Molecular complexity of visual mapping a challenge for regenerating therapy

NEURAL REGENERATION RESEARCH www.nrronline.orgREVIEW

Molecular complexity of visual mapping: a challenge for regenerating therapy

Mara Medori1, 2, Gonzalo Spelzini1, 2, Gabriel Scicolone1, 2, *

1 CONICET – Universidad de Buenos Aires, Instituto de Biología Celular y Neurociencias “Prof. E. De Robertis” (IBCN), Ciudad Autónoma de Buenos Aires, Argentina

2 Universidad de Buenos Aires, Facultad de Medicina, Departamento de Biología Celular, Histología, Embriología y Genética, Ciudad Autónoma de Buenos Aires, Argentina

Funding: This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 00441); Universidad de Buenos Aires (M 00526BA, 00769BA, both to GS).Abstract

*Correspondence to:

Investigating the cellular and molecular mechanisms involved in the development of topographically or-dered connections in the central nervous system constitutes an important issue in neurobiology because these connections are the base of the central nervous system normal function. The dominant model to study the development of topographic maps is the projection from the retinal ganglion cells to the optic tectum/colliculus. The expression pattern of Eph/ephrin system in opposing gradients both in the retina and the tectum, labels the local addresses on the target and gives specific sensitivities to growth cones ac-cording to their topographic origin in the retina. The rigid precision of normal retinotopic mapping has prompted the chemoaffinity hypothesis, positing axonal targeting to be based on fixed biochemical affini-ties between fibers and targets. However, several lines of evidence have shown that the mapping can adjust to experimentally modified targets with flexibility, demonstrating the robustness of the guidance process. Here we discuss the complex ways the Ephs and ephrins interact allowing to understand how the retinotec-tal mapping is a precise but also a flexible process.

Key Words: axon growth; axon guidance; development; Eph and ephrin; mapping; regeneration; retinal ganglion cells; retino-tectal systemGabriel Scicolone, PhD, MD, gscicolo@retina.ar. orcid:

0000-0002-3391-957X (Gabriel Scicolone)

doi: 10.4103/1673-5374.266044Received: February 26, 2019Accepted: May 14, 2019

Molecular Mechanisms of Mapping:

Retinotectal/Collicular System as a Model

Investigating the cellular and molecular mechanisms in-volved in the development of topographically ordered con-nections in the central nervous system (CNS) constitutes an important issue in neurobiology because these connections are the base of the CNS normal function. Axonal projec-tions between two populations of neurons, which preserve neighborhood relationships, are called topographic maps and they are ubiquitous in the brain. The dominant model to study the development of topographic maps is the projection from the retinal ganglion cells (RGCs) to its major midbrain target namely the optic tectum of fishes, frogs and chicks or its mammalian homolog, the superior colliculus. This map is organized in two orthogonally oriented axes. Nasal RGCs project to the caudal tectum and the temporal ones project to the rostral tectum, whereas dorsal RGCs project to the ventral (lateral) tectum and ventral RGCs project to the dorsal (medial) tectum (Figure 1). This organization is the cellular base by which the visual field inverted in the retina is correctly reconstituted on the tectal surface (Vanegas and Ito, 1983; Flanagan, 2006). Therefore, the regeneration of retinofugal maps is the final objective of any regenerative strategy applied in traumatic or degenerative pathologies af-fecting the RGCs or the optic nerve (Scicolone et al., 2009).Significant advances have been obtained in axonal re-growth of damaged RGCs, but no reconstitution of the ret-inotectal/collicular map could be obtained (Kim et al., 2018).

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Latest studies about retinotectal/collicular mapping have shown the existence of complex molecular mechanisms, some of which seems to present conflicting biological conse-quences. Analyzing these mechanisms is the purpose of this short review and their understanding could be applied to perform regeneration therapies.

We searched PubMed database. Selection criteria: activity dependent mapping; Eph ephrin binding; Eph ephrin clus-tering and binding; EphA4; Optic fiber regeneration and Eph ephrin; Optic nerve regeneration; Eph; Retinal projection regeneration; Retinotectal mapping; Visual system and Eph ephrin without data limits.

Eph/Ephrin System in Axon Guidance

Sperry’s classic theory predicted that RGC axons find their synaptic targets in the tectum through a process of interac-tion between recognition molecules, or chemoaffinity labels, differentially expressed on their growth cones and on tectal cells. Sperry suggested that each point in the tectum has a unique molecular address determined by the graded distri-bution of topographic guidance molecules along the two tec-tal axes. Each RGC has a unique profile of receptors for those molecules, resulting in a position-dependent, differential response to the guidance molecules by RGC axons (Sperry, 1963). Graded expression of Eph receptors and their ligands, the ephrins, both in the retina and the optic tectum/superior colliculus has suggested that these molecules could pro-vide the positional information that guides the topographic

Medori M, Spelzini G, Scicolone G (2020) Molecular complexity of visual mapping: a challenge for regenerating therapy.

movement of growth cones in the visual system (Scicolone et al., 2009). The analysis of the roles of Eph/ephrin system in mapping retinal projections onto the tectum/colliculus is the main issue of this review.

kinases comprising ten EphAs and six EphBs. They promis-Ephs are a family of widely expressed receptor tyrosine cuously bind the six glycosylphosphatidylinositol (GPI)-linked ephrin-As and the three transmembrane ephrin-Bs respectively. However, this apparent class-specificity may be an oversimplification, as EphA4 binds to ephrin-Bs, and EphB2 binds to ephrin-A5 (Gale et al., 1996; Himanen et al., 2004; Truitt and Freywald, 2011; Xia et al., 2013) (Figure 2).allows the Eph/ephrin system to signal in forward (Ephs The fact that the ephrins are membrane-bound proteins acting as receptors) and in reverse (ephrins acting as recep-tors) directions (Scicolone et al., 2009). It is thought that in the absence of cell-cell interactions, these molecules exist in loosely associated clusters (microdomains) within plasma membranes, which become much more compact upon Eph/ephrin complex formation, generating clearly defined signal-ing centers at the cell-cell interfaces (Vearing and Lackmann, 2005).The classic model of Eph and ephrin function in neighboring cells involves ephrins acting as in trans ligands of Eph receptors, resulting in cell repulsion (ephrin:Eph, or ‘forward’ signaling). However, Eph receptors can also act as in trans ligands for ephrins (Eph:ephrin or ‘reverse’ signal-ing), eliciting either cell repulsion or adhesion. In addition, both Eph proteins and ephrins can simultaneously act as receptors and ligands, leading to bidirectional or parallel and antiparallel signaling, depending on the distribution of Ephs and ephrins between interacting cells, as well as the direc-tion of signaling in single ephrin-Eph pairs. Ephrins can also induce signaling cascades independently of Eph proteins (Chin-Sang, 2002).

signaling mechanisms exist at present. In most cases, to elicit A number of detailed discussions of the multitude of Eph robust Eph receptor signaling, ephrins must be presented as multimers. This results in the formation of signaling clus-ters, in which Eph receptors form arrays intercalated with ephrins, the size of which correlates with the strength of the signal, and which might partly explain the diverse cellular responses that are elicited by Eph activation. Some studies argue that Ephs and ephrins can interact on the surface of the same cell (in cis), and that this attenuates Eph signal-pos-sibly by inhibiting the formation of Ephs clusters (Carvalho et al., 2006; Kao and Kania, 2011).

the activation of the kinase activity of the Eph (Kullander et One of the first forward ephrin: Eph signaling events is al., 2001) which results in the autophosphorylation of the juxtamembrane Tyr residues, an event that is crucial (Zisch et al., 1998) for Eph-directed cellular responses. In reverse Eph:ephrin signaling, the phosphorylation of the ephrin-B intracellular domain is also an important early event (Hol-land et al., 1996; Brückner et al., 1997) and is mediated by Src family kinases (Palmer et al., 2002). Signals generated by ephrin binding to Eph receptors involve their interaction with specific intracellular proteins, including the non-cata-

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lytic region of Tyr kinase adaptor protein 1 and Nck2, phos-phoinositide 3-kinase, Src family kinases, Vav2, Vav3 and ephexin. In turn, these effectors are coupled to Rho GTPases such as RhoA1, Cdc42 and Rac1, which can modulate the cytoskeleton (Kania and Klein, 2016).

for signaling mediated by both ephrin classes. In addition, For reverse signaling, Src family kinases seem to be crucial Ret and p75 are transmembrane effectors of class A ephrin signaling (Lim et al., 2008; Marler et al., 2008; Bonanomi et al., 2012), and the Grb4-Pak-1-Dock180 complex specifically interacts with the carboxyl terminus of B class ephrins (Xu and Henkemeyer, 2009). Many Eph-triggered cellular re-sponses eventually lead to cytoskeletal rearrangements, such as the collapse of the cytoskeleton, by controlling the balance between small GTPase activation and inactivation (Kania and Klein, 2016). Once intracellular signaling is initiated, the repulsive cellular responses seem to rely on the dissociation of ephrins from Ephs through proteolytic cleavage of ephrins (Hattori et al., 2000; Janes et al., 2005) and/or Eph receptors (Lin et al., 2008), an event that has been proposed to termi-nate ephrin-Eph signaling.

Rigid and Precise Mapping Rigid and precise mapping: the chemoaffinity hypothesis Robust Mapping

versus Flexible and based on Eph/ephrin system

The rigid precision of normal retinotopic mapping has prompted the chemoaffinity hypothesis, positing axonal targeting to be based on fixed biochemical affinities between fibers and targets (Scicolone et al., 2009). However, sever-al lines of evidence have been gathered that the mapping can adjust to experimental modified targets with flexibility demonstrating the robustness of the guidance process. The identification of ephrins and Eph-receptors as the underlying molecular cues has mostly been interpreted as supporting the fiber-target chemoaffinity hypothesis, while the evidence on mapping robustness has been neglected (Weth et al., 2014).

in gradients in both the retina and the tectum, and it was Eph receptors and their ephrin ligands are expressed shown that they represent the main molecular system con-trolling the mapping of retinal projections onto the tectum/colliculus (Scicolone et al., 2009). EphAs and ephrin-As define the topographic retinotectal connections along the rostro-caudal axis, whereas EphBs and ephrin-Bs have been implicated along the dorso-ventral axis. This is achieved through opposing gradients of Ephs and ephrins in both the retina and the tectum (Scicolone et al., 2009) (Mapping along rostro-caudal axis was focused in this review Figure 1). because the molecular mechanisms in this axis are better un-derstood than in the dorso-ventral one.

dient in the tectum are growth cone repellents (Drescher et Ephrin-As expressed in an increasing rostro-caudal gra-al., 1995; Nakamoto et al., 1996; Monschau et al., 1997) and interstitial branching inhibitors (Yates et al., 2001; Sakurai et al., 2002) that preferentially affect temporal RGC axons

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Medori M, Spelzini G, Scicolone G (2020) Molecular complexity of visual mapping: a challenge for regenerating therapy. Neural Regen Res 15(3):382-389. doi:10.4103/1673-5374.266044

by activating their EphA receptors (Brown et al., 2000; Feld-heim, 2004). Thus, tectal ephrin-As prevent temporal RGCs from branching caudally to their appropriate termination zone. It was shown that axonal ephrin-As diminish the repulsive response of axonal EphA receptors to tectal eph-rin-As, preventing repulsion of nasal RGC axons from the caudal tectum (Hornberger et al., 1999). However, these data do not explain why nasal RGC axons grow toward the caudal tectum without branching rostrally to their appropriate tar-get area. Two opposing forces are required, so that each axon branches off where these forces balance (Yates et al., 2001; Flanagan, 2006; Gosse et al., 2008; Scicolone et al., 2009).mapping force. One model proposes a bifunctional activ-Conflicting models have been postulated about the second ity of tectal ephrin-As, showing an attractant effect at low concentrations in the rostral tectum and a repulsive effect at higher concentrations in the caudal tectum (Hansen et al., 2004; Honda, 2004). The transition from attraction to repul-sion varied systematically with both ephrin concentration and retinal position, providing topographic specificity. These results support a model in which map position could be specified as a point where positive and negative forces bal-ance out for each specific position of origin of RGCs (Hansen et al., 2004; Naoki, 2017) (branching was evaluated. Indeed, others have posited that Figure 3A). However, no effect on branch formation is induced where the balance of EphA/ephrin-A signaling in the RGC is achieved to allow for Brain-derived neurotrophic factor (BDNF)-induced branch-ing (Triplett, 2014). Besides, these experiments employed membrane vesicles from chicken rostral tecta to provide a permissive substrate mixed with different concentrations of 293T cell membranes transfected with ephrin-As. Since an attractant effect was demonstrated for the EphA3 expressed in chicken rostral tectum (Ortalli et al., 2012), the former work cannot exclude the possibility that the attractant effect attributed to lower concentrations of ephrin-As could be partially due to parallel increasing concentrations of EphA3 (Scicolone et al., 2009) (poses that this second force is produced by a decreasing ros-Figure 3A). Another model pro-tro-caudal gradient of EphA7 which repels nasal optic fibers and prevent them from branching in the rostral tectum/col-liculus throughout ephrin-As reverse signaling (Rashid et al., 2005; Lim et al., 2008) (invade the tectum/colliculus throughout the highest part Figure 3B). However, as optic fibers of this gradient, this model cannot explain how the axons invade the tectum/colliculus without being repelled from it. On the other hand, we demonstrated that the decreas-ing rostro-caudal tectal gradient of EphA3 promotes nasal RGC axon growth toward the caudal tectum and inhibits them from branching rostrally (Ortalli et al., 2012). Thus, its positive effect on axon growth, instead of a repellent effect, allows explaining the axonal invasion of the tectum and the axon growth of nasal RGCs toward the caudal tectum (Ortalli et al., 2012) (the bifuntional effect of tectal ephrin-As on RGC axons plus Figure 3C). It is possible that a combination of the promoting axon growth and branching inhibition of tec-tal EphA3 could act as partially redundant systems together

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with BDNF branching promoting effect (Figure 3D).Plasticity in axon guidance and retinotectal mapping are consistent with precise distributed cues

The concept of rigid mapping was challenged by experiments. In mature goldfish, a disconnected temporal in vivo half-retina resulting from nerve transaction and nasal abla-tion regenerated an expanded, proper projection covering the whole tectum (expansion experiments) (Schmidt et al., 1978). Similar results were also obtained in Xenopus development. Double temporal or double nasal retinae mi-crosurgically assembled in the larvae, instead of forming doubly occupied maps on their respective tectal halves, matured into expanded projections of each half-retina on the whole target (compound eye experiments) (Gaze et al., 1963). Conversely, tectal ablations (rostral or caudal half) in the goldfish, irrespective of concomitant nerve transaction, yielded compressed maps of the full retina on the remaining half-target (compression experiments) (Sharma, 1972). No-tably, without nerve transaction, this implies mobilization of maturely connected axons of the remaining half-tectum. Thus, topographic maps can flexibly adjust to the target. As Ephs/ephrins are stable, while axons can flexibly map, che-moaffinity might not be absolute but relative (Weth et al., 2014). Neither expansion nor normal topographic mapping depends on neural activity, but they are genetically hard-wired. Pre-vision spontaneous activity, like early experi-ence-driven activity is important for circuit refinement (Weth et al., 2014; Thompson et al., 2017).

eye and compression experiments indicate, in addition to Weth et al. (2014) concluded that expansion, compound fiber-target chemoaffinity, the existence of a second guidance influence, which they called fiber-fiber chemoaffinity. Thus, the ephrin/Eph forward and reverse signaling throughout trans and cis interactions in fiber-fiber interactions can ex-plain the maintaining of the retinotectal map with changes in the target (binding between EphAs and ephrin-As in trans and in cis, Figure 4A). Thus, interaxonal and intraaxonal could add plasticity to the retinotectal mapping. This as-sumption can reconcile the seemingly conflicting findings on rigid and flexible topographic mapping. Accordingly, some works have suggested that interaxonal competition partici-pates in the establishment of topographic ordered connec-tions in fishes, chicks and mice (Feldheim, 2004; Honda, 2004; Yates et al., 2004; Pfeiffenberger et al., 2006; Triplett et al., 2011). Nevertheless, Gosse et al. (2008) postulated that interaxonal competition is not required for retinotopic tar-geting along the rostro-caudal axis in zebrafish, but serves to restrict arbor size and shape.

ed that retinal growth cones (GCs) robustly adapt towards On the other hand, novel adaptation assays demonstrat-ephrin-A/EphA forward and reverse signals (Fiederling et al., 2017). Ineffect of a molecular cue is recognized by producing growth in vitro collapse assays (in which the repulsive cone collapse) typically temporal GCs collapse with eph-rin-As. Surprisingly, temporal GCs recover their morpholo-gy, despite the presence of the repulsive cue ephrin-As after

Medori M, Spelzini G, Scicolone G (2020) Molecular complexity of visual mapping: a challenge for regenerating therapy.

prolonged incubation. This indicates desensitization of RGC GCs towards forward signals. The same phenomenon was observed for reverse ephrin-A/EphA signaling when EphAs were applied as ligands after prolonged incubation. This was puzzling, because topographic guidance was believed to rely on precise quantitative sensing. Computational modeling suggested that topographic accuracy and adaptability could be reconciled by a novel mechanism of coupled adaptation of forward and reverse Eph/ephrins signaling. Thus, ephrin-As ligands in the substrate are able to desensitize GCs from EphA forward and ephrin-As reverse signaling, and EphAs acting as ligands in the substrate are able to desensitize GCs from ephrin-As reverse and EphAs forward signaling (4Bbetween the surface membrane and recycling endosomes. ). Co-adaptation involves trafficking of unbound sensors Figure Authors proposed that co-adaptative desensitization eventu-ally relies on guidance sensor translocation into cis-signaling endosomes (Fiederling et al., 2017). Together, these findings proved the existence of a novel mechanism of signal modula-tion (co-adaptation), which allows for topographic mapping in the presence of GC adaptation. Co-adaptation could ex-plain the ingrowth of optic fibers through the repulsive effect of EphA7 in the rostral tectum/colliculus (no molecule was discovered Figure 3B), but optic fibers from rostral tectal EphAs.

in vivo which could desensitize allows explaining how RGC axons invade the tectum and Finally, we have shown a new molecular mechanism which how axon growth is regulated over tectal surface (Fiore et al., 2019). Thus, we demonstrated that ephrin-As-dependent EphA4 forward signaling decreases axon growth in a target independent way. We showed that cis interactions and per-haps trans interaxonal interactions produce EphA4 forward signaling. This effect is higher in nasal RGC axons (Fiore et al., 2019). This is a long lasting effect in which EphA4 bear-ing axons are not retracted by axonal ephrin-As, instead, they decrease the level of axon growth. This effect has some similarities with the adaptation process described by Fie-derling et al. (2017). Therefore, when nasal axons arrive the tectum, EphA3 decreases EphA4 forward signaling by com-peting for axonal ephrin-As (increases axon growth by reducing ephrin-A-dependent Figure 4C). Thus, target EphA3 EphA4 forward signaling. When nasal RGC axons arrive the caudal tectum, the decreasing level of EphA3 allows increas-ing ephrin-A-dependent EphA4 signaling which produces a decrease in the level of axon growth. Besides, the increas-ing levels of tectal ephrin-As stop axon growth throughout EphA forward signaling (Fiore et al., 2019).

also a flexible process. Several molecular ways of interac-In summary, the retinotectal mapping is a precise but tions between EphAs and ephrin-As have been described which could explain the coexistence of these two apparently opposite properties of mapping. Thus, fiber-target EphAs/ephrin-As forward and reverse signaling, competition be-tween fiber and target EphAs for fibers ephrin-As, fiber-fi-ber EphAs/ephrin-As forward and reverse signaling, as a consequence of trans and cis interactions have been shown. Besides, different ways of cis interactions –in parallel with

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masking properties (Kania and Klein, 2016) or in anti-par-allel orientation on GC surface (Fiore et al., 2019) or inside endosomes (Fiederling et al., 2017) inducing signaling have been shown (part of the complex molecular network which regulates axon Figure 4). All of these interactions represent a guidance and retinotectal mapping.

Can These Findings Collaborate for Designing Regeneration Therapies?

No clinical treatments are available to help those who suffer from loss of function due to axonal injuries associated with optic neuropathy. The optic nerve crush rodent model of traumatic optic neuropathy is a well-established system for tackling the fundamental problem of long-distance axon regeneration failure in the CNS and for determining poten-tially novel treatments. Few studies showed regeneration of RGC axons beyond the optic chiasm (Kim et al., 2018).

generation: enhancing the intrinsic growth capacity of RGCs, There are several hurdles to overcome in optic nerve re-overcoming the extrinsic growth-inhibitory environment of the optic nerve and optimizing the reinnervation of their targets. Some degree of optic nerve regeneration has been achieved by factors associated with inducing intraocular in-flammation or by providing exogenous neurotrophic factors. Reactivating intrinsic growth capacity of mature RGCs has enabled experimental optic nerve regeneration by inhibition of cell-intrinsic suppressors of axon growth, or by activation of the intracellular signaling pathways (Chun and Cestari, 2017). Stimulation of neural activity enhanced RGC axon regeneration. However, RGC axons that recover back to their target fails during myelination and consequently undergo slower conduction of electrical potentials (Laha et al., 2017). Modifying the extrinsic growth-inhibitory environment of the optic nerve has also achieved some degree of optic nerve regeneration by suppressing receptors to cell extrinsic inhibitors, inhibiting RhoA/ROCK pathway, by chelation of mobile zinc, or by administration of calcium channel block-ers (Chun and Cestari, 2017). Peripheral nerve grafts have been used to bridge tissue defects from retina to colliculus (You et al., 2016). In some experiments, axons have shown to reinnervate their targets, but they generally showed a lack of topographic order. Therefore, a major aim of visual system repair is the restoration of neural maps (You et al., 2016; Chun and Cestari, 2017). For this purpose, retinal projections must be topographically organized onto targets according to the gradients of Eph/ephrin system. It has been suggested that this system persist in the mature mammalian visual system or become upregulated after optic nerve injury, but more research is required in this area (Chun and Cestari, 2017).

avian generally do not regenerate. By contrast, in anamniotes Severed axonal connections in the CNS of mammals and (fishes and amphibians) many axonal tracts, including the optic nerve, spontaneously regrow leading to functional re-covery (Stuermer et al., 1992; Bernhardt, 1999). Accordingly, these animals present a continuous retinal growth during

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Medori M, Spelzini G, Scicolone G (2020) Molecular complexity of visual mapping: a challenge for regenerating therapy. Neural Regen Res 15(3):382-389. doi:10.4103/1673-5374.266044

Figure 1 Representation of retinotectal/collicular projections and the expression patterns of EphAs and ephrin-As along its rostro-caudal axis and the EphBs and ephrin-Bs along its dorso-ventral axis.

Retinal ganglion cells (RGCs) project to contralateral tectum in chicks whereas they project bilaterally to colliculus in mice. Nasal (N) RGCs axons project to caudal (C) tectum/colliculus whereas temporal (T) RGC axons project to rostral (R) tectum/colliculus. Dorsal (D) RGCs axons project to ventral(V)-lateral (L) tectum/colliculus meanwhile ventral (V) RGC axons project to dorsal (D)-medial (M) tectum/colliculus. Ephs and ephrins are expressed in gradients both in the retina and the tectum/col-liculus. EphAs (3, 5, 6) (light blue) are expressed in an increasing naso-temporal gradient in the retina, whereas EphA4 presents an even expression along the retina (purple), but it presents a decreasing nasodorsal to temporoventral gradient of phosphor-ylation (p) (blue). EphAs (3, 6, 7) are expressed in a decreasing rostro-caudal gradient in the tectum/colliculus. Ephrin-As (2, 5, 6) are expressed in a decreasing naso-temporal gradient in the retina and in an increasing rostro-caudal gradient in the tectum/colliculus (red). EphBs are expressed in increasing dorso-ventral gradients both in the retina and the tectum/colliculus (orange) whereas ephrin-Bs are expressed in-decreasing dorso-ventral gra-dients both in the retina and the tectum/colliculus (green).

Figure 2 Structural classes of Eph receptors and ephrins and theirbinding specificities.

Despite some described high affinity ligand/receptor interactions (red arrows), binding is mostly promiscuous within each of the ephrin/Eph specificity classes (black arrows). In addition, there are two exceptions that show low affinities between members of distinct subclasses (green dashed arrows). Ligands for EphA9 and EphB5 receptors have still not been described. Ligands and receptors have been characterized in mammals (light red), chick (blue) or both (light red/blue).

Figure 3 Schematic representations of different biological models that try to explain the retinotectal mapping along the rostro-caudal axis.

(A) Ephrin-A2 has an attractant role (green) at low concentra-tions in the rostral tectum and a repellent effect (red) at high concentrations in the caudal tectum. Termination zones (TZ) are formed where both forces are balanced according to the different sensitivity of the optic fibers. (B) EphA7 repels (red) optic fibers from the rostral colliculus meanwhile ephrin-As (red) repel them from the caudal colliculus. How are the optic fibers (OF) able to invade the colliculus throughout the re-pellent activity of EphA7? (C) EphA3 expressed in the rostral tectum pushes optic fibers to the caudal tectum (green) mean-while ephrin-As repel them (red). (D) Combination of A and C. Green: Positive effect on axon growth; red: repulsive effect.

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