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).
Neural crest cells migrate segmentally through the rostral half of each trunk somite due to inhibitory influences of ephrins and other molecules present in the caudal-half of somites. To examine the potential role of Notch/Delta signaling in establishing the segmental distribution of ephrins, neural crest migration and ephrin expression were examined in Delta-1 mutant mice. Using Sox-10 as a marker, it was noted that neural crest cells moved through both rostral and caudal halves of the somites in mutants, consistent with the finding that ephrinB2 levels are significantly reduced in the caudal-half somites. Later, mutant embryos had aberrantly fused and/or reduced dorsal root and sympathetic ganglia, with a marked diminution in peripheral glia. These results show that Delta-1 is essential for proper migration and differentiation of neural crest cells. Interestingly, absence of Delta-1 leads to diminution of both neurons and glia in peripheral ganglia, suggesting a general depletion of the ganglion precursor pool in mutant mice (De Bellard, 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).
During development of the vertebrate hindbrain, regulatory gene expression is confined to precise segmental domains. Studies of cell lineage and gene expression suggest that establishment of these domains may involve a dynamic regulation of cell identity and restriction of cell movement between segments. A dominant negative approach has been taken to interfere with the function of Sek-1, a member of the Eph-related receptor tyrosine kinase family expressed in rhombomeres r3 and r5. In Xenopus and zebrafish embryos expressing truncated Sek-1, which lack kinase sequences, expression of r3/r5 markers occurs in adjacent even-numbered rhombomeres, in domains contiguous with r3 or r5. This disruption is rescued by full-length Sek-1, indicating a requirement for the kinase domain in the segmental restriction of gene expression. These data suggest that Sek-1, perhaps with other Eph-related receptors, is required for interactions that regulate the segmental identity or movement of cells (Xu, 1995).
A dominant-negative approach was taken in the zebrafish embryo to interfere with the function of Rtk1, an Eph-related RTK expressed in the developing diencephalon. Expression of a truncated receptor leads to expansion of the eye field into diencephalic territory and loss of diencephalic structures, indicating a role for Rtk1 in patterning the developing forebrain (Xu, 1996).
Eph family receptor tyrosine kinases have been proposed to control axon guidance and fasciculation. To address the biological functions of the Eph family member Nuk, two mutations in the mouse germline have been generated: a protein null allele (Nuk1) and an allele that encodes a Nuk-beta gal fusion receptor lacking the tyrosine kinase and C-terminal domains (Nuk[lacZ]). In Nuk1 homozygous brains, the majority of axons forming the posterior tract of the anterior commissure migrate aberrantly to the floor of the brain, resulting in a failure of cortical neurons to link the two temporal lobes. These results indicate that Nuk, a receptor that binds transmembrane ligands, plays a critical and unique role in the pathfinding of specific axons in the mammalian central nervous system (Henkemeyer, 1996).
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).
The visual cortex in primates is distributed into distinct areas: cytoarchitectonic, physiologic, and connectional and include the striate cortex (V1) and the extrastriate cortex, consisting of V2 and numerous higher association areas. The innervation of distinct visual cortical areas by the thalamus is especially segregated in primates, such that the lateral geniculate (LG) nucleus specifically innervates striate cortex, whereas pulvinar projections are confined to extrastriate cortex. The molecular bases for the parcellation of the visual cortex and thalamus, as well as the establishment of reciprocal connections between distinct compartments within these two structures, are largely unknown. Prospective visual cortical areas and corresponding thalamic nuclei in the embryonic rhesus monkey can be defined by combinatorial expression of genes encoding Eph receptor tyrosine kinases and their ligands, the ephrins, prior to obvious cytoarchitectonic differentiation within the cortical plate and before the establishment of reciprocal connections between the cortical plate and thalamus. These results indicate that molecular patterns of presumptive visual compartments in both the cortex and thalamus can form independently of one another and suggest a role for EphA family members in both compartment formation and axon guidance within the visual thalamocortical system (Sestan, 2001).
To evaluate the influence of reciprocal connections between the cortex and thalamus on differential gene expression in these structures, Eph family expression was evaluated at embryonic day 65 (E65), before all of the neurons that eventually receive input from the thalamus have been generated (prospective layer IV), and, thus, before patterned thalamocortical connectivity has been established. Patterning of expression within the CP is obvious at E65: EphA6 and EphA7 are expressed in overlapping but distinct gradients that selectively label the posterior half of the cortical plate (CP), a region that corresponds spatially to future visual cortex. Furthermore, the expression of another receptor, EphA3, and a ligand, ephrin-A5, demarcates distinct compartments within the EphA6- and EphA7-positive prospective visual CP: EphA3 expression comprises a plus-minus pattern, with high anterior levels, being absent in the posterior, whereas ephrin-A5 expression respects the same border, but its expression varies according to prospective laminae. At this stage, levels of ephrin-A5 expression are constant within the most superficial CP along the anteroposterior axis but increase selectively in the deepest strata of the posterior CP. AChE historeactivity was used to visualize the location of pulvinar fibers, which have entered the intermediate zone but have not yet invaded the CP at E65. Ephrin-A5 is uniformly expressed in the CP overlying pulvinar fibers but is restricted to the deepest strata of the CP in AChE-poor cerebral wall (prospective layers V and VI). This data, in combination with extrapolation from localization at older ages, led to the conclusion that differences in expression of ephrin-A5 and EphA3 at E65 correspond to distinctions between presumptive striate and extrastriate cortex. Thus, Eph family gene expression distinguish between the two regions at a time when there are no other known areal landmarks within the visual CP. In addition, these molecular differences exist in the absence of reciprocal synaptic connections between the CP and thalamus, indicating that they emerge independently (Sestan, 2001).
Analyzed next was whether corresponding patterns of Eph family expression exist in the thalamus at E65. Despite the lack of reciprocal connections between the CP and thalamus at this age, patterning of ephrin-A5 and select Eph receptors was observed within the developing thalamus. Ephrin-A5 is predominantly expressed within the ventrolateral nucleus that eventually innervates somatosensory CP, whereas EphA3, EphA6, and EphA7 are most abundant in the pulvinar and to a lesser extent in the geniculate body that eventually innervate visual CP. Moreover, distinct gradients of EphA6 and EphA7 expression are present in the pulvinar. This patterned expression within the thalamus, in conjunction with the complex neocortical expression that has been documented, supports a model in which combinatorial Eph family expression independently establishes cellular groupings within the thalamus and cortex and then influences reciprocal innervation between these structures (Sestan, 2001).
Eph receptors have been implicated in cell-to-cell interaction during embryogenesis. EphA2 mutant mice were generated using a gene trap method. Homozygous mutant mice develop short and kinky tails. In situ hybridization using a Brachyury probe found the notochord to be abnormally bifurcated at the caudal end between 11.5 and 12.5 days post coitum. EphA2 is expressed at the tip of the tail notochord, while one of its ligands, ephrinA1, is expressed at the tail bud in normal mice. In contrast, EphA2-deficient notochordal cells are spread broadly into the tail bud. These observations suggest that EphA2 and its ligands are involved in the positioning of the tail notochord through repulsive signals between cells expressing these molecules on the surface (Naruse-Nakajima, 2001).
EphB receptor tyrosine kinases and ephrin-B ligands regulate several types of cell-cell interactions during brain development, generally by modulating the cytoskeleton. EphB/ephrinB genes are expressed in the developing neural tube of early mouse embryos with distinct overlapping expression in the ventral midbrain. To test EphB function in midbrain development, mouse embryos compounds homozygous for mutations in the EphB2 and EphB3 receptor genes were examined for early brain phenotypes. These mutants display a morphological defect in the ventral midbrain, specifically an expanded ventral midline evident by embryonic day E9.5-10.5, which forms an abnormal protrusion into the cephalic flexure. The affected area is comprised of cells that normally express EphB2 and ephrin-B3. A truncated EphB2 receptor causes a more severe phenotype than a null mutation, implying a dominant negative effect through interference with EphB forward (intracellular) signaling. In mutant embryos, the overall number, size, and identity of the ventral midbrain cells are unaltered. Therefore, the defect in ventral midline morphology in the EphB2;EphB3 compound mutant embryos appears to be caused by cellular changes that thin the tissue, forcing a protrusion of the ventral midline into the cephalic space. These data suggests a role for EphB signaling in morphological organization of specific regions of the developing neural tube (Altick, 2005).
The Eph-related receptor tyrosine kinase gene, Cek-8, is expressed in mesenchyme at the tip of chick limb buds, with high levels of transcripts posteriorly and apically but fading out anteriorly. Expression of Cek-8 in distal mesenchyme is regulated by apical ridge- and FGF-polarizing signals and retinoic acid, and is uniform across the anteroposterior axis in talpid3 mutants. These data indicate that Cek-8 expression responds to regulatory signals during limb patterning and suggest that this receptor tyrosine kinase may have a role in coordinating responses to signals in the progress zone of early buds. Later on in limb development, Cek-8 expression is associated with cell condensations that form tendons and their attachments to cartilage rudiments and then in developing feather buds (Patel, 1996).
A highly dynamic expression pattern of the chick EphA7 gene occurs in the developing limb. Expression is detected in discrete domains of the dorsal mesenchyme from 3 days of incubation. The expressing cells are adjacent to the routes where axons grow to innervate the limb at several key points: the region of plexus formation, the bifurcation between dorsal and ventral fascicles, and the pathway followed by axons innervating the dorsal muscle mass. These results suggested a role for EphA7 in cell-cell contact-mediated signaling in dorsal limb patterning and/or axon guidance. Experimental manipulations were carried in the chick embryo wing bud to alter the dorsoventral patterning of the limb. The analyses of EphA7 expression and innervation in the operated wings indicate that a signal emanating from the dorsal ectoderm regulates EphA7 in such a way that, in its absence, the wing bud lacks EphA7 expression and shows innervation defects at the regions where the gene is downregulated. EphA7 downregulation in the dorsal mesenchyme after dorsal ectoderm removal is more rapid than that of Lmx-1, the gene known to mediate dorsalization in response to the ectodermal signal. These results add a new gene to the dorsalization signaling pathway in the limb. Moreover, they implicate the Eph receptor family in the patterning and innervation of the developing limb, extending its role in axon pathfinding to the distal periphery (Araujo, 1998).
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).
B61, a cytokine-inducible endothelial gene product, is the ligand for the Eck receptor protein tyrosine kinase (RPTK). Expression of a B61-immunoglobulin chimera shows that B61 could act as an angiogenic factor in vivo and a chemoattractant for endothelial cells in vitro. The Eck RPTK is activated by tumor necrosis factor-alpha (TNF-alpha) through induction of B61, and an antibody to B61 attenuates angiogenesis induced by TNF-alpha but not by basic fibroblast growth factor. This finding suggests the existence of an autocrine or paracrine loop involving activation of the Eck RPTK by its inducible ligand B61 after an inflammatory stimulus, the net effect of which would be to promote angiogenesis, a hallmark of chronic inflammation (Pandey, 1995a) .
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).
Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. A phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicates a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system (Adams, 1999).
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).
During development, Eph receptors mediate the repulsive axon guidance function of ephrins, a family of membrane attached ligands with their own receptor-like signaling potential. In cultured glutamatergic neurons, EphB2 receptors associate with NMDA receptors at synaptic sites and have been suggested to play a role in synaptogenesis. Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways. Mice lacking EphB2 have normal hippocampal synapse morphology, but display defects in synaptic plasticity. In EphB2-/- hippocampal slices, protein synthesis-dependent long-term potentiation (LTP) is impaired, and two forms of synaptic depression are completely extinguished. Interestingly, targeted expression of a carboxy-terminally truncated form of EphB2 rescues the EphB2 null phenotype, indicating that EphB2 kinase signaling is not required for these EphB2-mediated functions (Grunwald, 2001).
Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain show wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor have reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current is reduced in dentate granule neurons in EphB2 null mice, as is synaptically localized NR1 as revealed by immunogold localization. EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function (Henderson, 2001).
Mutations in the C. elegans vab-1 gene disrupt the coordinated movements of cells during two periods of embryogenesis. vab-1 mutants are defective in the movement of neuroblasts during closure of the ventral gastrulation cleft and in the movements of epidermal cells during ventral enclosure of the embryo by the epidermis. vab-1 encodes a receptor tyrosine kinase of the Eph family. Disruption of the kinase domain of VAB-1 causes weak mutant phenotypes, indicating that VAB-1 may have both kinase-dependent and kinase-independent activities. VAB-1 is expressed in neurons during epidermal enclosure and is required in these cells for normal epidermal morphogenesis, demonstrating that cell-cell interactions are required between neurons and epidermal cells for epidermal morphogenesis (George, 1998).
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).
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).
In vertebrate embryos, neural crest cell migration and motor axon outgrowth are restricted to rostral somite halves by repulsive factors located in the caudal somite compartment. Two Eph family transmembrane ligands, Lerk2 and HtkL, are expressed in caudal somite halves, and crest cells and motor axons express receptors for these ligands. In several independent in vitro assays, preclustered ligand-Fc fusion proteins can repulsively guide both crest migration and motor axon outgrowth. These repulsive activities depend on a graded or discontinuous presentation of the ligands when tested in the context of permissive substrates, such as laminin or fibronectin. These results identify Lerk2 and HtkL as potential determinants of segmental pattern in the peripheral nervous system (Wang, 1997).
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).
The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation suggests that there is a role for long range signaling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. This could be achieved by the expression of the BMP antagonist, Follistatin. Local application of Follistatin leads to the development of ectopic feather buds. A suggestion is made that Follistatin locally antagonizes the action of the BMPs and so permits the cellular changes associated with feather placode formation. Evidence is provided for the role of short range signaling during feather formation. Changes in cellular morphology in feather placodes are correlated with the expression of the gene Eph-A4 that encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. Expression of this gene precedes cellular reorganization required for feather bud formation (Patel, 1999).
Transgenic mice over-expressing the EphB4 receptor tyrosine kinase in the kidney were established. The EphB4 protein localizes to the developing tubular system of both control and transgenic newborn mice. In transgenic adults, transgene expression persists in the proximal tubules and the Bowman's capsules, structures that were not stained in control kidneys. The glomeruli of control animals consist of regular, round vascular baskets with clearly discernable afferent and efferent arterioles. In contrast, approximately 40% of the transgenic glomeruli had an irregular shrivelled appearance and many exhibited fused, horse shoe-like afferent and efferent arterioles bypassing the glomerulus. These abnormal glomerular structures are very reminiscent of aglomerular vascular shunts, a human degenerative glomerulopathy of unknown aetiology (Andres, 2003).
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).
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