Recent studies have implicated Eph-related receptor tyrosine kinases and their membrane-bound ligands in restricting or stimulating the movement of cells and axons. Members of these large families of receptors and ligands fall into two major binding specificity classes, in which the GPI-anchored subgroup of ligands can each bind to all members of a subgroup of receptors, whereas the transmembrane ligands interact with a distinct subgroup of receptors. Analysis of expression patterns is therefore important in order to understand which receptor-ligand interactions occur in vivo. Mouse orthologs of five members of the ligand family have been cloned and their developmental expression has been analysed in detail, both in comparison with one other, and with the receptor specificity class with which they interact. B61, AL-1/RAGS, LERK4, and ELF-1, members of the GPI-anchored subgroup of ligands, have both distinct and overlapping aspects to their expression in early mesoderm, somites, and branchial arches; in complex, dynamic patterns in the limb, and in spatial domains and specific neurons in the CNS. Similarly, Elk-L is expressed in hindbrain segments, the roof plate, and floor plate, overlapping in these locations the expression of other transmembrane ligands, but Elk-L has distinct expression in somites. The expression domains of ligands are complementary to those of the corresponding receptors in a number of tissues, including the midbrain, hindbrain, and differentiating limbs, consistent with potential roles in restricting cell movement. There are some overlaps in the expression of receptors and ligands, for example, in somites and the early limb. Taken together with previous studies showing that Eph-related receptors also have distinct but overlapping expression patterns, these data indicate that each ligand may have stage- and tissue-specific interactions with an individual member or multiple members of the receptor family (Flenniken, 1996).
Neuron-target interaction is a key feature in the establishment of neuronal networks. However, the underlying mechanism remains unclear. At the time of target innervation, Bsk, an eph family receptor, is expressed at high levels in several brain regions, including the hippocampus, olfactory bulb, and retina. To study whether the ligands are expressed in the target tissues, the expression of Bsk ligands was examined using a ligand-affinity probe, Bsk-AP, which consisted of the extracellular domain of Bsk fused in frame with a human placental alkaline phosphatase. These analyses show that the ligands are expressed at high levels in the developing septum, hypothalamus, olfactory neural epithelium, and tectum. In situ hybridization studies reveal that at least three different factors are responsible for the Bsk-AP binding. In the septum, Elf-1, Lerk3 (Eff-2), and AL-1/Lerk7 are transcribed. In the hypothalamus, AL-1/Lerk7 is the ligand detected by Bsk-AP. In the olfactory system, high levels of Lerk3 are detected in the sensory neurons. Both Elf-1 and AL-1/Lerk7 are present in the tectum. These ligand-positive areas are known to be anatomically connected to Bsk-expressing regions. These observations strongly suggest that Bsk and the ligands participate in neuron-target interactions in multiple systems and provide support for their involvement in topographic projection (Zhang, 1996).
The task of organizing the vast array of longitudinal axons that constitute the ventral and lateral funiculi, which project alongside or near the vertebrate midline of the developing vertebrate spinal cord, is likely to be relegated to a number of discrete repellent guidance cues that act upon axons emanating from multiple populations of functionally distinct interneurons. The Eph family of receptor tyrosine kinases are particularly good candidates since they interact exclusively with membrane-associated ligands, exhibit dynamic and spatially restricted expression patterns in the developing central nervous system, and together with the ephrins, mediate axonal patterning and pathway selection through contact-dependent repulsion in a variety of neural systems. Given their transmembrane structure, B-class ephrins are especially suited to function as highly localized, contact-dependent repellents. Although previous studies have documented a role for these proteins in repulsive axon guidance, the function of the transmembrane ephrins in the spinal cord proper are presently unknown. mRNA encoding all three transmembrane ligands is expressed in the floor plate during commissural axon pathfinding. Furthermore, B-class ephrin protein is tightly localized to the lateral floor plate margins, in immediate proximity to longitudinal fiber tracts formed by decussated commissural axons. Strikingly, expression of B-class Eph receptors is detected on only those segments of commissural axons that have crossed the floor plate and turned into the longitudinal axis. It is proposed that the onset of EphB receptor expression subsequent to midline crossing plays an important role in maintaining the longitudinal trajectory of commissural axons through a repulsive interaction with transmembrane ephrins situated at the lateral floor plate boundaries. In support of this interpretation, it is shown that all three B-class ephrins can induce the collapse of a subset of commissural growth cones in vitro. This is the first identification of a membrane-associated factor that directly promotes the collapse of vertebrate commissural growth cones (Imondi, 2000).
To identify molecules involved in neurogenesis, monoclonal antibodies were raised against embryonic day 12.5 mouse telencephalon. One antibody, monoclonal antibody 25H11, stains predominantly the ventricular zone of the anterior and lateral telencephalon. Purification of the 25H11 antigen, a 47 kDa integral membrane protein, from mouse telencephali reveals its identity with ephrin B1. Ephrin B1 appears at the onset of neocortical neurogenesis, being first expressed in neuron-generating neuroepithelial cells and rapidly thereafter in virtually all neuroepithelial cells. Expression of ephrin B1 persists through the period of neocortical neurogenesis and is downregulated thereafter. Ephrin B1 is present on the ventricular as well as basolateral plasma membrane of neuroepithelial cells and exhibits a ventricular-high to pial-low gradient across the ventricular zone. Expression of ephrin B1 is also detected on radial glial cells, extending all the way to their pial endfeet, and on neurons in the mantle/intermediate zone but not in the cortical plate. These results suggest that ephrin B1, presumably via ephrin-Eph receptor signaling, has a role in neurogenesis. Given the ventricular-to-pial gradient of ephrin B1 on the neuroepithelial cell surface and its known role in cell migration in other systems mediated by its repulsive properties, it is proposed that ephrin B1 may be involved in the migration of newborn neurons out from the ventricular zone toward the neocortex (Stuckmann, 2001).
Development of the tectum and the cerebellum is induced by a reciprocal inductive signaling between their respective primordia, the midbrain and the midbrain/hindbrain boundary (MHB). It is of interest to identify molecules that function in and downstream of this reciprocal signaling. Overexpression of LIM domain of the transcription factor Islet-3 (LIM Isl-3) leads to inhibition of this reciprocal signaling and to resultant defects in tectal and cerebellar development. Genes were sought that may be either up- or down-regulated by overexpression of LIMIsl-3 by comparing the gene expression profiles in the midbrain and the MHB of normal embryos and embryos in which Islet-3 function is repressed, using a combination of ordered differential display and whole-mount in situ hybridization. Among genes identified in this search, two cDNA fragments encode Wnt1 and FGF8, which are already known to be essential for the reciprocal signaling between the midbrain and the MHB, confirming the effectiveness of this strategy. Four other partial cDNA clones were identified that were specifically expressed around the MHB, ten cDNAs specifically expressed in the tectum, and three cDNAs expressed in neural crest cells, including those derived from the midbrain level. The ephrin-A3 gene is specifically expressed in posterior tectum in a gradient that decreases anteriorly. Although ephrin-A2 and ephrin-A5 have been reported to be expressed in the corresponding region in mouse embryos (the superior/inferior colliculi), mouse ephrin-A3 is not expressed prominently in this region, suggesting that the role of ephrin-A3 in brain development may have been altered in the process of brain evolution (Hirate, 2001).
In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. Here it has been shown that ß-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3-/- mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. It is concluded that in the intestinal epithelium ß-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system (Batlle, 2002).
Chick brain factor 1 (CBF1), a nasal retina-specific winged-helix transcription factor, is known to prescribe the nasal specificity that leads to the formation of the precise retinotectal map, especially along the anteroposterior (AP) axis. However, its downstream topographic genes and the molecular mechanisms by which CBF1 controls the expression of them have not been elucidated. Misexpression of CBF1 represses the expression of EphA3 and CBF2, and induces that of SOHo1, GH6, ephrin A2 and ephrin A5. CBF1 controls ephrin A5 by a DNA binding-dependent mechanism, ephrin A2 by a DNA binding-independent mechanism, and CBF2, SOHo1, GH6 and EphA3 by dual mechanisms. BMP2 expression begins double-gradiently (varying in both naso-temporal and ventral-dorsal axes) in the retina from E5 in a complementary pattern to Ventroptin expression. Ventroptin antagonizes BMP2 as well as BMP4. CBF1 interferes in BMP2 signaling and thereby induces expression of ephrin A2. These data suggest that CBF1 is located at the top of the gene cascade for the regional specification along the nasotemporal (NT) axis in the retina and distinct BMP signals play pivotal roles in the topographic projection along both axes (Takahashi, 2003).
The transmembrane (TM) subfamily of Eph ligands and their receptors have been implicated in axon pathfinding and in pattern formation during embryogenesis. These functions are thought to involve repulsive interactions but this has not been demonstrated directly. A growth cone collapse assay has been used to determine if the TM ligands Lerk2 and HtkL have repellant guidance activity. Lerk2, but not HtkL, is a collapsing factor for a subset of embryonic cortical neurons. Analysis of the effects of Lerk2 on both the morphology and the cytoskeleton of cortical neurons suggests a mechanism of action different from that of AL-1, a GPI-linked Eph ligand having similar repellant activity. Treatment with Lerk2 disrupts the organization of both the actin cytoskeleton and the microtubules and induces the formation of swellings in the center of the growth cone and along the axon. Measurement of the relative F-actin concentrations in the neurites and soma indicate that F-actin levels in the neurites decrease while those in the soma increase, with the net F-actin content of the neuron remaining unchanged. In contrast, prolonged treatment with AL-1 leads to a net loss of F-actin, consistent with the hypothesis that AL-1 acts by perturbing actin polymerization. These results provide evidence that the ectodomain of Lerk2 functions as a repellant guidance cue and show that, despite overlapping specificities in vitro, the biological activities of related ligands are not necessarily overlapping. Further, TM and GPI-linked Eph ligands appear to exert repellant activity by different mechanisms, opening up the possibility that they may have different effects on growth cones in vivo (Meima, 1997).
During the past few years, evidence has accumulated that activation of EphA kinases on the axons of retinal ganglion cells by ephrins-A on cells of the optic tectum (superior colliculus) plays a critical role in mapping the rostrocaudal axis of the retina onto the corresponding axis of the tectum. The similarities between topographic maps in the motor and visual systems has suggested that similar molecular mechanisms might underlie them. Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Subfamily A ephrins are implicated in this topographic mapping because: (1) developing muscles express all five of the ephrin-A genes; (2) rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5; (3) the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles; (4) topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms. If the conclusion that ephrins are directly involved in nerve-muscle matching is accepted, new questions arise about how they act. These can be divided into issues of mapping and synaptogenesis. In terms of mapping, one plausible model is that muscle fibers express different levels or combinations of ephrins in accordance with their positions and that motor axons express different levels or combinations of Eph kinases (the likely ephrin receptors) in accordance with their positions. For example, levels of expression might be graded along the rostrocaudal axis, as appears to be the case in the retinotectal system. Alternatively, patterns of ephrin and Eph kinase expression might describe a more complex combinatorial code, as suggested by the discrete identities of motor pools and muscles. Indeed, whereas Eph kinase and ephrin levels are graded along the rostrocaudal axis in the visual system, there is currently no evidence for graded expression of ligands or receptors in the motor system. Another possiblity is that interactions between ephrins and their receptors are permissive rather than instructive, enabling positional information to be transmitted by other molecules. It is important to emphasize that the data do not distinguish permissive versus instructive roles for ephrins (Feng, 2000).
In the embryonic visual system, EphA receptors are expressed on both temporal and nasal retinal ganglion cell axons. Only the temporal axons, however, are sensitive to the low concentrations of ephrin-A ligands found in the anterior optic tectum. The poor responsiveness of nasal axons to ephrin-A ligands, which allows them to traverse the anterior tectum and reach their targets in the posterior tectum, has been attributed to constitutive activation of the EphA4 receptor expressed in these axons. EphA4 is highly expressed throughout the retina, but is preferentially phosphorylated on tyrosine (activated) in nasal retina. In a screen for EphA4 ligands expressed in chicken embryonic retina, a novel ephrin, ephrin-A6, has been identified. Like ephrin-A5, ephrin-A6 has high affinity for EphA4 and activates this receptor in cultured retinal cells. In the embryonic day 8 (E8) chicken visual system, ephrin-A6 is predominantly expressed in the nasal retina and ephrin-A5 in the posterior tectum. Thus, ephrin-A6 has the properties of a ligand that activates the EphA4 receptor in nasal retinal cells. Ephrin-A6 binds with high affinity to several other EphA receptors as well and causes growth cone collapse in retinal explants, demonstrating that it can elicit biological responses in retinal neurons. Ephrin-A6 expression is high at E6 and E8, when retinal axons grow to their tectal targets, and gradually declines at later developmental stages. The asymmetric distribution of ephrin-A6 in retinal cells, and the time course of its expression, suggest that this new ephrin plays a role in the establishment of visual system topography (Menzel, 2001).
The role of the Eph family of receptor tyrosine kinases and their ligands in the establishment of the vomeronasal projection in the mouse has been investigated. The data show intriguing differential expression patterns of ephrin-A5 on vomeronasal axons and of EphA6 in the accessory olfactory bulb (AOB), such that axons with high ligand concentration project onto regions of the AOB with high receptor concentration and vice versa. These data suggest a mechanism for development of this projection that is the opposite of the repellent interaction between Eph receptors and ligands observed in other systems. In support of this idea, when given the choice of whether to grow on lanes containing EphA-Fc/laminin or Fc/laminin protein (in the stripe assay), vomeronasal axons prefer to grow on EphA-Fc/ laminin. Analysis of ephrin-A5 mutant mice reveal a disturbance of the topographic targeting of vomeronasal axons to the AOB. In summary, these data, which are derived from in vitro and in vivo experiments, indicate an important role of the EphA family in setting up the vomeronasal projection (Knoll, 2001a).
Growing axons follow highly stereotypical pathways, guided by a variety of attractive and repulsive cues, before establishing specific connections with distant targets. A particularly well-known example that illustrates the complexity of axonal migration pathways involves the axonal projections of motor neurons located in the motor cortex. These projections take a complex route during which they first cross the midline, then form the corticospinal tract, and ultimately connect with motor neurons in the contralateral side of the spinal cord. These obligatory contralateral connections account for why one side of the brain controls movement on the opposing side of the body. The netrins and slits provide well-known midline signals that regulate axonal crossings at the midline. A member of the ephrin family, ephrin-B3, also plays a key role at the midline to regulate axonal crossing. In particular, ephrin-B3 acts as the midline barrier that prevents corticospinal tract projections from recrossing when they enter the spinal gray matter. In ephrin-B3-/- mice, corticospinal tract projections freely recross in the spinal gray matter, such that the motor cortex on one side of the brain now provides bilateral input to the spinal cord. This neuroanatomical abnormality in ephrin-B3-/- mice correlates with loss of unilateral motor control, yielding mice that simultaneously move their right and left limbs and thus have a peculiar hopping gait quite unlike the alternate step gait displayed by normal mice. The corticospinal and walking defects in ephrin-B3-/- mice resemble those recently reported for mice lacking the EphA4 receptor, which binds ephrin-B3 as well as other ephrins, suggesting that the binding of EphA4-bearing axonal processes to ephrin-B3 at the midline provides the repulsive signal that prevents corticospinal tract projections from recrossing the midline in the developing spinal cord (Kullander, 2001).
To investigate Eph-ephrin bidirectional signaling, a series of mutations were generated in the ephrin-B3 locus. The absence of both forward and reverse signaling results in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract is defective since axons fail to respect the midline bound high-fidelity connection between the motor cortex and contralateral and ipsilateral motor neuron populations. A second mutation that expresses a truncated ephrin-pathfinding protein lacking Ephrin-B3's cytoplasmic domain does not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal cortico-spinal neurons as their fibers pathfind down the contralateral spinal cord. These data indicate ephrin-B3 functions as a midline-anchored repellent to stimulate forward signaling in EphA4-expressing axons (Yokoyama, 2001).
In both invertebrate and lower vertebrate species, decussated commissural axons travel away from the midline and assume positions within distinct longitudinal tracts. In the developing chick and mouse spinal cord, most dorsally situated commissural neuron populations extend axons across the ventral midline and through the ventral white matter along an arcuate trajectory on the contralateral side of the floor plate. Within the dorsal (chick) and intermediate (mouse) marginal zone, commissural axons turn at a conserved boundary of transmembrane ephrin expression, adjacent to which they form a discrete ascending fiber tract. In vitro perturbation of endogenous EphB-ephrinB interactions results in the failure of commissural axons to turn at the appropriate dorsoventral position on the contralateral side of the spinal cord; consequently, axons inappropriately invade more dorsal regions of B-class ephrin expression in the dorsal spinal cord. Taken together, these observations suggest that B-class ephrins act locally during a late phase of commissural axon pathfinding to specify the dorsoventral position at which decussated commissural axons turn into the longitudinal axis (Imondi, 2001).
Ascending sensory information reaches primary sensory cortical areas via thalamic relay neurons that are organized into modality-specific compartments or nuclei. Although the sensory relay nuclei of the thalamus show consistent modality-specific segregation of afferents, in a wild-type mouse strain it has been shown that the visual pathway can be surgically 'rewired' so as to induce permanent retinal innervation of auditory thalamic cell groups. Applying the same rewiring paradigm to a transgenic mouse lacking the EphA receptor family ligands ephrin-A2 and ephrin-A5 results in more extensive rewiring than in the wild-type strain. Ephrin-A2 and ephrin-A5 define a distinct border between visual and auditory thalamus. In the absence of this ephrin-A2/A5 border and after rewiring surgery, retinal afferents are better able to invade and innervate the deafferented auditory thalamus. These data suggest that signals that induce retinal axons to innervate the denervated auditory thalamus may compete with barriers, such as the ephrins, that serve to contain retinal axons within the normal target. The present findings thus show that the targeting of retinothalamic projections can be surgically manipulated in the mouse and that such plasticity can be controlled by proteins known to regulate topographic mapping (Lyckman, 2001).
The development of connections between thalamic afferents and their cortical target cells occurs in a highly precise manner. Thalamic axons enter the cortex through deep cortical layers, then stop their growth in layer 4 and elaborate terminal arbors specifically within this layer. The mechanisms that underlie target layer recognition for thalamocortical projections are not known. The growth patterns have been compared of thalamic explants cultured on membrane substrates purified from cortical layer 4, the main recipient layer for thalamic axons, and cortical layer 5, a non-target layer. Thalamic axons exhibit a reduced growth rate and an increased branching density on their appropriate target membranes compared with non-target substrate. When confronted with alternating stripes of both membrane substrates, thalamic axons grow preferentially on their target membrane stripes. Enzymatic treatment of cortical membranes has revealed that growth, branching and guidance of thalamic axons are independently regulated by attractive and repulsive cues differentially expressed in distinct cortical layers. These results indicate that multiple membrane-associated molecules collectively contribute to the laminar targeting of thalamic afferents. Furthermore, it was found that interfering with the function of Eph tyrosine kinase receptors and their ligands (ephrins) abolishes the preferential branching of thalamic axons on their target membranes, and that recombinant ephrin-A5 ligand elicites a branch-promoting activity on thalamic axons. It is concluded that interactions between Eph receptors and ephrins mediate branch formation of thalamic axons and thereby may play a role in the establishment of layer-specific thalamocortical connections (Mann, 2002a).
Neural maps in the vertebrate central nervous system often show discontinuously segregated, domain-to-domain patterns. However, the molecular mechanism that establishes such maps is not well understood. In the chicken olivocerebellar system, EphA receptors and ephrin-As are expressed with distinct levels and combinations in mapping domains. When ephrin-A2 is retrovirally overexpressed in the cerebellum, the olivocerebellar map is disrupted, excluding axons with high receptor activity from ectopic expression domains. Conversely, overexpression of a truncated EphA3 receptor in the cerebellum reduces endogenous ligand activity to undetectable levels and causes aberrant mapping, with high receptor axons invading high ligand domains. In vitro, ephrin-A2 inhibits outgrowth of inferior olive axons in a region-specific manner. These results suggest that Eph receptors and ephrins constitute domain-specific positional information, and the spatially accurate receptor-ligand interaction is essential to guide inferior olive axons to their correct target domains (Nishida, 2002).
Olfactory sensory neurons expressing a given odorant receptor (OR) project with precision to specific glomeruli in the olfactory bulb, generating a topographic map. Neurons expressing different ORs express different levels of ephrin-A protein on their axons. Moreover, alterations in the level of ephrin-A alter the glomerular map. Deletion of the ephrin-A5 and ephrin-A3 genes posteriorizes the glomerular locations for neurons expressing either the P2 or SR1 receptor, whereas overexpression of ephrin-A5 in P2 neurons results in an anterior shift in their glomeruli. Thus the ephrin-As are differentially expressed in distinct subpopulations of neurons and are likely to participate, along with the ORs, as one of a complement of guidance receptors governing the targeting of like axons to precise locations in the olfactory bulb (Cutforth, 2003).
The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, in vivo evidence is provided that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. These results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas (Dufour, 2003).
Molecular mechanisms generating the topographic organization of corticothalamic (CT) circuits, which comprise more than three-quarters of the synaptic inputs onto sensory relay neurons, and their interdependence with thalamocortical (TC) axon development, are unknown. Using in utero electroporation-mediated gene transfer, EphA7-mediated signaling on neocortical axons was shown to control the within-nucleus topography of CT projections in the thalamus. Notably, CT axons that misexpress EphA7 do not shift the relative positioning of their pathway within the subcortical telencephalon (ST), indicating that they do not depend upon EphA7/ephrin-A signaling in the ST for establishing this topography. Moreover, misexpression of cortical EphA7 results in disrupted topography of CT projections, but unchanged inter- and intra-areal topography of TC projections. These results support a model in which EphA/ephrin-A signaling controls independently the precision with which CT and TC projections develop, yet is essential for establishing their topographic reciprocity (Torii, 2005).
How can TC and CT projections achieve their reciprocal organization without interdependent controls? Based on the present results and studies suggesting that the intra-areal topography of TC projections is regulated by an EphA gradient in thalamic nuclei and an ephrin-A gradient in target cortical areas, a model is proposed in which complementary gradients of EphA and ephrin-A activity, located between as well as within specific cortical areas and their corresponding thalamic nuclei, produce reciprocal signaling between these structures. In this model, however, the EphA/ephrin-A signaling controls the topography of TC and CT projections independently, yet establishes essential functional topographic reciprocity (Torii, 2005).
In this model, functional gradients of EphA receptors and ephrin-A ligands are required to be complementary both between and within each reciprocal target. Specific expression patterns of EphAs and ephrin-As appear before CT and TC axons reach their targets. Together with recent evidence for intrinsic regulation of EphA7 and ephrin-A5 expression within the neocortex by transcription factors or morphogens, gradients of EphAs and ephrin-As are likely established by intrinsic regulation within the neocortex and DT independently. Complementary patterns might be achieved by reciprocal transcriptional regulation for each by the same transcription factor. For example, in dorsoventral patterning of the retina, Tbx5 enhances transcription of ephrin-Bs and represses EphBs. In contrast, Vax2 enhances the transcription of EphBs and represses ephrin-Bs. In addition to transcriptional regulation, repressive modulation of EphA function by coexpressed ephrin-A, as shown on retinal ganglion cell axons, might sharpen their functional complementarity where their expression overlaps. Reciprocal and topographic neural connections are found in many other systems throughout the brain. It will be important to determine whether this independent, but reciprocal, EphA/ephrin-A signaling model is applicable generally to other systems (Torii, 2005).
Visual connections to the mammalian forebrain are known to be patterned by neural activity, but it remains unknown whether the map topography of such higher sensory projections depends on axon guidance labels. Complementary expression and binding are shown for the receptor EphA5 in mouse retina and its ligands ephrin-A2 and ephrin-A5 in multiple retinal targets, including the major forebrain target, the dorsal lateral geniculate nucleus (dLGN). These ligands can act in vitro as topographically specific repellents for mammalian retinal axons and are necessary for normal dLGN mapping in vivo. The results suggest a general and economic modular mechanism for brain mapping whereby a projecting field is mapped onto multiple targets by repeated use of the same labels. They also indicate the nature of a coordinate system for the mapping of sensory connections to the forebrain (Feldheim, 1999).
In the retinotectal projection, the Eph receptor tyrosine kinase ligands ephrinA2 and ephrinA5 are differentially expressed, not only in the tectum, but also in a high-nasal-to-low-temporal pattern in the retina. Recently, retrovirally driven overexpression of ephrinA2 on retinal axons leads to topographic targeting errors of temporal axons in that they overshoot their normal termination zones in the rostral tectum and project onto the mid- and caudal-tectum. The behavior of nasal axons, however, is only marginally affected. Overexpression of ephrinA5 affects the topographic targeting behavior of both temporal and nasal axons. These data reinforce the idea that differential ligand expression on retinal axons contributes to topographic targeting in the retinotectal projection. Additionally, it has been found that ectopic expression of ephrinA2 and ephrinA5 frequently leads to pathfinding errors at the chiasm, resulting in an increased stable ipsilateral projection (Dutting, 1999).
The Eph family is thought to exert its function through the complementary expression of receptors and ligands. Here, EphA receptors colocalize on retinal ganglion cell (RGC) axons with EphA ligands, which are expressed in a high-nasal-to-low-temporal pattern. In the stripe assay, only temporal axons are normally sensitive for repellent axon guidance cues of the caudal tectum. However, overexpression of ephrinA ligands on temporal axons abolishes this sensitivity, whereas treatment with PI-PLC both removes ephrinA ligands from retinal axons and induces a striped outgrowth of formerly insensitive nasal axons. In vivo, retinal overexpression of ephrinA2 leads to topographic targeting errors of temporal axons. These data suggest that differential ligand expression on retinal axons is a major determinant of topographic targeting in the retinotectal projection (Hornberger, 1999).
Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinal axonal projection to the tectum or to its mammalian equivalent, the superior colliculus (SC). In this paper, gene disruptions for both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes (Feldheim, 2000).
Currently, a leading model is that both attractant and repellent gradients may be prespecified within the target. Each axon then identifies its correct termination zone as the point where repellent and attractant forces cancel out. In this model, wild-type nasal axons map to posterior SC because they are attracted there. A prediction of this model is that in the ephrin-A2-/-; ephrin-A5-/- double mutant, axons would tend to map more posteriorly than normal, or in the case of extreme nasal axons might show no shift. This appears to be contrary to the results obtained in this study, especially considering that the opposite shifts in the nasal and temporal axons are of similar magnitudes. For similar reasons these results do not seem consistent with a variant of the counterbalanced gradient model, where a posterior to anterior ephrin gradient would be balanced by a prespecified repellent in an anterior to posterior gradient. An alternative possibility that could perhaps be reconciled with these results would be for ephrins in the target to act as either repellents or attractants at different concentrations, although there is currently no direct evidence to support such an effect from axon guidance assays (Feldheim, 2000 and references therein).
As an alternative to models with two counterbalanced gradients prespecified in the target, these results could be explained by a model involving a repellent gradient of ephrins, in combination with axon-axon competition. This competition could be for limiting positive factors in the target or could also involve direct axon-axon interactions. A model of this type has been suggested in an analysis of the LGN in ephrin-A5-/- mice, and the model now receives strong support from the more comprehensive analysis in the SC presented here. The repulsion/competition model would account for normal mapping as follows. Temporal axons would be unable to terminate in the posterior SC, because they are repelled by the ephrins, so they would be forced to arborize in the anterior SC. Nasal axons are less sensitive to ephrin repulsion and so would be able to terminate in the posterior SC. In the anterior SC, nasal axons face greater competition for limiting amounts of permissive factor(s), so they prefer to avoid this competition and arborize only in the posterior SC (Feldheim, 2000).
Incorporating competition in the model can explain several aspects of the data. (1) Nasal axons shift anteriorly in the ephrin-A5-/- single mutant (although this could be explained by the axon sensitivity model outlined above), and shift even further to the anterior in the ephrin-A2-/-; ephrin-A5-/- double mutant. According to the competition model, posterior repellents are removed, so axons from temporal or central retina are now able to compete more effectively in posterior SC; nasal axons therefore face increased competition in the posterior SC, so they lose their strong preference for this region and spread out into more anterior regions. The model thus seems to fit well with the opposite and quantitatively similar shifts of axons from temporal and nasal extremes of the retina. Labelings at intermediate retinal positions are more difficult to characterize because of the lack of fixed landmarks but, consistent with the competition model, such labelings revealed multiple arborizations, seemingly shifted in both anterior and posterior directions. (2) Axons are not respecified to a specific ectopic position. Instead, arborizations are scattered over an abnormally broad zone in the mutants, including both normal and abnormal regions. This is the result predicted by the competition model: as the topographically specific repellents are removed, axons would spread into abnormal regions, but there is no reason for them to disfavor the correct region. (3) Even in the ephrin-A2-/-; ephrin-A5-/- mutant, retinal axons fill the entire SC and axons from both nasal and temporal extremes of the retina form connections within the target. Models that involve a strict matching of values in the projecting and target field would predict that the mutant should have unmatched areas, whereas the competition model predicts that the entire projecting and target fields should still match up (Feldheim, 2000).
In the mammalian visual system, retinal axons undergo temporal and spatial rearrangements as they project bilaterally to targets on the brain. Retinal axons cross the neuraxis to form the optic chiasm on the hypothalamus in a position defined by overlapping domains of regulatory gene expression. However, the downstream molecules that direct these processes remain largely unknown. A novel in vitro paradigm was used to study possible roles of the Eph family of receptor tyrosine kinases in chiasm formation. In vivo, Eph receptors and their ligands distribute in complex patterns in the retina and hypothalamus. In vitro, retinal axons are inhibited by reaggregates of isolated hypothalamic, but not dorsal diencephalic or cerebellar cells. Furthermore, temporal retinal neurites are more inhibited than nasal neurites by hypothalamic cells. Addition of soluble EphA5-Fc to block Eph 'A' subclass interactions decreases both the inhibition and the differential response of retinal neurites by hypothalamic reaggregates. These data show that isolated hypothalamic cells elicit specific, position-dependent inhibitory responses from retinal neurites in culture. Moreover, these responses are mediated, in part, by Eph interactions. Together with the in vivo distributions, these data suggest possible roles for Eph family members in directing retinal axon growth and/or reorganization during optic chiasm formation (Marcus, 2000).
Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. The distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon has been examined during the period when visual connections are being established. Both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor-ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph-ephrin molecules is observed. A similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway (Marin, 2001).
The idea has been put forward that molecules and mechanisms acting during development are re-used during regeneration in the adult, for example in response to traumatic injury in order to re-establish the functional integrity of neuronal circuits. Members of the Eph family of receptor tyrosine kinases and their 'ligands', the ephrins, play a prominent role during development of the retinocollicular projection in rodents, where EphA receptors and ephrin-As are expressed in gradients in both the retina and the superior colliculi (SC). Whether EphA family members are also expressed or re-expressed in the adult after optic nerve lesion was examined, since the presence of axon guidance information is an important prerequisite for a topographically appropriate re-connection by retinal ganglion cell (RGC) axons. This analysis was encouraged by results showing that RGC axons do not exert guidance preferences in response to membranes from adult unlesioned SC, but in response to membranes from the adult deafferented SC. A graded expression pattern of ephrin-As was found in the SC both before and after deafferentation, which is remarkably similar to those found during development. EphA receptor levels are reduced in the SC after deafferentation and the expression patterns of the EphB family are not changed. In particular, the presence of a graded ephrin-A expression in the deafferented SC suggests that -- if robust regeneration of RGC axons can be achieved -- topographic guidance information as a likely requirement for a functionally successful re-establishment of the retinocollicular projection is available (Knoll, 2001b).
The retinotectal projection is the predominant model for studying molecular mechanisms controlling development of topographic axonal connections. Analyses of topographic mapping of retinal ganglion cell (RGC) axons in chick optic tectum indicate that a primary role for guidance molecules is to regulate topographic branching along RGC axons, a process that imposes unique requirements on the molecular control of map development. Topographically appropriate connections are established exclusively by branches that form along the axon shaft. Initially, RGC axons overshoot their appropriate termination zone (TZ) along the anterior-posterior (A-P) tectal axis; temporal axons overshoot the greatest distance and nasal axons the least: this correlates with the nonlinear increasing A-P gradient of ephrin-A repellents. In contrast, branches form along the shaft of RGC axons with substantial A-P topographic specificity. Topography is enhanced through the preferential arborization of appropriately positioned branches and elimination of ectopic branches. Using a membrane stripe assay and time-lapse microscopy, it has been shown that branches form de novo along retinal axons. Temporal axons preferentially branch on their topographically appropriate anterior tectal membranes. After the addition of soluble EphA3-Fc, which blocks ephrin-A function, temporal axons branch equally on anterior and posterior tectal membranes, indicating that the level of ephrin-As in posterior tectum is sufficient to inhibit temporal axon branching and generate branching specificity in vitro. These findings indicate that topographic branch formation and arborization along RGC axons are critical events in retinotectal mapping. Ephrin-As inhibit branching along RGC axons posterior to their correct TZ, but alone cannot account for topographic branching and must cooperate with other molecular activities to generate appropriate mapping along the A-P tectal axis (Yates, 2001).
Ephrin-B and EphB are distributed in matching dorsoventral gradients in the embryonic Xenopus visual system with retinal axons bearing high levels of ligand (dorsal axons) projecting to tectal regions with high receptor expression (ventral regions). In vitro stripe assays show that dorsal retinal axons prefer to grow on EphB receptor stripes supporting an attractive guidance mechanism. In vivo disruption of EphB/ephrin-B function by application of exogenous EphB or expression of dominant-negative ephrin-B ligand in dorsal retinal axons causes these axons to shift dorsally in the tectum, while misexpression of wild-type ephrin-B in ventral axons causes them to shift ventrally. These dorsoventral targeting errors are consistent with the hypothesis that an attractive mechanism that requires ephrin-B cytoplasmic domain is critical for retinotectal mapping in this axis (Mann, 2002b).
The retinotectal map is the best characterized model system to study how axons respond to guidance cues during the formation of the nervous system. The critical event in forming this map is topographic-specific axon branching. To elucidate the in vivo role of the repulsive cue ephrin-A5 in this event, chromophore-assisted laser inactivation (CALI) was used to generate acute loss of ephrin-A5 function in localized areas of the posterior tectum of chick embryos in ovo and the resulting changes of retinal projections were analyzed during initial outgrowth (E11) and when retinal axons arborize in the deep layers in the tectum (E12). Ephrin-A5 functions to restrict initial axon outgrowth at E11. At E12, CALI of ephrin-A5 did not affect the extent of axon outgrowth on the tectal surface but instead caused ectopic arborization posterior to the topographically correct site in deeper layers of the tectum. This shows that ephrin-A5 restricts arborization during this critical process for developing the retinotopic map. CALI provides an approach to inactivate in vivo function in higher vertebrates with high temporal and spatial specificity that may have wide application (Sakurai, 2002).
The EphB receptor ligand, ephrin-B1, may act bifunctionally as both a branch repellent and attractant to control the unique mechanisms in mapping the dorsal-ventral (DV) retinal axis along the lateral-medial (LM) axis of the optic tectum. EphB receptors are expressed in a low to high DV gradient by retinal ganglion cells (RGCs), and ephrin-B1 is expressed in a low to high LM gradient in the tectum. RGC axons lack DV ordering along the LM tectal axis, but directionally extend interstitial branches that establish retinotopically ordered arbors. Recent studies show that ephrin-B1 acts as an attractant in DV mapping and in controlling directional branch extension. Modeling indicates that proper DV mapping requires that this attractant activity cooperates with a repellent activity in a gradient that mimics ephrin-B1. Ectopic domains of high, graded ephrin-B1 expression created by retroviral transfection repel interstitial branches of RGC axons and redirect their extension along the LM tectal axis, away from their proper termination zones (TZs). In contrast, the primary RGC axons are unaffected and extend through the ectopic domains of ephrin-B1 and arborize at the topographically correct site. However, when the location of a TZ is coincident with ectopic domains of ephrin-B1, the domains appear to inhibit arborization and shape the distribution of arbors. These findings indicate that ephrin-B1 selectively controls, through either attraction or repulsion, the directional extension and arborization of interstitial branches extended by RGC axons arising from the same DV position: branches that arise from axons positioned lateral to the correct TZ are attracted up the gradient of ephrin-B1 and branches that arise from axons positioned medial to the same TZ are repelled down the ephrin-B1 gradient. Alternatively, EphB receptor signaling may act as a 'ligand-density sensor' and titrate signaling pathways that promote branch extension toward an optimal ephrin-B1 concentration found at the TZ; branches located either medial or lateral to the TZ would encounter a gradient of increasingly favored attachment in the direction of the TZ (McLaughlin, 2003).
The Eph family of receptor tyrosine kinases and their ligands, the ephrins, play important roles during development of the nervous system. Frequently they exert their functions through a repellent mechanism, so that, for example, an axon expressing an Eph receptor does not invade a territory in which an ephrin is expressed. Eph receptor activation requires membrane-associated ligands. This feature discriminates ephrins from other molecules that sculpt the nervous system such as netrins, slits and class 3 semaphorins, which are secreted molecules. While the ability of secreted molecules to guide axons, i.e., to change their growth direction, is well established in vitro, little is known about this for the membrane-bound ephrins. Using Xenopus laevis retinal axons the properties were investigated of substratum-bound and (artificially) soluble forms of ephrin-A5 (ephrin-A5-Fc) to guide axons. When immobilized in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from the chick. The analysis of growth cone behavior in a gradient of soluble ephrin-A5 in the 'turning assay' reveals a substratum-dependent reaction of Xenopus retinal axons. On fibronectin, a repulsive response is observed, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations, indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, these data indicate that ephrin-As are able to guide axons in immobilized bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown (Wein, 2003).
In animals with binocular vision, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. Ephrin-Bs in the chiasm region direct the divergence of retinal axons through the selective repulsion of a subset of RGCs that express EphB1. Ephrin-B2 is expressed in radial glia at the mouse chiasm midline as the ipsilateral projection is generated and is selectively inhibitory to axons from ventrotemporal (VT) retina, where ipsilaterally projecting RGCs reside. Moreover, blocking ephrin-B2 function in vitro rescues the inhibitory effect of chiasm cells and eliminates the ipsilateral projection in the semiintact mouse visual system. A receptor for ephrin-B2, EphB1, is found exclusively in regions of retina that give rise to the ipsilateral projection. EphB1 null mice exhibit a dramatically reduced ipsilateral projection, suggesting that this receptor contributes to the formation of the ipsilateral retinal projection, most likely through its repulsive interaction with ephrin-B2. This study provides the first direct evidence that ephrin-B2 is more inhibitory to ipsilateral than contralateral retinal axons and demonstrates that ephrin-B2 is necessary for the ipsilateral projection to form. Using in situ hybridization and analyses of mutant mice, EphB1, a receptor for ephrin-B2, is shown to be expressed specifically in regions of the retina that give rise to the ipsilateral projection, and in mice lacking EphB1. Together these data identify ephrin-B2 and EphB1 as key players in retinal axon divergence and suggest that the function of B-class Ephs and ephrins in patterning binocular vision is conserved between species (Williams, 2003).
Ephrin-As act as retinal topographic mapping labels, but the molecular basis for two key aspects of mapping remains unclear. (1) Although mapping is believed to require balanced opposing forces, ephrin-As have been reported to be retinal axon repellents, and the counterbalanced force has not been molecularly identified. (2) Although graded responsiveness across the retina is required for smooth mapping, a sharp discontinuity has instead been reported. An axon growth assay has been developed to systematically vary both retinal position and ephrin concentration and test responses quantitatively. Responses varied continuously with retinal position, fulfilling the requirement for smooth mapping. Ephrin-A2 inhibits growth at high concentrations but promoted growth at lower concentrations. Moreover, the concentration producing a transition from promotion to inhibition varied topographically with retinal position. These results lead directly to a mapping model where position within a concentration gradient may be specified at the neutral point between growth promotion and inhibition (Hansen, 2004).
A quantitative axon outgrowth assay has been developed that allows both retinal position and ephrin concentration to be varied systematically. This outgrowth assay, unlike the stripe assay, is not a growth cone steering assay. However, anterior-posterior mapping primarily involves the regulation of the final extent of axon growth across the tectum/SC rather than growth cone steering. Moreover, ephrins are known to regulate several types of mapping-related axon growth response, including growth cone steering, collateral branching, and extent of outgrowth. While the output of this assay (or any other assay) is not taken as precisely matching the biology of normal mapping, the system used in this study provides a quantitative and controllable test of the growth response of retinal axons to added ephrins and displays topographic specificity (Hansen, 2004).
Initial experiments that tested the response to tectal membranes found that both nasal and temporal axons show selectivity; both grow preferentially on anterior membranes, with a stronger preference being shown by temporal axons. This finding differs from the in vitro stripe assay, where axons from the entire nasal side of the retina were reported to be unresponsive to posterior tectal membranes. The results here can therefore provide a resolution to the previously puzzling question of how axons across the nasal half of the retina are mapped if they are unresponsive to posterior tectal labels (Hansen, 2004).
In subsequent experiments that tested responses to ephrins, the results show that responsiveness across the retinal N-T axis does not fit a two-step discontinuous model and instead appear to be smoothly graded. It is not clear why previous assays detected a sharp cutoff between nasal and temporal axons. One possibility is a species difference between the mouse axons that were used in this study versus the chick axons that were used in earlier studies. In chick, normal in vivo mapping involves an initial phase with a simple nasal versus temporal discrimination, before the full graded map develops, and the in vitro assays might reflect this initial phase. An alternative explanation is suggested by the finding that, at the highest ephrin-A2 concentrations that were tested in this study (comparable to ephrin-A levels in posterior tectum), outgrowth across the retina did not appear to be graded and instead fit a two-step model of responsiveness, with a sharp cutoff between the two halves of the retina. This result may help explain why previous studies have shown a nasal versus temporal discontinuity if, as seems likely, those studies used a growth substrate with ephrin activity that was similar to or higher than the 100% ephrin-A2 substrate. Whatever the reason for the discontinuity in previous studies, the graded responsiveness that was seen here fits the predictions of the chemoaffinity theory, as required to form a smooth topographic map (Hansen, 2004).
The results show that the shape of the responsiveness curve along the N-T axis of the retina differs, depending on whether the axons were tested with ephrin-A5 or ephrin-A2. In response to ephrin-A5, retinal positions showed a continuous monotonic variation in response, from high outgrowth at the nasal extreme to low outgrowth at the temporal extreme. In contrast, ephrin-A2 elicits a biphasic response curve, with maximum outgrowth from axons midway across the nasal half of the retina, decreasing in both nasal and temporal directions. These responsiveness profiles in the retina show an interesting correspondence with the expression profiles in the superior colliculus (SC). Ephrin-A5 expression increases in a monotonic gradient from the anterior to the posterior SC, whereas ephrin-A2 shows a biphasic distribution with a high point midway across the posterior half of the SC, decreasing in both anterior and posterior directions. Thus, the retinal position that gave the highest outgrowth on each ligand maps to the SC region with the highest concentration of that particular ligand (Hansen, 2004).
In terms of the overall significance for mapping, the distinctive gradients of ephrin-A2 and ephrin-A5 could be interpreted by two models. One model could be that ephrin-A2 plays no role in mapping the far posterior SC and declines there simply because it is not needed. This model could be consistent with genetic studies that observed map disruptions in the far posterior SC in ephrin-A5 but not ephrin-A2 gene-targeted mice. However, those studies would not necessarily have detected a more subtle ephrin-A2 phenotype. An alternative model is that ephrin-A2 may make some contribution as a reverse-orientation mapping gradient in the far posterior SC. This second model could be consistent with proposals by Sperry that in mammals the mapping labels are not likely to be simple orthogonal gradients and were predicted to have a central-to-peripheral radial component (Hansen, 2004).
Regarding the molecular basis for the difference in response curves to ephrin-A2 and ephrin-A5, presumably the two ligands are differentially recognized by specific Eph receptors. This idea appears to be consistent with genetic evidence suggesting that a preferential functional relationship exists between specific ligand-receptor pairs, such as ephrin-A5 and EphA5. Such preferential relationships could arise from differential binding affinities, which have been observed among the ephrin-A ligands and EphA receptors. Although binding interactions within ephrin and Eph receptor subfamilies are relatively promiscuous, preferential quantitative aspects of the interactions may be functionally important, especially for a quantitative process such as mapping (Hansen, 2004).
Ephrin-As and -Bs have been shown in a number of assay systems to have either repellent/inhibitory effects or attractant/adhesive effects. In some cases, positive and negative effects have been seen in a single biological system. Transient growth promotion followed by axon fragmentation, has been observed when hippocampal axons are grown on ephrin-A-expressing fibroblast monolayers. These observations suggest a potential relevance to hippocamposeptal development, although they did not lead to a model to specify map position, since both positive and negative actions were highest on axons from the same side of the projecting area. The current study found no evidence that the growth-promoting effect was transient, and a possible explanation for the transient effect that was seen in the co-culture system could be a continuing rise in expression during long-term culture of fibroblast monolayers. Another study has reported that soluble ephrin-As can be either attractant or repellent for retinal axons, depending on the substrate upon which the axons are growing. While very interesting as a mechanism for axon regulation, this again does not lead to any obvious model to specify position within a topographic map (Hansen, 2004).
Another example is provided by the ephrin-Bs in dorsoventral retinotectal/retinocollicular mapping. Expression patterns initially led to the prediction that the interaction must be attractant. Consistent with this prediction, in vitro assays, in vivo overexpression, and dominant-negative experiments in Xenopus, as well as gene knockout analysis in mouse, all concluded that ephrin-B/EphB interactions do indeed have attractant effects. However, a more recent chick overexpression study found repellent effects, with patches of ectopic ephrin-B1 always being avoided by retinal axons. The observation of attraction in some assays and repulsion in other assays led to a suggestion that normal mapping by ephrin-Bs might involve a transition between attraction and repulsion. However, such a transition has not actually been observed for ephrin-Bs, and ephrin-As are still assumed to act in retinotectal position specification only by repulsion (Hansen, 2004).
The initial goal in developing a quantitative assay for axon outgrowth was to test for graded responses, and it was expected that ephrin-As would only have inhibitory effects on retinal axons. However, the results showed that ephrin-A2 could also promote retinal axon growth. There was no indication that either the positive or negative responses were transient, with outgrowth following a similar time course at all concentrations that were tested (Hansen, 2004).
The positive and negative effects that were observed in this study have three crucial features in relation to map specification: (1) they are concentration dependent, with a transition between positive and negative depending on the ephrin-A2 concentration; (2) whether the response is positive or negative is also dependent on retinal position, and (3) the direction of these two dependences fits the orientation of the retinotectal map, with appropriate topographic specificity (Hansen, 2004).
Based on these results, the following model is proposed for topographic map development. Axon growth would be promoted by low ephrin-A concentrations anterior to the topographically correct position and inhibited by higher ephrin-A concentrations posterior to the correct position. Each axon would ultimately form a termination zone at the neutral point between these positive and negative influences. The transition point between positive and negative effects would vary according to retinal position, occurring at higher ephrin-A concentrations for nasal axons, which terminate posteriorly, and lower ephrin-A concentrations for temporal axons, which terminate anteriorly. The result would be the formation of a topographic map. It is proposed that axons originating from different positions across the retina have different sensitivities to ephrin, presumably due to the graded distribution of EphA receptors in the retina, so that the neutral inflection point between positive and negative effects in the tectum/SC varies with retinal position. The result is the production of a smooth topographic map (Hansen, 2004).
During development of the retinocollicular projection in mouse, retinal axons initially overshoot their future termination zones (TZs) in the superior colliculus (SC). The formation of TZs is initiated by interstitial branching at topographically appropriate positions. Ephrin-As are expressed in a decreasing posterior-to-anterior gradient in the SC, and they suppress branching posterior to future TZs. This study investigates the role of an EphA7 gradient in the SC, which has the reverse orientation compared to the ephrin-A gradient. In EphA7 mutant mice the retinocollicular map is disrupted, with nasal and temporal axons forming additional or extended TZs, respectively. In vitro, retinal axons are repelled from growing on EphA7-containing stripes. These data support the idea that EphA7 is involved in suppressing branching anterior to future TZs. These findings suggest that opposing ephrin-A and EphA gradients are required for the proper development of the retinocollicular projection (Rashid, 2005).
Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act to organize thalamic inputs. This study mapped the visual cortex (V1) in mice deficient for ephrin-A2, -A3, and -A5 functionally [ephrin-A triple knock out (TKO)], using intrinsic signal optical imaging and microelectrode recording, and structurally, by anatomical tracing of thalamocortical projections. V1 is shifted medially, rotated, and compressed and its internal organization is degraded. Expressing ephrin-A5 ectopically by in utero electroporation in the lateral cortex shifts the map of V1 medially, and expression within V1 disrupts its internal organization. These findings indicate that interactions between gradients of EphA/ephrin-A in the cortex guide map formation, but that factors other than redundant ephrin-As are responsible for the remnant map. Together with earlier work on the retinogeniculate map, the current findings show that the same molecular interactions may operate at successive stages of the visual pathway to organize maps (Cang, 2005).
The visual system has been a long-standing model in the study of topographic map development. EphA receptors and ephrin-A ligands have been shown to be necessary for the development of topographic maps from the retina to two of its subcortical targets, the superior colliculus (SC) in the midbrain and dorsal lateral geniculate nucleus (dLGN) in the thalamus. The dLGN then projects to the visual cortex. In this study, it was found that the topographic mapping of geniculocortical projections is disrupted both structurally and functionally in an ephrin-A TKO. The expression patterns of EphA receptors and ephrin-A ligands in the geniculocortical projection are consistent with EphA/ephrin-A interactions acting in thalamocortical (TC) projections in the same manner as that proposed for EphA/ephrin-A interactions in subcortical mapping. In the cases in which ephrin-A ligand was successfully misexpressed within the developing visual cortex, the functional topographic maps were defective, consistent with ephrin-A ligands acting as molecular labels in the cortex. Together, these experiments suggest that the same mapping mechanisms are used at subcortical and cortical stages, acting in each projection and target area to maintain the topographic order of visual information as it is transferred to the next stage (Cang, 2005).
Described here is the isolation and characterization of two zebrafish Eph receptor ligand cDNAs that have been called zfEphL3 and zfEphL4. These genes are expressed in the presumptive midbrain of developing embryos from 6 somites. By 24 hours, L3 is expressed throughout the midbrain including the region of the presumptive tectum, whereas L4 is strongly expressed in the midbrain caudal to the presumptive tectum. At later stages of development L3 is expressed in a graded fashion throughout the tectum and L4 is maintained at its posterior margin. Growth cone collapse and pathway selection assays demonstrate that both these proteins have a collapse activity for retinal ganglion cells. When faced with a choice of substrate on which to grow, temporal axons from chick retinal ganglion cells selectively avoid membranes from Cos cells transfected with L3, whereas nasal axons do not. Both temporal and nasal axons avoid membranes from Cos cells transfected with L4. The expression patterns, together with the functional data, suggest that although both ligands may be able to guide retinal ganglion cells axons in vitro, they have different roles in the guidance of retinotectal projections in vivo. The expression of L3 is consistent with a role in the guidance of retinal ganglion cells to their targets on the tectum, whereas that of L4 suggests a role in delineating the posterior boundary of the optic tectum (Brennan, 1997).
Rhombomeres are segmental units of the developing vertebrate hindbrain that underlie the reiterated organization of cranial neural crest migration and neuronal differentiation. valentino (val), a zebrafish homolog of the mouse bzip transcription factor-encoding gene, kreisler (potential Drosophila homolog: CG10034), is required for segment boundary formation caudal to rhombomere 4 (r4). val is normally expressed in r5/6 and is required for cells to contribute to this region. In val minus mutants, rX, a region one rhombomere in length and of mixed identity, lies between r4 and r7. While a number of genes involved in establishing rhombomeric identity are known, it is still largely unclear how segmental integrity is established and boundaries are formed. Members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, are candidates for functioning in rhombomere boundary formation. Indeed, expression of the receptor ephB4a coincides with val in r5/6, while ephrin-B2a, which encodes a ligand for EphB4a, is expressed in r4 and r7, complementary to the domain of val expression (Cooke, 2001).
In val minus embryos, ephB4a expression is downregulated and ephrin-B2a expression is upregulated between r4 and r7, indicating that Val is normally required to establish the mutually exclusive expression domains of these two genes. Juxtaposition of ephB4a-expressing cells and ephrin-B2a-expressing cells in the hindbrain leads to boundary formation. Loss of the normal spatial regulation of eph/ephrin expression in val mutants correlates not only with absence of boundaries but also with the inability of mutant cells to contribute to wild-type r5/6. Using a genetic mosaic approach, it has been shown that spatially inappropriate Eph signaling underlies the repulsion of val minus cells from r5/6. It is proposed that Val controls eph expression and that interactions between EphB4a and Ephrin-B2a mediate cell sorting and boundary formation in the segmenting caudal hindbrain (Cooke, 2001).
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