Topographic mapping: Organising by repulsion and competition? David G. Wilkinson

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1 R447 Topographic mapping: Organising by repulsion and competition? David G. Wilkinson The establishment of topographic maps of neuronal connections is believed to involve graded repulsion mediated by EphA receptors and ephrin-a ligands. Gene knockouts show that ephrin-a ligands do indeed have a crucial role in mapping, and that mechanisms in addition to graded repulsion must also be at work. of repulsiveness of the target tissue depends on its level of ephrin-a ligands [8,9]. Retinal axons enter the anterior tectum, and it is proposed that, as they navigate posteriorly up the ephrin-a gradient, growth cone arrest occurs when Figure 1 Address: Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. dwilkin@nimr.mrc.ac.uk Current Biology 2000, 10:R447 R /00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. One way that information is transferred from one place to another in the nervous system is via circuitry in which spatial relationships within a layer of neuronal cell bodies are maintained in their connections to target tissue. A striking example of such a topographic map is the projection of retinal ganglion cell axons to a region of the midbrain, known as the tectum in the chick and as the superior colliculus in rodents. Axons from the temporal (posterior) retina project to the anterior tectum, and those from increasingly nasal (anterior) regions of the retina project to increasingly posterior parts of the tectum (Figure 1a). Similarly, there is an orderly map of projections from dorsal retina to ventral tectum, and from ventral retina to dorsal tectum. Topographic mapping also occurs in the projection of retinal axons to target areas in the forebrain. As a consequence, the spatial organisation of information received by each eye is maintained upon transfer to the brain. A key question is how topographic maps are set up during development. From theoretical considerations, Sperry [1] reasoned that maps may be established by a graded distribution of guidance molecules in the target tissue and of their receptor(s) in neurons. Indeed, there is now extensive evidence that such a mechanism has an important role along the anteroposterior axis of the retinotectal projection. This involves graded repulsion [2,3] that is mediated by EphA receptor tyrosine kinases and their membraneanchored ephrin-a ligands. In the chick embryo, the EphA3 receptor is expressed in a gradient decreasing from temporal to nasal in the retina, and ephrin-a5 and ephrin-a2 are each expressed in a gradient decreasing from posterior to anterior in the tectum [4,5]. Evidence that ephrin-a5 and ephrin-a2 trigger repulsion [5 7] led to a model in which the sensitivity of axons to repulsion depends on their level of EphA3, and the degree Topographic mapping and EphA/ephrin-A expression. (a) Retinal axons form a map in which temporal axons (T) project to the anterior (A), and nasal axons (N) to the posterior (P) tectum/superior colliculus. They do not form connections with the inferior colliculus. (b) In the chick, there is graded expression of EphA3 in the retina, and of ephrin-a2 plus ephrin-a5 in the tectum. These gradients may underlie topographic mapping by mediating graded repulsion, such that temporal axons (high EphA3) are restricted to the anterior tectum (low ephrin-a), and nasal axons (low EphA3) can enter posterior tectum (high ephrin-a). (c) In the mouse, there is graded expression of EphA5 (rather than EphA3) in the retina, and of ephrin-a5 and ephrin-a2 in the superior colliculus. Ephrin-A5 is also expressed in the inferior colliculus. (d) Graded expression of ephrin-a5 also occurs in the chick and mouse retina, overlapping with uniform EphA4 expression. (e) This overlap in expression leads to desensitisation of retinal axons. This is represented as a gradient of sensitive Eph receptors (EphA*).

2 R448 Current Biology Vol 10 No 12 repulsion counterbalances an attractive influence of the tectum. As a consequence, temporal axons (high EphA3) are restricted to the anterior tectum (low ephrin-a), whereas more nasal axons (lower EphA3) can enter the more posterior tectum (higher ephrin-a) (Figure 1b). A crucial test of this appealing model involves the effects of single and double null mutations of the ephrin-a5 and ephrin-a2 genes. This has now been accomplished and the results show that there is more to mapping than graded repulsion [10]. In the mouse embryo, ephrin-a2 expression is highest within the posterior superior colliculus and decreases anteriorly and posteriorly. Ephrin-A5 is expressed in the inferior colliculus the tissue immediately posterior to the superior colliculus and in a posterior-to-anterior gradient in the superior colliculus that is somewhat steeper than the ephrin-a2 gradient (Figure 1c; see [10] for references). This pattern, and those in other species, is consistent with ephrin-a2 and ephrin-a5 having additive roles in graded repulsion, and with ephrin-a5 being predominant in preventing overshooting of retinal axons into territory posterior to the tectum/superior colliculus [7,11]. In the mouse, unlike in the chick, EphA5 rather than EphA3 is expressed in a temporal-to-nasal gradient in retinal axons [12]. As reported in an earlier paper [13], in homozygous null mutants for ephrin-a5, some temporal axons project to a more posterior location in the superior colliculus, and some transiently overshoot into the inferior colliculus (Figure 2a). Similarly, more posterior terminations of temporal axons in the superior colliculus are seen in ephrin-a2 homozygous mutants, and in ephrin-a5/ephrin-a2 double heterozygotes [10] (Figure 2a). In the ephrin-a2 mutants, however, there is no overshooting into the inferior colliculus, supporting the idea that this role is fulfilled by ephrin-a5. Notably, in all of these mutants, many axons still terminate in the correct region, rather than having a posterior shift in the projection of all axons that would leave the anterior superior colliculus unfilled. Nasal axons are not affected in ephrin-a2 mutants, consistent with a dominant role of ephrin-a5 in the posterior superior colliculus. In ephrin-a5 mutants, however, rather than overshooting into posterior territory, some nasal axons project to more anterior regions of the superior colliculus [10]; an equivalent situation also occurs for targets of retinal axons in the forebrain [12]. In double ephrin-a5/ephrin-a2 homozygous mutants, topographic mapping is almost (but not quite) abolished, and a greater proportion of temporal axons project more posteriorly, and nasal axons more anteriorly, than in the ephrin-a5 single mutant [10] (Figure 2a). Remarkably, the projections of retinal axons still fill the superior colliculus, rather than there being a global overshooting in the double mutant. These results seem contrary to the idea that mapping involves the arrest of retinal growth cones when they arrive at a threshold level of ephrin-a ligands. A partial explanation for the mutant phenotype may come from intriguing findings that implicate the expression of ephrin-a ligands in retinal axons in retinotectal mapping [14]. In addition to being expressed in the tectum, ephrin-a5 and ephrin-a2 are expressed, in a decreasing nasal-to-temporal gradient, in the chick retina [11,15,16] (Figure 1d). This overlaps with the expression of EphA receptors, including EphA4, which occurs uniformly across the retina, leading to persistent activation of EphA4 in the region of overlap [16]. In in vitro assays, stripes of cell membranes derived from the posterior tectum, which contain high levels of ephrin-a ligands, were found to repel temporal but not nasal retinal axons [3]. But when ephrin-a ligands were removed from nasal axons by enzymatic cleavage of their glycophosphatidyl inositol (GPI) membrane anchors, the axons now became sensitive to repulsion in the stripe assay [14]. Conversely, when ephrin-a5 was ectopically expressed in temporal axons, they became insensitive to repulsion in the stripe assay, and overexpression in temporal and nasal axons in vivo caused them to overshoot in the tectum [14,17]. These results suggest that overlapping expression of EphA receptors with ephrin-a ligand in the nasal retina decreases the sensitivity of the retinal axons to repulsion by the ephrin-a gradient encountered in the tectum. It is not known whether this is because of persistent EphA receptor activation or some other mechanism, but it can be thought of as creating a decreasing temporal-to-nasal gradient of sensitive EphA receptors (EphA*; Figure 1e). The differential sensitivity of retinal axons may therefore involve a combination of graded levels of Eph receptor expression and an overlap of graded ephrin and uniform receptor expression that underlies graded Eph receptor sensitivity. In support of a role of retinal ephrin expression in desensitisation, membrane stripe assays showed that nasal axons from ephrin double mutant mice, unlike those from wild-type mice, are sensitive to repulsion by membranes from the posterior superior colliculus of wild-type mice [10]. A desensitisation function for ephrin-a5 expression can neatly explain why nasal axons arrest in the more anterior superior colliculus in ephrin-a5 null mutants: even though the total ephrin level in the superior colliculus is decreased in the absence of ephrin-a5, nasal axons are now more strongly repelled by the ephrin-a2 gradient (Figure 2b). A similar situation does not occur in the ephrin-a2 null mutant, despite ephrin-a2 being expressed in the retina of wild-type mouse embryos (D. Feldheim and J. Flanagan, personal communication), and this may indicate that ephrin-a5 has a dominant role in desensitisation. Indeed, overexpression of ephrin-a5 has

3 R449 a more potent effect on nasal axons than ephrin-a2 [17], perhaps because of its higher affinity for EphA4. Figure 2 The desensitisation model does not explain why there are even more anterior projections of nasal axons in ephrin-a5/ephrin-a2 double mutants than in the ephrin-a5 single mutant. As shown by using an Eph receptor affinity probe, no other ephrin-a ligands are present in the tectum that could compensate for the absence of ephrin-a5 and ephrin-a2 [10], so why don t all retinal axons overshoot in the double mutant? This puzzle can be explained by a competition mechanism [10] similar to those proposed based on the results of classical tissue rotation and transplantation experiments (reviewed in [18]). Furthermore, this model can explain why the projections of retinal axons still fill the superior colliculus when ephrin gradients are altered. The idea is that nasal and temporal axons compete to terminate in the superior colliculus, and their relative success is biased by differences in sensitivity to repulsion. In wild-type embryos, temporal axons are confined to the anterior superior colliculus and outcompete nasal axons that are able to enter more posterior territory. In the double mutant, there is little or no bias between nasal and temporal axons, and therefore each set of axons spreads to largely similar domains. An intermediate situation occurs when ephrin gradients are decreased in the single ephrin mutants, leading to less bias and thus a spreading of axons over a wider area than in the wild type. In this model, the finding that nasal axon projections spread more anteriorly in the ephrin-a5 mutant, but not the ephrin-a2 mutant, may be explained by a dominant role of the steeper ephrin-a5 gradient in the posterior tectum. In addition, a decrease in the gradient of EphA receptor desensitisation resulting from loss of retinal ephrin-a5 could lead to less bias in responsiveness between nasal axons. The recent work highlights new (and some old) questions, not only for mapping along the anteroposterior axis, but for other aspects of retinal axon pathfinding. Unexpectedly, it was found that dorsoventral mapping to the superior colliculus is also affected in the ephrin-a5/ephrin-a2 double mutants [10]. It was previously assumed that mapping occurs independently along the two axes, and circumstantial evidence had implicated the other class of interacting Eph receptors and ephrins the EphB and transmembrane ephrin-b proteins in dorsoventral mapping, perhaps by mediating adhesion [19,20]. The new results may be hinting that graded EphA [21] and ephrin-a [10] expression along the dorsoventral axis, although not prominent, contributes to mapping along this axis too. Another possibility is that mapping along the anteroposterior and dorsoventral axes is actually interdependent. A further advance has come from a recent study of another crucial aspect of retinal axon pathfinding [22]. In organisms Gradients of gene expression and projections of retinal axons after ephrin-a gene knockouts, or ephrin-a5 overexpression. The termination zone(s) of retinal axons are illustrated, with the direction of changes in projection compared with wild type indicated by arrows. (a) Repulsion of retinal axons mediated by EphA5 can explain why some temporal axons overshoot posteriorly in ephrin-a2 and ephrin-a5 mutant mice. It does not, however, explain why some nasal axons project to more anterior regions in ephrin-a5 mutants (!). The graded repulsion model also does not explain why nasal and temporal axons still project to the superior colliculus in the ephrin-a5/ephrin-a2 double mutant, rather than all overshooting (!). (b) Graded sensitivity of EphA receptors (EphA*), resulting from overlapping EphA and graded ephrin-a5 expression in the retina, can explain some aspects of topographic mapping. After overexpression of ephrin-a5 in the chick retina, temporal and nasal axons overshoot because of the increased desensitisation of EphA receptors (lower EphA*). In ephrin-a5 mutant mice, the desensitisation of nasal axons is removed (higher EphA*), and they project more anteriorly because the increased sensitivity to repulsion by ephrin-a2 more than compensates for the absence of ephrin-a5 in the tectum. with laterally placed eyes, all retinal axons project to the contralateral side of the brain (from left to right, and vice versa). When the two eyes face in the same direction, however, they have overlapping fields of vision, and specific axons project ipsilaterally (to the same side) so that retinal cells sharing the same visual space connect to the

4 R450 Current Biology Vol 10 No 12 same area. The pattern in Xenopus development is a fascinating example, because the laterally placed eyes of the tadpole shift during metamorphosis to a dorsal location in the adult. This is accompanied by a switch from a purely contralateral to both contralateral and ipsilateral connections to the forebrain. The choice of projection is made at the optic chiasm, where the axons from each eye converge. It is therefore intriguing that ephrin-b expression occurs at the chiasm during, but not before metamorphosis in Xenopus, and that the spatial location of EphB expression in retinal axons correlates partly (albeit not entirely) with an ipsilateral choice [22]. Furthermore, ectopic expression of ephrin-b2 in the chiasm of tadpoles was found to cause premature formation of ipsilateral projections [22]. These results suggest that the normal up-regulation of ephrin-b protein in the chiasm during metamorphosis diverts specific axons from a contralateral to an ipsilateral projection. An important message from the observations on ephrin gene knockout mice is that graded repulsion mediated by Eph receptors and ephrins is essential, but not sufficient, to explain topographic mapping. The presence of some degree of mapping in the ephrin-a5/ephrin-a2 double mutant be explained by the existence of other repellent molecules with graded distributions that remain to be identified [23]. A crucial further step towards understanding topographic mapping is likely to be the identification of attractive factors in the tectum that may counterbalance repulsion and/or underlie competition between axons. It also seems likely that deeper understanding of how Eph receptors and ephrins control cellular responses will give new insights into mapping mechanisms. Intriguingly, adhesive or de-adhesive responses can occur when Eph receptors are activated in endothelial cells. When plated on extracellular matrix mixed with increasing densities of ephrin, endothelial cells increasingly attach via integrins, but above a certain density they detach [24]. This suggests that cells may be capable of switching from adhesive to de-adhesive or repulsive responses depending on the density of Eph receptor clustering. Although adhesive responses of neuronal growth cones have not been detected in in vitro assays, it may be significant that ephrins can prevent branching of some axons, but induce branching of other axons [25]. A threshold-dependent switch between promoting and preventing growth cone migration in vivo seems worth exploring, as it would provide an economical system of attraction and repulsion that explains some aspects of topographic mapping [26]. But as retinal growth cones do not simply project to a specific level of ephrins [10], it would seem necessary to propose that such threshold responses are dynamically modulated by other factors present in the tectum. A further avenue for investigation is suggested by the recent demonstration that the GPI-anchored ephrin-a proteins can themselves transduce signals, leading to increased cellular attachment via focal adhesion complexes [27]. As EphA3 is expressed in an anterior-to-posterior gradient in the tectum [16], could signalling through retinal ephrin-a proteins affect growth cone behaviour, and perhaps contribute to the desensitisation of nasal axons? Whether or not these and the other current speculations and models are correct, it is safe to predict that there will be many further fascinating developments in eludication of the molecular basis of topographic mapping. References 1. Sperry RW: Chemoaffinity in the orderly growth of nerve fiber patterns and connections. 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5 R Goodhill GJ, Richards LJ: Retinotectal maps: molecules, models and misplaced data. Trends Neurosci 1999, 22: Holash JA, Soans C, Chong LD, Shao H, Dixit VM, Pasquale EB: Reciprocal expression of the Eph receptor Cek5 and its ligand(s) in the early retina. Dev Biol 1997, 182: Braisted JE, McLaughlin T, Wang HU, Friedman GC, Anderson DJ, O Leary DDM: Graded and lamina-specific distributions of ligands of EphB receptor tyrosine kinases in the developing retinotectal system. Dev Biol 1997, 191: Sefton M, Arujo M, Nieto MA: Novel expression gradients of Eph-like receptor tyrosine kinases in the developing chick retina. Dev Biol 1997, 188: Nakagawa S, Brennan C, Johnson KG, Shewan D, Harris WA, Holt CE: Ephrin-B regulates the ipsilateral routing of retinal axons at the optic chiasm. Neuron 2000, 25: Muller BK, Jay DG, Bonhoeffer F: Chromophore-assisted laser inactivation of a repulsive axonal guidance molecule. Curr Biol 1996, 6: Huyn-Do U, Stein E, Lane AA, Liu H, Cerretti DP, Daniel TO: Surface densities of ephrin-b1 determine EphB1-coupled activation of cell attachment through α V β 3 and α 5 β 1 integrins. EMBO J 1999, 18: Castellani V, Yue Y, Gao P-P, Zhou R, Bolz J: Dual action of a ligand for Eph receptor tyrosine kinases on specific populations of axons during the development of cortical circuits. J Neurosci 1998, 18: Honda H: Topographic mapping in the retinotectal projection by means of complementary ligand and receptor gradients: a computer simulation study. J Theor Biol 1998, 192: Davy A, Gale NW, Murray EW, Klinghoffer RA, Soriano P, Feuerstein C, Robbins SM: Compartmentalized signaling by GPI-anchored ephrin-a5 requires the fyn tyrosine kinase to regulate cellular adhesion. Genes Dev 1999, 13: If you found this dispatch interesting, you might also want to read the February 2000 issue of Current Opinion in Neurobiology which included the following reviews, edited by Chris Q Doe and Joshua R Sanes, on Development: Asymmetric division of Drosophila neural stem cells: a basis for neural diversity Fumio Matsuzaki Get to know your stem cells Stefan Momma, Clas B Johansson and Jonas Frisén Notch and presenilins in vertebrates and invertebrates: implications for neuronal development and degeneration Dennis J Selkoe Axonal signals in the assembly of neural circuitry Sam Kunes Vnd/nkx, ind/gsh, and msh/msx: conserved regulators of dorsoventral neural patterning? Robert A Cornell and Tonia Von Ohlen Transcriptional mechanisms in the development of motor control Linda W Jurata, John B Thomas and Samuel L Pfaff From Abl to actin: Abl tyrosine kinase and associated proteins in growth cone motility Lorene M Lanier and Frank B Gertler Semaphorins and their receptors in vertebrates and invertebrates Jonathan A Raper Slit proteins: key regulators of axon guidance, axonal branching, and cell migration Katja Brose and Marc Tessier-Lavigne The GDNF family ligands and receptors implications for neural development Robert H Baloh, Hideki Enomoto, Eugene M Johnson Jr and Jeffrey Milbrandt Active killing of neurons during development and following stress: a role for p75 NTR and Fas? Cédric Raoul, Brigitte Pettmann and Christopher E Henderson Rapid dendritic movements during synapse formation and rearrangement Wai T Wong and Rachel OL Wong Development of neuron neuron synapses Sang Hyoung Lee and Morgan Sheng Molecular mechanisms for activity-regulated protein synthesis in the synapto-dendritic compartment David G Wells, Joel D Richter and Justin R Fallon Critical periods during sensory development Nicoletta Berardi, Tommaso Pizzorusso and Lamberto Maffei The full text of Current Opinion in Neurobiology is in the BioMedNet library at