension of four anterior leading cells to the ventral midline, and (2) enclosure of the posterior epidermis. A catenin/cadherin system is required for normal movement of the anterior epidermal cells, and likely functions within epidermal cells to modulate cytoskeletal behavior. The Eph receptor VAB-1 is required in neurons for epidermal morphogenesis during C. elegans embryogenesis. Two models have been proposed for the nonautonomous role of VAB-1: neuronal VAB-1 might signal directly to epidermis, or VAB-1 signaling between neurons might be required for epidermal development. The ephrin VAB-2 (also known as EFN-1) is a ligand for VAB-1 and can function in neurons to regulate epidermal morphogenesis. In the absence of VAB-1 signaling, ephrin-expressing neurons are disorganized. vab-2/efn-1 mutations synergize with vab-1 kinase alleles, suggesting that VAB-2/EFN-1 may partly function in a kinase-independent VAB-1 pathway. These data indicate that ephrin signaling between neurons is required nonautonomously for epidermal morphogenesis in C. elegans (Chin-Sang, 1999).
How might lack of VAB-1 in neurons cause defects in the epidermis? Two models have been proposed for this nonautonomy of VAB-1: the 'steric hindrance' model and the 'reverse signaling' model; these models are not mutually exclusive. In the steric hindrance model, VAB-1 signaling operates between neuroblasts and neurons, and lack of VAB-1 causes neurons to be disorganized; enclosure is defective because the epidermal cells cannot move over the abnormal neuronal substrate. In the reverse signal model, VAB-1 signals directly from neurons to the epidermal cells; in vab-1 mutants such cues are absent and epidermal cells fail to migrate normally. Analysis of vab-2/efn-1 supports a steric hindrance model for the nonautonomous role of ephrin signaling. VAB-2/EFN-1 is mostly expressed in neuroblasts or neurons, and neuron-specific expression of VAB-2/EFN-1 can partly rescue epidermal defects of vab-2/efn-1 mutants, showing that VAB-2/EFN-1 can function in neurons to regulate epidermal development. Furthermore, mutations in the VAB-1 receptor cause disorganization of the VAB-2/EFN-1-expressing neurons. The spreading of VAB-2/EFN-1-expressing cells in vab-1 mutants likely reflects abnormal cell migration or adhesion, since vab-1 mutants do not display cell fate transformations (Chin-Sang, 1999).
This role of VAB-1 in preventing cell spreading is strikingly reminiscent of the cell sorting functions of Eph signaling in vertebrate neurogenesis. It has been concluded that VAB-2/EFN-1 signaling to VAB-1 occurs between neuronal precursors, and regulates cell adhesion or movement. In the absence of VAB-1 or VAB-2/EFN-1, neuroblasts fail to close up the ventral gastrulation cleft and as a result, descendant neurons are disorganized. Disorganized neurons might block epidermal movements directly (true 'steric hindrance') or indirectly: for example, the boundary between VAB-1 and VAB-2/EFN-1-expressing cells might attract migrating epidermal cells; if this boundary is disorganized, epidermal migrations would be disrupted. An analogous situation might occur in vertebrate angiogenesis, where ephrin signaling between endocardial cells is required for the development of overlying myocardial trabeculae. Such models do not rule out signaling from neurons to epidermis, possibly via other ephrins; however, of the three other C. elegans ephrins, at least two are expressed mainly in neurons. The small number of Eph receptors and ephrins in C. elegans suggests that it will be feasible to dissect the complete network of Eph/ephrin signaling in a simple animal (Chin-Sang, 1999).
The C. elegans genome encodes a single Eph receptor tyrosine kinase, VAB-1, which functions in neurons to control epidermal morphogenesis. Four members of the ephrin family of ligands for Eph receptors have been identified in C. elegans. Three ephrins (EFN-1/VAB-2, EFN-2 and EFN-3) have been previously shown to function in VAB-1 signaling. Mutations in the gene mab-26 affect the fourth C. elegans ephrin, EFN-4. efn-4 also functions in embryonic morphogenesis, and it is expressed in the developing nervous system. Interestingly, efn-4 mutations display synergistic interactions with mutations in the VAB-1 receptor and in the EFN-1 ephrin, indicating that EFN-4 may function independently of the VAB-1 Eph receptor in morphogenesis. Mutations in the LAR-like receptor tyrosine phosphatase PTP-3 and in the Semaphorin-2A homolog MAB-20 disrupt embryonic neural morphogenesis. efn-4 mutations synergize with ptp-3 mutations, but not with mab-20 mutations, suggesting that EFN-4 and Semaphorin signaling could function in a common pathway or in opposing pathways in C. elegans embryogenesis (Chin-Sang, 2002).
Ephrins and semaphorins regulate a wide variety of developmental processes, including axon guidance and cell migration. The roles of the ephrin EFN-4 and the semaphorin MAB-20 have been studied in patterning cell-cell contacts among the cells that give rise to the ray sensory organs of Caenorhabditis elegans. In wild-type, contacts at adherens junctions form only between cells belonging to the same ray. In efn-4 and mab-20 mutants, ectopic contacts form between cells belonging to different rays. Ectopic contacts also occur in mutants in regulatory genes that specify ray morphological identity. efn-4 and mab-20 reporters were used to investigate whether these ray identity genes function through activating expression of efn-4 or mab-20 in ray cells. mab-20 reporter expression in ray cells is unaffected by mutants in the Pax6 homolog mab-18 and the Hox genes egl-5 and mab-5, suggesting that these genes do not regulate mab-20 expression. mab-18 is found to be necessary for activating efn-4 reporter expression, but this activity alone is not sufficient to account for mab-18 function in controlling cell-cell contact formation. In egl-5 mutants, efn-4 reporter expression in certain ray cells is increased, inconsistent with a simple repulsion model for efn-4 action. The evidence indicates that ray identity genes primarily regulate ray morphogenesis by pathways other than through regulation of expression of semaphorin and ephrin (Hahn, 2003).
Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migration processes during development. The molecular cloning and tissue distribution are reported 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. These observations suggest Eph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation (Chan, 2001).
Eph receptor tyrosine kinases (RTK) and their ephrin ligands are involved in the transmission of signals that regulate cytoskeletal organization and cell migration, and are expressed in spatially restricted patterns at discrete phases during embryogenesis. Loss of function mutants of Eph RTK or ephrin genes result in defects in neuronal pathfinding or cell migration. Soluble forms of human EphA3 and ephrin-A5, acting as dominant negative inhibitors, interfere with early events in zebrafish embryogenesis. Exogenous expression of both proteins results in dose-dependent defects in somite development and organization of the midbrain-hindbrain boundary and hindbrain. The nature of the defects as well as the distribution and timing of expression of endogenous ligands/receptors for both proteins suggest that Eph-ephrin interaction is required for the organization of embryonic structures by coordinating the cellular movements of convergence during gastrulation (Oates, 1999).
Somitogenesis involves the segmentation of the paraxial mesoderm into units along the anteroposterior axis. A role for Eph and ephrin signaling in the patterning of presomitic mesoderm and formation of the somites is shown. Ephrin-A-L1 and ephrin-B2 are expressed in an iterative manner in the developing somites and presomitic mesoderm, as is the Eph receptor EphA4. The role of these proteins was examined by injection of RNA, encoding dominant negative forms of Eph receptors and ephrins. Interruption of Eph signaling leads to abnormal somite boundary formation and reduced or disturbed myoD expression in the myotome. Disruption of Eph family signaling delays the normal down-regulation of her1 and Delta D expression in the anterior presomitic mesoderm and disrupts myogenic differentiation. It is suggested that Eph signaling has a key role in the translation of the patterning of presomitic mesoderm into somites (Durbin, 1998).
Targeted inactivation of the Eph receptor ligand ephrinB1 in mouse causes perinatal lethality, edema, defective body wall closure, and skeletal abnormalities. In the thorax, sternocostal connections are arranged asymmetrically and sternebrae are fused -- defects that are phenocopied in EphB2/EphB3 receptor mutants. In the wrist, loss of ephrinB1 leads to abnormal cartilage segmentation and the formation of additional skeletal elements. It is concluded that ephrinB1 and B class Eph receptors provide positional cues required for the normal morphogenesis of skeletal elements. Another malformation, preaxial polydactyly, is exclusively seen in heterozygous females in which expression of the X-linked ephrinB1 gene is mosaic, so that ectopic EphB-ephrinB1 interactions led to restricted cell movements and the bifurcation of digital rays. These findings suggest that differential cell adhesion and sorting might be relevant for an unusual class of X-linked human genetic disorders, in which heterozygous females show more severe phenotypes than hemizygous males (Compagni, 2003).
How can ephrinB1-EphB interactions regulate segmentation and patterning processes? One possibility might be that enhanced proliferation of chondrogenic cells leads to a size expansion of cartilaginous condensations and eventually triggers the bifurcation of digits. Arguing against such a passive growth-controlled mechanism, ephrinB1 mutant limbs do not contain enlarged skeletal elements, and numbers of proliferating cells are comparable between mosaic areas. The A class receptor EphA7 facilitates chondrogenic condensation, but ephrinB1 KO, KO/+, and control mesenchyme show very similar chondrogenic capacities. Furthermore, the polydactyly in ephrinB1 KO/+ limbs is independent from changes in the Shh pathway (Compagni, 2003).
Interdigital zones forming between bifurcated digits in ephrinB1 KO/+ mutants are largely devoid of programmed cell death. BMPs and their receptors regulate apoptosis of interdigital cells, and, indeed, transcripts for bmp2, bmpr-1a, and msx-1 were initially absent in ectopic IDZs, which instead showed residual expression of the chondrogenic marker sox9. This indicates that morphological alterations induced by ectopic EphB-ephrinB1 signaling precede and are, to a certain extent, independent from changes in the genetic programs controlling the development of digits and IDZs (Compagni, 2003).
Previous work has shown that the Eph/ephrin system plays important roles in the guidance of neuronal growth cones and migrating cells by providing repulsive cues. Moreover, complementary patterns of Eph and ephrin expression in adjacent hindbrain rhombomeres are critically involved in restricting cell movement and intermingling, as confirmed by studies in zebrafish embryos. In mosaic KO/+ limbs, evidence was found for reduced cell mixing between mosaic areas of mesenchyme expressing EphB receptors and ephrinB1, respectively. It is hypothesized that EphB-ephrinB1 signaling interfaces provide interacting cells with repulsive cues, which, when properly located, lead to diverging cell movements and the splitting of growing digits (Compagni, 2003).
During somitogenesis, segmental patterns of gene activity provide the instructions by which mesenchymal cells epithelialize and form somites. Various members of the Eph family of transmembrane receptor tyrosine kinases and their Ephrin ligands are expressed in a segmental pattern in the rostral presomitic mesoderm. This pattern establishes a receptor/ligand interface at each site of somite furrow formation. This study uses the fused somites (fss) mutant as an in vivo system to study the role of Eph/Ephrin signaling during somite morphogenesis. fss encodes Tbx24, a T box transcription factor involved in maturation of the presomitic mesoderm. fss mutants lack anterior-posterior polarity within presumptive segments of the rostral PSM and fail to form somites. In the fused somites mutant, lack of intersomitic boundaries and epithelial somites is accompanied by a lack of Eph receptor/Ephrin signaling interfaces. These observations suggest a role for Eph/Ephrin signaling in the regulation of somite epithelialization. Restoration of Eph/Ephrin signaling in the paraxial mesoderm of fss mutants rescues most aspects of somite morphogenesis. (1) Restoration of bidirectional or unidirectional EphA4/Ephrin signaling results in the formation and maintenance of morphologically distinct boundaries. (2) Activation of EphA4 leads to the cell-autonomous acquisition of a columnar morphology and apical redistribution of ß-catenin, aspects of epithelialization characteristic of cells at somite boundaries. (3) Activation of EphA4 leads to nonautonomous acquisition of columnar morphology and polarized relocalization of the centrosome and nucleus in cells on the opposite side of the forming boundary. These nonautonomous aspects of epithelialization may involve interplay of EphA4 with other intercellular signaling molecules. These results demonstrate that Eph/Ephrin signaling is an important component of the molecular mechanisms driving somite morphogenesis. A new role is proposed for Eph receptors and Ephrins as intercellular signaling molecules that establish cell polarity during mesenchymal-to-epithelial transition of the paraxial mesoderm (Barrios, 2003).
The definitive retinal progenitors of the eye field are specified by transcription factors that both promote a retinal fate and control cell movements that are critical for eye field formation. However, the molecular signaling pathways that regulate these movements are largely undefined. Both the FGF and ephrin pathways impact eye field formation. Activating the FGF pathway before gastrulation represses cellular movements in the presumptive anterior neural plate and prevents cells from expressing a retinal fate, independent of mesoderm induction or anterior-posterior patterning. Inhibiting the FGF pathway promotes cell dispersal and significantly increases eye field contribution. EphrinB1 reverse signaling is required to promote cellular movements into the eye field, and can rescue the FGF receptor-induced repression of retinal fate. These results indicate that FGF modulation of ephrin signaling regulates the positioning of retinal progenitor cells within the definitive eye field (Moore, 2004).
Retinal development consists of a series of steps that progressively restrict the available cell fates. First, a subset of embryonic cells are prevented from expressing a retinal fate by inherited maternal factors, whereas others become biased toward retinal fates due to their position within the neural inductive field of the animal hemisphere. As the CNS is regionalized, part of the anterior neural plate is specified as the eye field. Potential retinal progenitors need to be positioned within the eye field to receive the local environmental signals that will direct their ultimate fates. Only after these steps are accomplished do the steps of eye organogenesis, cellular lamination, and phenotype specification occur. Although there has been great progress in understanding how retinal cell type specification occurs, the molecular mechanisms that control which embryonic cells become specified as the definitive retinal progenitors in the eye field remain largely undefined (Moore, 2004).
An accepted hypothesis of how the eye field forms is that signals from surrounding anterior structures regionalize the anterior neural plate. The presumptive eye field then expresses several transcription factors that initiate the retina developmental program, e.g., rx1, pax6, and six3. However, cellular movements during gastrulation and neurulation, directed in part by eye field transcription factors, also are critical, and the signaling factors involved in these early steps of eye field formation have not been identified (Moore, 2004).
Several FGF family members have been implicated in affecting cell movements during gastrulation, and the anterior expression patterns of some FGFs and their receptors are consistent with a role in the morphogenetic movements of eye field cells. Therefore, whether FGF signaling prior to gastrulation plays a role in determining which embryonic cells form the eye field was investigated. Using a constitutively active FGF receptor, enhanced FGF signaling was demonstrated to prevent the normal retinal progenitors from populating the presumptive eye field, suggesting that low levels of FGF signaling are normally required for cells to adopt a retinal fate. This was confirmed by demonstrating that reduced FGF signaling, accomplished by expression of a dominant-negative receptor, enhances the number of cells that become retinal progenitors. It is further reported that ephrinB1 signaling during gastrulation is required for retinal progenitors to move into the eye field, and that this movement can be modified by activating the FGF pathway. These results demonstrate that FGF modulation of ephrin signaling is important for establishing the bona fide retinal progenitors in the anterior neural plate (Moore, 2004).
The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of embryonic blood vascular morphogenesis. However, the molecular mechanisms required for ephrinB2 transduced cellular signaling in vivo have not been characterized. To address this question, two sets of knock-in mice have been generated: ephrinB2DeltaV mice expressed ephrinB2 lacking the C-terminal PDZ interaction site, and ephrinB25F mice expressed ephrinB2 in which the five conserved tyrosine residues were replaced by phenylalanine to disrupt phosphotyrosine-dependent signaling events. This analysis revealed that the homozygous mutant mice survive the requirement of ephrinB2 in embryonic blood vascular remodeling. However, ephrinB2DeltaV/DeltaV mice exhibit major lymphatic defects, including a failure to remodel their primary lymphatic capillary plexus into a hierarchical vessel network, hyperplasia, and lack of luminal valve formation. Unexpectedly, ephrinB25F/5F mice display only a mild lymphatic phenotype. These studies define ephrinB2 as an essential regulator of lymphatic development and indicate that interactions with PDZ domain effectors are required to mediate its functions (Makinen, 2005).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.