Graded expression patterns of ephrin-As in the superior colliculus after lesion of the adult mouse optic nerve

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). We were interested in investigating whether EphA family members are also expressed or re-expressed in the adult after optic nerve lesion, 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. We found a graded expression pattern of ephrin-As in the SC both before and after deafferentation, which was remarkably similar to those found during development. EphA receptor levels were reduced in the SC after deafferentation and the expression patterns of the EphB family were 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.     

Dynamic Alterations of Retinal EphA5 Expression in Retinocollicular Map Plasticity

The topographically ordered retinocollicular projection is an excellent system for studying the mechanism of axon guidance. Gradients of EphA receptors in the retina and ephrin‐As in the superior colliculus (SC) pattern the anteroposterior axis of the retinocollicular map, but whether they are involved in map plasticity after injury is unknown. Partial damage to the caudal SC at birth creates a compressed, complete retinotopic map in the remaining SC without affecting visual response properties. Previously, we found that the gradient of ephrin‐A expression in compressed maps is steeper than normal, suggesting an instructive role in compression. Here we measured EphA5 mRNA and protein levels after caudal SC damage in order to test the hypothesis that changes in retinal EphA5 expression occur that are complementary to the changes in collicular ephrin‐A expression. We find that the nasotemporal gradient of EphA5 receptor expression steepens in the retina and overall expression levels change dynamically, especially in temporal retina, supporting the hypothesis. This change in receptor expression occurs after the change in ephrin‐A ligand expression. We propose that changes in the retinal EphA5 gradient guide recovery of the retinocollicular projection from early injury. This could occur directly through the change in EphA5 expression instructing retino‐SC map compression, or through ephrin‐A ligand signaling instructing a change in EphA5 receptor expression that in turn signals the retinocollicular map to compress. Understanding what molecular signals direct compensation for injury is essential to developing rehabilitative strategies and maximizing the potential for recovery.     

Regulation of axial patterning of the retina and its topographic mapping in the brain

Topographic maps are a fundamental organizational feature of axonal connections in the brain. A prominent model for studying axial polarity and topographic map development is the vertebrate retina and its projection to the optic tectum (or superior colliculus). Linked processes are controlled by molecules that are graded along the axes of the retina and its target fields. Recent studies indicate that ephrin-As control the temporal-nasal mapping of the retina in the optic tectum/superior colliculus by regulating the topographically-specific interstitial branching of retinal axons along the anterior-posterior tectal axis. This branching is mediated by relative levels of EphA receptor repellent signaling. A major recent advance is the demonstration that EphB receptor forward signaling and ephrin-B reverse signaling mediate axon attraction to control dorsal-ventral retinal mapping along the lateral-medial tectal axis. In addition, several classes of regulatory proteins have been implicated in the control of the axial patterning of the retina, and its ultimate readout of topographic mapping.     

Domain-specific olivocerebellar projection regulated by the EphA-ephrin-A interaction

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. Here we show that 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.     

Regulation of ephrin‐A expression in compressed retinocollicular maps

Retinotopic maps can undergo compression and expansion in response to changes in target size, but the mechanism underlying this compensatory process has remained a mystery. The discovery of ephrins as molecular mediators of Sperry's chemoaffinity process allows a mechanistic approach to this important issue. In Syrian hamsters, neonatal, partial (PT) ablation of posterior superior colliculus (SC) leads to compression of the retinotopic map, independent of neural activity. Graded, repulsive EphA receptor/ephrin‐A ligand interactions direct the formation of the retinocollicular map, but whether ephrins might also be involved in map compression is unknown. To examine whether map compression might be directed by changes in the ephrin expression pattern, we compared ephrin‐A2 and ephrin‐A5 mRNA expression between normal SC and PT SC using in situ hybridization and quantitative real‐time PCR. We found that ephrin‐A ligand expression in the compressed maps was low anteriorly and high posteriorly, as in normal animals. Consistent with our hypothesis, the steepness of the ephrin gradient increased in the lesioned colliculi. Interestingly, overall levels of ephrin‐A2 and ‐A5 expression declined immediately after neonatal target damage, perhaps promoting axon outgrowth. These data establish a correlation between changes in ephrin‐A gradients and map compression, and suggest that ephrin‐A expression gradients may be regulated by target size. This in turn could lead to compression of the retinocollicular map onto the reduced target. These findings have important implications for mechanisms of recovery from traumatic brain injury. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Eph and ephrin signaling in the formation of topographic maps

The axonal connections between the retina and its midbrain target, the superior colliculus (SC), is mapped topographically, such that the spatial relationships of cell bodies in the retina are maintained when terminating in the SC. Topographic map development uses a Cartesian mapping system such that each axis of the retina is mapped independently. Along the nasal-temporal mapping axis, EphAs and ephrin-As, are graded molecular cues required for topographic mapping while the dorsal-ventral axis is mapped in part via EphB and ephrin-Bs. Because both Ephs and ephrins are cell surface molecules they can signal in the forward and reverse directions. Eph/ephrin signaling leads to changes in cytoskeletal dynamics that lead to actin depolymerization and endocytosis guiding axons via attraction and repulsion.     

Ephrin-as guide the formation of functional maps in the visual cortex

Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act to organize thalamic inputs. Here, we map the visual cortex (V1) in mice deficient for ephrin-A2, -A3, and -A5 functionally, 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.     

Visual map development: bidirectional signaling, bifunctional guidance molecules, and competition

Topographic maps are a two-dimensional representation of one neural structure within another and serve as the main strategy to organize sensory information. The retina's projection via axons of retinal ganglion cells to midbrain visual centers, the optic tectum/superior colliculus, is the leading model to elucidate mechanisms of topographic map formation. Each axis of the retina is mapped independently using different mechanisms and sets of axon guidance molecules expressed in gradients to achieve the goal of representing a point in the retina onto a point within the target. An axon's termination along the temporal-nasal mapping axis is determined by opposing gradients of EphAs and ephrin-As that act through their forward and reverse signaling, respectively, within the projecting axons, each of which inhibits interstitial branching, cooperating with a branch-promoting activity, to generate topographic specific branching along the shaft of the parent axons that overshoot their correct termination zone along the anterior-posterior axis of the target. The dorsal-ventral termination position is then determined using a gradient of ephrin-B that can act as a repellent or attractant depending on the ephrin-B concentration relative to EphB levels on the interstitial branches to guide them along the medial-lateral axis of the target to their correct termination zone, where they arborize. In both cases, axon-axon competition results in axon mapping based on relative rather than absolute levels of repellent or attractant activity. The map is subsequently refined through large-scale pruning driven in large part by patterned retinal activity.     

Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling

Topographic maps are a fundamental feature of sensory representations in nervous systems. The formation of one such map, defined by the connection of ganglion cells in the retina to their targets in the superior colliculus of the midbrain, is thought to depend upon an interaction between complementary gradients of retinal EphA receptors and collicular ephrin-A ligands. We have tested this hypothesis by using gene targeting to elevate EphA receptor expression in a subset of mouse ganglion cells, thereby producing two intermingled ganglion cell populations that express distinct EphA receptor gradients. We find that these two populations form separate maps in the colliculus, which can be predicted as a function of the net EphA receptor level that a given ganglion cell expresses relative to its neighbors.     

Balancing of ephrin/Eph forward and reverse signaling as the driving force of adaptive topographic mapping

The retinotectal projection, which topographically maps retinal axons onto the tectum of the midbrain, is an ideal model system with which to investigate the molecular genetics of embryonic brain wiring. Corroborating Sperry's seminal hypothesis, ephrin/Eph counter-gradients on both retina and tectum were found to represent matching chemospecificity markers. Intriguingly, however, it has never been possible to reconstitute topographically appropriate fiber growth in vitro with these cues. Moreover, experimentally derived molecular mechanisms have failed to provide explanations as to why the mapping adapts to grossly diverse targets in some experiments, while displaying strict point-to-point specificity in others. In vitro, ephrin-A/EphA forward, as well as reverse, signaling mediate differential repulsion to retinal fibers, instead of providing topographic guidance. We argue that those responses are indicative of ephrin-A and EphA being members of a guidance system that requires two counteracting cues per axis. Experimentally, we demonstrate by introducing novel double-cue stripe assays that the simultaneous presence of both cues indeed suffices to elicit topographically appropriate guidance. The peculiar mechanism, which uses forward and reverse signaling through a single receptor/ligand combination, entails fiber/fiber interactions. We therefore propose to extend Sperry's model to include ephrin-A/EphA-based fiber/fiber chemospecificity, eventually out-competing fiber/target interactions. By computational simulation, we show that our model is consistent with stripe assay results. More importantly, however, it not only accounts for classical in vivo evidence of point-to-point and adaptive topographic mapping, but also for the map duplication found in retinal EphA knock-in mice. Nonetheless, it is based on a single constraint of topographic growth cone navigation: the balancing of ephrin-A/EphA forward and reverse signaling.     

Computational modeling of retinotopic map development to define contributions of EphA-ephrinA gradients, axon-axon interactions, and patterned activity

The topographic projection of retinal ganglion cell (RGC) axons to mouse superior colliculus (SC) or chick optic tectum (OT) is formed in three phases: RGC axons overshoot their termination zone (TZ); they exhibit interstitial branching along the axon that is topographically biased for the correct location of their future TZ; and branches arborize preferentially at the TZ and the initial exuberant projection refines through axon and branch elimination to generate a precise retinotopic map. We present a computational model of map development that demonstrates that the countergradients of EphAs and ephrinAs in retina and the OT/SC and bidirectional repellent signaling between RGC axons and OT/SC cells are sufficient to direct an initial topographic bias in RGC axon branching. Our model also suggests that a proposed repellent action of EphAs/ephrinAs present on RGC branches and arbors added to that of EphAs/ephrinAs expressed by OT/SC cells is required to progressively restrict branching and arborization to topographically correct locations and eliminate axon overshoot. Simulations show that this molecular framework alone can develop considerable topographic order and refinement, including axon elimination, a feature not programmed into the model. Generating a refined map with a condensed TZ as in vivo requires an additional parameter that enhances branch formation along an RGC axon near sites that it has a higher branch density, and resembles an assumed role for patterned neural activity. The same computational model generates the phenotypes reported in ephrinA deficient mice and Isl2-EphA3 knockin mice. This modeling suggests that gradients of counter-repellents can establish a substantial degree of topographic order in the OT/SC, and that repellents present on RGC axon branches and arbors make a substantial contribution to map refinement. However, competitive interactions between RGC axons that enhance the probability of continued local branching are required to generate precise retinotopy.     

Analysis of mouse EphA knockins and knockouts suggests that retinal axons programme target cells to form ordered retinotopic maps

I present a novel analysis of abnormal retinocollicular maps in mice in which the distribution of EphA receptors over the retina has been modified by knockin and/or knockout of these receptor types. My analysis shows that in all these cases, whereas the maps themselves are discontinuous, the graded distribution of EphA over the nasotemporal axis of the retina is recreated within the pattern of axonal terminations across rostrocaudal colliculus. This suggests that the guiding principle behind the formation of ordered maps of nerve connections between vertebrate retina and superior colliculus, or optic tectum, is that axons carrying similar amounts of Eph receptor terminate near to one another on the target structure. I show how the previously proposed marker induction model embodies this principle and predicts these results. I then describe a new version of the model in which the properties of the markers, or labels, are based on those of the Eph receptors and their associated ligands, the ephrins. I present new simulation results, showing the development of maps between two-dimensional structures, exploring the role of counter-gradients of labels across the target and confirming that the model reproduces the retinocollicular maps found in EphA knockin/knockout mice. I predict that abnormal distributions of label within the retina lead to abnormal distributions of label over the target, so that in each of the types of knockin/knockout mice analysed, there will be a different distribution of labels over the target structure. This mechanism could be responsible for the flexibility with which neurons reorganise their connections during development and the degree of precision in the final map. Activity-based mechanisms would play a role only at a later stage of development to remove the overlap between individual retinal projection fields, such as in the development of patterns of ocular dominance stripes.     

Selective disruption of one Cartesian axis of cortical maps and receptive fields by deficiency in ephrin-As and structured activity

The topographic representation of visual space is preserved from retina to thalamus to cortex. We have previously shown that precise mapping of thalamocortical projections requires both molecular cues and structured retinal activity. To probe the interaction between these two mechanisms, we studied mice deficient in both ephrin-As and retinal waves. Functional and anatomical cortical maps in these mice were nearly abolished along the nasotemporal (azimuth) axis of the visual space. Both the structure of single-cell receptive fields and large-scale topography were severely distorted. These results demonstrate that ephrin-As and structured neuronal activity are two distinct pathways that mediate map formation in the visual cortex and together account almost completely for the formation of the azimuth map. Despite the dramatic disruption of azimuthal topography, the dorsoventral (elevation) map was relatively normal, indicating that the two axes of the cortical map are organized by separate mechanisms.     

Opposing gradients of ephrin-As and EphA7 in the superior colliculus are essential for topographic mapping in the mammalian visual system

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. Here we investigate the role of an EphA7 gradient in the SC, which has the reverse orientation to the ephrin-A gradient. We find that 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. Our 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.     

Two homeobox genes define the domain of EphA3 expression in the developing chick retina

Graded expression of the Eph receptor EphA3 in the retina and its two ligands, ephrin A2 and ephrin A5 in the optic tectum, the primary target of retinal axons, have been implicated in the formation of the retinotectal projection map. Two homeobox containing genes, SOHo1 and GH6, are expressed in a nasal-high, temporal-low pattern during early retinal development, and thus in opposing gradients to EphA3. Retroviral misexpression of SOHo1 or GH6 completely and specifically repressed EphA3 expression in the neural retina, but not in other parts of the central nervous system, such as the optic tectum. Under these conditions, some temporal ganglion cell axons overshot their expected termination zones in the rostral optic tectum, terminating aberrantly at more posterior locations. However, the majority of ganglion cell axons mapped to the appropriate rostrocaudal locations, although they formed somewhat more diffuse termination zones. These findings indicate that other mechanisms, in addition to differential EphA3 expression in the neural retina, are required for retinal ganglion axons to map to the appropriate rostrocaudal locations in the optic tectum. They further suggest that the control of topographic specificity along the retinal nasal-temporal axis is split into several independent pathways already at a very early time in development.     

The Caenorhabditis elegans Six/sine oculis class homeobox gene ceh-32 is required for head morphogenesis

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. We have analyzed the distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon during the period when visual connections are being established. We have found that 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 was observed. Finally, we also show that 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.     

Endocytosis of EphA receptors is essential for the proper development of the retinocollicular topographic map

Endocytosis of Eph-ephrin complexes may be an important mechanism for converting cell-cell adhesion to a repulsive interaction. Here, we show that an endocytosis-defective EphA8 mutant forms a complex with EphAs and blocks their endocytosis in cultured cells. Further, we used bacterial artificial chromosome transgenic (Tg) mice to recapitulate the anterior>posterior gradient of EphA in the superior colliculus (SC). In mice expressing the endocytosis-defective EphA8 mutant, the nasal axons were aberrantly shifted to the anterior SC. In contrast, in Tg mice expressing wild-type EphA8, the nasal axons were shifted to the posterior SC, as predicted for the enhanced repellent effect of ephrinA reverse signalling. Importantly, Rac signalling was shown to be essential for EphA-ephrinA internalization and the subsequent nasal axonal repulsion in the SC. These results indicate that endocytosis of the Eph-ephrin complex is a key mechanism by which axonal repulsion is generated for proper guidance and topographic mapping.     

Expression patterns of Ephs and ephrins throughout retinotectal development in Xenopus laevis

The Eph family of receptor tyrosine kinases and their ligands the ephrins play an essential role in the targeting of retinal ganglion cell axons to topographically correct locations in the optic tectum during visual system development. The African claw-toed frog Xenopus laevis is a popular animal model for the study of retinotectal development because of its amenability to live imaging and electrophysiology. Its visual system undergoes protracted growth continuing beyond metamorphosis, yet little is known about ephrin and Eph expression patterns beyond stage 39 when retinal axons first arrive in the tectum. We used alkaline phosphatase fusion proteins of EphA3, ephrin-A5, EphB2, and ephrin-B1 as affinity probes to reveal the expression patterns of ephrin-As, EphAs, ephrin-Bs, and EphBs, respectively. Analysis of brains from stage 40 to adult frog revealed that ephrins and Eph receptors are expressed throughout development. As observed in other species, staining for ephrin-As displayed a high caudal to low rostral expression pattern across the tectum, roughly complementary to the expression of EphAs. In contrast with the prevailing model, EphBs were found to be expressed in the tectum in a high dorsal to low ventral gradient in young animals. In animals with induced binocular tectal innervation, ocular dominance bands of alternating input from the two eyes formed in the tectum; however, ephrin-A and EphA expression patterns were unmodulated and similar to those in normal frogs, confirming that the segregation of axons into eye-specific stripes is not the consequence of a respecification of molecular guidance cues in the tectum.     

Genetic analysis of EphA-dependent signaling mechanisms controlling topographic mapping in vivo

Ephrin/Eph ligands and receptors are best known for their prominent role in topographic mapping of neural connectivity. Despite the large amount of work centered on ephrin/Eph-dependent signaling pathways in various cellular contexts, the molecular mechanisms of action of Eph receptors in neural mapping, requiring dynamic interactions between complementary gradients of ephrins and Eph receptors, remain largely unknown. Here, we investigated in vivo the signaling mechanisms of neural mapping mediated by the EphA4 receptor, previously shown to control topographic specificity of thalamocortical axons in the mouse somatosensory system. Using axon tracing analyses of knock-in mouse lines displaying selective mutations for the Epha4 gene, we determined for the first time which intracellular domains of an Eph receptor are required for topographic mapping. We provide direct in vivo evidence that the tyrosine kinase domain of EphA4, as well as a tight regulation of its activity, are required for topographic mapping of thalamocortical axons, whereas non-catalytic functional modules, such as the PDZ-binding motif (PBM) and the Sterile-alpha motif (SAM) domain, are dispensable. These data provide a novel insight into the molecular mechanisms of topographic mapping, and constitute a physiological framework for the dissection of the downstream signaling cascades involved.     

p75(NTR) mediates ephrin-A reverse signaling required for axon repulsion and mapping

Reverse signaling by ephrin-As upon binding EphAs controls axon guidance and mapping. Ephrin-As are GPI-anchored to the membrane, requiring that they complex with transmembrane proteins that transduce their signals. We show that the p75 neurotrophin receptor (NTR) serves this role in retinal axons. p75(NTR) and ephrin-A colocalize within caveolae along retinal axons and form a complex required for Fyn phosphorylation upon binding EphAs, activating a signaling pathway leading to cytoskeletal changes. In vitro, retinal axon repulsion to EphAs by ephrin-A reverse signaling requires p75(NTR), but repulsion to ephrin-As by EphA forward signaling does not. Constitutive and retina-specific p75(NTR) knockout mice have aberrant anterior shifts in retinal axon terminations in superior colliculus, consistent with diminished repellent activity mediated by graded ephrin-A reverse signaling induced by graded collicular EphAs. We conclude that p75(NTR) is a signaling partner for ephrin-As and the ephrin-A- p75(NTR) complex reverse signals to mediate axon repulsion required for guidance and mapping.