Neural map specification by gradients John G Flanagan

Neural map specification by gradients John G Flanagan Topographic maps, in which the spatial order of neurons maps smoothly onto their axonal target, are a central feature of neural wiring. Ephrins and Eph receptors are well accepted as graded labels for map development, enabling current studies into molecular principles of mapping. Ephrins regulate axon growth either positively or negatively, leading to models in which axons terminate at a neutral or optimum point in the gradient. Axonal competition ensures the target is filled. Ephrins and Ephs are typically expressed in complex overlapping patterns, with implications for signaling mechanisms, scale of internal map features, and coordinated interconnection of multiple mapping modules. Recent studies of Wnt3 and En-2 show that topographic axon guidance cues may be as diverse as molecules previously regarded as morphogens and transcription factors. Addresses Department of Cell Biology and Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA Corresponding author: Flanagan, John G (flanagan@hms.harvard.edu) This review comes from a themed issue on Development Edited by Anirvan Ghosh and Christine E Holt Available online 18th January 2006 0959-4388/$ see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2006.01.010 Introduction A key principle of neural connectivity is the organization of axonal projections into topographic maps in which the spatial order of a sheet of projecting neurons maps smoothly and continuously onto the spatial order of their axonal connections in a target region, with nearest-neighbor relationships preserved. For example, in the visual system, topographically mapped axonal connections enable images of the outside world to be transferred from the retina to the higher visual centers of the brain in a spatially conserved form. Although visual maps have been studied the most extensively, maps of this type are found throughout the nervous system, and are presumably an efficient way to transfer information from one place to another. The idea that neural map development could be explained by positional labels in gradients across the projecting and target areas was first proposed by Sperry [1]. For several decades the molecular identity of the graded labels remained elusive, until the ephrin-as and their binding partners the EphA receptor tyrosine kinases were identified as Sperry-type labels for the anteroposterior (A P) axis of the projection from the retina to the tectum of the midbrain, the retinotectal projection [2,3]. This function for the ephrin-as was supported by the major categories of evidence applied to developmental guidance molecules including: expression patterns, which are in complementary gradients (Figure 1); in vitro assays of axon guidance activity, showing differential effects on axons from different parts of the projecting area, an essential requirement for a topographic label; and in vivo gain- and loss-of-function experiments, showing changes in map organization [2 6]. Ephrin-Bs and EphB receptors were subsequently identified as graded mapping molecules for the orthogonal dorsoventral (D V) axis of the retinotectal map [7,8] (Figure 1). This review focuses on basic principles of topographic mapping by guidance molecules in gradients, particularly advances in the past two to three years. Examples are taken primarily from ephrin-a function in retinotectal mapping, although other molecules and neural projections are also discussed to address the generality of the mechanisms, or where they reveal additional principles. A later section will cover recent work identifying Wnt3 and En-2 as graded proteins that can guide retinal axons [9,10 ](Figure 1), providing an exciting new vista into the potentially broad nature of positional information available for mapping. Positive and negative gradient actions In the years following Sperry s proposal of graded labels, theoretical studies led to the proposal that one gradient per axis is not sufficient, because a single gradient of an attractant or repellent would simply cause all axons to migrate to one end of the map. Instead, counter-balanced forces are thought to be required, with each axon coming to rest at the point at which these opposing forces balance out [11]. These counterbalanced forces could take the form of separate repellent and attractant molecules in gradients, or a graded guidance molecule with both positive and negative actions. Ephrin-As were initially described as having repellent activity on retinal axons. This was originally based on results from the in vitro stripe assay, in which axons given a choice of alternating stripes avoided lanes containing ephrin-a [3,6]. Further support came from in vivo gain-offunction studies, in which localized elevation of either www.sciencedirect.com

60 Development Figure 1 Gradients of guidance cues and their receptors in the retinotectal system. All the illustrated tectal guidance molecules display topographic specificity (producing distinct responses by axons from different parts of the retina) although only the ephrins and Ephs are known to be required as tectal guidance labels in vivo. (a) Map topography. Axons map from the retina to the tectum of the midbrain, also known as the superior colliculus in mammals. Abbreviations: A, anterior; D, dorsal; N, nasal; P, posterior; T, temporal; V, ventral (N and T are embryologically equivalent to anterior and posterior, respectively; D and V in the tectum are sometimes termed medial and lateral). (b) Ephrin and Eph gradients. The diagram is formed on the basis of studies of chick, mouse and Xenopus. Each gradient illustrated is typically a composite of more than one molecule (for details see [55]); for example the tectal ephrin-a gradient contains ephrin-a2 and -A5, both of which are required for normal mapping. The gradients do not necessarily function as purely independent orthogonal systems along A P and D V axes. Interestingly, in humans the retinal EphA gradient is central-to-peripheral [56], consistent with Sperry s prediction for binocular species [1], so that maps from the two eyes overlap. Moreover, there can be signaling cross-talk between the A and B ephrins [57], possibly explaining mild DV abnormalities in mice with gene-targeted ephrin-as [14]. (c) RGM and its receptor neogenin. (d) Wnt3 and its receptors. Ryk is a tyrosine kinase receptor that mediates repulsion in this system, whereas Frizzled (Fz) proteins are seven-transmembrane receptors and mediate the attraction seen at lower Wnt3 concentrations [10 ]. (e) En-2 gradient. En-2 attracts nasal and repels temporal axons, apparently by crossing the cell membrane, inferring that it has one or more graded intracellular retinal receptors [9 ]. tectal ephrin-a [6] or retinal EphA [12] caused axons with high receptor levels to avoid target regions with high ligand levels. Because the ephrin-as appeared to function as repellents, a widely appealing idea was that they might be balanced by an unidentified independent counter-gradient [13]. If so, a prediction would be that removal of the repellent tectal ephrin gradient would tend to cause all axons to move in a posterior direction. However, the results of gene targeting of the major tectal ephrin-as, ephrin-a2 and -A5, instead showed an approximately equal and opposite shift of temporal axons in a posterior direction, and nasal axons in an anterior direction. These and other genetic results suggested against the independent countergradient model [14,15]. A limitation of the initial experiments detecting only repellent effects is that they were all essentially binary perturbations: comparing cells with or without overexpressed ephrin-a or EphA in vivo [6,12], or stripes with or without ephrin-a in vitro [3,6]. Because mapping is inherently a continuously variable phenomenon, an alternative in vitro assay was developed, in which both retinal position and ephrin concentration could be varied continuously, and the resulting effect on axon outgrowth was compared quantitatively [16 ]. The results showed that ephrin-as can have both positive and negative effects on axon growth, ranging widely from complete inhibition at high concentrations to several-fold promotion at lower concentrations (Figure 2). Importantly, the transition from attraction to repulsion varied systematically with both ephrin concentration and retinal position, providing topographic specificity and leading to a model in which map position could be specified as the point where positive and negative forces balance out [16 ](Figure 2). Similar principles may also apply to ephrin-b action along the dorsoventral retinotectal axis. In this case, the gradient orientations (Figure 1) suggested that the interactions should be attractant, not repellent [17]. Consistent with this expectation, ephrin-bs and EphBs were found to have attractant effects on Xenopus retinal axons in vitro and in vivo [8], while gene disruption of mouse EphBs produced a shift in mapping also consistent with an attractant role [7]. However, ectopic tectal expression of ephrin-b1 was more www.sciencedirect.com

Gradients in neural mapping Flanagan 61 Figure 2 Concentration-dependent positive and negative effects of topographic guidance factors. (a) Model for mapping, formed on the basis of positive and negative responses to ephrin-a2. In the model, axon growth in the target is promoted by low ephrin-a, and inhibited by higher ephrin-a concentrations, with axons terminating at the neutral position. Axons from different positions across the retina have different sensitivities to ephrin, so that the inflection point between positive and negative effects in the tectum varies with retinal position. The result is a smooth topographic map [16 ]. (b,c) Retinal axon outgrowth in response to varying concentrations of ephrin-a2 or Wnt3. In the middle of each box in the Figure is a piece of explanted retina, taken from varying positions across the retina. Axon fibers are seen growing out from the explant onto a substrate containing variable concentrations of ephrin-a2 or Wnt3. (b) Effects of ephrin-a2 on retinal axon growth in vitro. Promotion is seen at lower concentrations and more nasal retinal positions, whereas inhibition is seen at higher concentrations and more temporal positions [16 ]. (c) Effects of Wnt3 on retinal axon growth in vitro. Promotion is seen at lower concentrations and dorsal retinal positions, whereas inhibition is seen at higher concentrations and more ventral positions [10 ]. Although ephrin-a2 and Wnt3 are in different molecular families, and form gradients along different retinal axes, there is a striking parallel in their concentration- and position-dependent positive and negative effects. recently found to cause avoidance by retinal axons [18] and, taken together with the earlier studies, this suggested that ephrin-bs might function through both attractant and repellent effects [18]. For ephrin-bs, a concentrationdependent change from positive to negative responses has not yet been directly shown for retinal axons in vitro. However, early studies on endothelial cell adhesion showed an optimum concentration of ephrin-b, with less adhesion at higher or lower concentrations [19]. Extrapolated to migration, this suggested a model in which axons might settle at an optimum point in the ephrin-b gradient at which adhesion or attraction is highest [18,20]. More recently, cell migration was assessed in response to ephrin- B, showing a sharp transition from strong chemoattraction at low concentrations to strong chemorepulsion at high concentrations [21 ]. These results all seem to fit the idea that concentration-dependent positive and negative effects are likely to be a general principle in specifying the termination point within an ephrin gradient. New evidence shows that both positive and negative influences on retinal axon growth are shown by two novel guidance factors, Wnt3 and En-2 (Figures 1 and 2; also described further below) [9,10 ]. Thus, having dual topographically specific positive and negative effects is a property found not only in ephrins, and may be a more general principle of gradient action in mapping. Axon competition: reliably filling the target In addition to a role for topographically specific graded labels, a second key principle in mapping is axon axon competition [22,23]. Initial support came from embryological manipulations showing that if part of the retina is ablated, the remaining axons tend to expand to fill the tectal space; whereas if part of the tectum is ablated the axons tend to compress [24]. Further evidence now comes from genetic manipulations of ephrin-as in anteroposterior mapping. Loss-of-function of ephrin-a2 or -A5 results in disrupted map precision yet, despite changes in the ephrin gradients, dye filling of the retina shows that the target space remains smoothly filled with retinal axons [14]. In gain-of-function studies in which EphA receptors are overexpressed in a subset of retinal neurons, the overexpressing neurons shift more posteriorly in the www.sciencedirect.com

62 Development target, whereas the neurons with no change in EphA expression map more anteriorly than normal [12,25 ]. These genetic studies clearly show that ephrins specify relative not absolute position, and provide new evidence of axon axon competition. The molecular basis for this competition remains unknown, although it might involve one or more limiting factors in the target, such as neurotrophins [26] or adhesion molecules [27]. Competition is likely to be an important principle in mapping because it can ensure that despite changes in gradient shape or map scaling during development or evolution the target remains smoothly filled with axons. Multiple overlapping gradients and countergradients Within an individual map, multiple Ephs and ephrins are typically expressed in complex overlapping gradients (Figure 1), including co-expression of both ephrins and Ephs within the same cells. Several distinct theories, which may not be mutually exclusive, have been proposed for the functional significance of this overlap. One proposal is that when EphAs and ephrin-as are coexpressed they may interact in cis, within individual cells or neighboring cells, and that this cis interaction downregulates signaling function. This idea is supported by several lines of evidence. Gain-of-function experiments show that localized ephrin-a5 overexpression in retina causes temporal axons to lose sensitivity to ephrins in vitro, and map to abnormally posterior tectal positions, consistent with functional downregulation of EphA receptors [28]. Correspondingly, loss-of-function of ephrin-a2 and -A5 causes a strong increase in the in vitro repellent response of nasal retinal axons to ephrins, indicating functional upregulation in EphA receptors [14]. In individual cultured cells, ephrin-as and EphA receptors were found to interact in cis via their binding domains, as indicated by formation of coincident cell surface patches, and this cis interaction inhibited trans interactions [29 ]. Because this cis interaction downregulates signaling, the countergradients might serve to accentuate the steepness of the functional gradients of Eph receptors and ephrins, perhaps enabling greater precision in map specification. Another role for the overlapping gradients stems from the ability of ephrins to signal bi-directionally, with a forward signal transduced into the cell bearing the Eph receptor, and a reverse signal into the cell bearing the ephrin ligand. Reverse signaling through the transmembrane ephrin-b ligands appears to mediate dorsoventral mapping in the Xenopus retinotectal system, on the basis of in vivo misexpression studies and in vitro stripe assays showing a topographically specific attractant effect of EphB receptors [8]. Although reverse signaling through the lipid-anchored ephrin-a ligands is less well characterized molecularly, reverse signaling through ephrin-as appears to be involved in mapping the vomeronasal system [30] and in anteroposterior retinotectal mapping. In retinotectal mapping retinal axons are repelled by EphA7-Fc in vitro, and EphA7 gene targeting results in abnormally anterior termination of both temporal and nasal axons [31 ]. The preceding two paragraphs raise a question. If cis interactions of ephrins and Eph receptors downregulate signaling, do their actions inevitably cancel out, or is it possible for forward and reverse signaling to co-exist in the same cell? This question is addressed in a recent study of mapping in the motor system, in which treatment of motor axons with ephrin-a-fc (forward signaling) or EphA-Fc (reverse signaling) fusion proteins elicited distinct axon growth responses in vitro. Moreover, ephrin-a and EphA proteins on the growth cone surface were found to localize in distinct membrane microdomains [32 ]. The relationship of these results to the studies showing cis interactions is not yet clear. However, the results might not be mutually exclusive, as it seems possible that cis interactions cause some degree of receptor ligand downregulation, whereas other receptor and ligand molecules remain located in distinct subcellular locations where they mediate forward and reverse signaling independently. In the context of overlapping gradients, it is important to note that individual mapped areas of the nervous system typically serve as both targets for incoming projections, and the origin for outgoing projections, as discussed further in the next section. The arrangement of ephrins and Eph receptors in countergradients within individual areas, and their ability to signal in both forward and reverse directions, seem likely to be functionally important by providing a logical way to coordinate the mapping of both incoming and outgoing connections (Figure 3c). Finally, multiple overlapping gradients may serve to determine the relative scale of representation of features within a map. In the cortical somatosensory map, it has long been known that the surface area devoted to different regions, such as parts of the face or hands, varies between species and even between individuals in a manner that reflects functional importance and neural abilities. In mice in which ephrin-a5 is disrupted by gene targeting, leaving other graded ephrin-as in the cortical somatosensory area, the representation of some parts of the body are expanded and others are contracted [33,34]. These results show that in addition to their function in setting up the basic nearest-neighbor organization of axons in a map, the specific combination of overlapping ephrin gradients can shape the relative scale of representation of different features within a map. Repeated use of gradients: modular organization of maps There are approximately 10 12 neurons in the human brain, interconnected by roughly 10 15 synapses. By comparison, www.sciencedirect.com

Gradients in neural mapping Flanagan 63 Figure 3 Modular organization of topographic maps. The same labels are used repeatedly in multiple maps, providing an economic modular scheme for map development and evolution. Ephrin-A gradients are shown in blue, EphA in cyan; countergradients are not shown in all cases. (a) Multiple targets of a single projecting area. The retina forms maps in several targets including the dlgn and vlgn (dorsal and ventral lateral geniculate nucleus) and the SC, each containing an ephrin-a gradient [4]. Another target region, the pretectal nuclei (PT), also expresses ephrin-as, although a role in mapping is not clear. (b) Maps in multiple modalities. Ephrin-A gradients are found in maps in distinct neural systems including visual and auditory projections. When visual axons are surgically rewired, they can form ephrin-a-dependent topographic maps in auditory targets [45]. (c) Multiple levels of a neural pathway, including reciprocal projections. In addition to mapping projections up to relay nuclei in the thalamus, such as the dlgn, ephrin-as also map higher projections from the thalamus to cortical areas [33,34,36], in addition to reciprocal projections from the cortex to the thalamus [37]. In addition to their role in mapping intra-area topography, ephrin-as also map broader inter-area organization in the cortex, apparently mediated by early developmental gradients in the ventral diencephalon, an intermediate target for thalamocortical projections [33]. The organization of ephrins and Ephs in counter-gradients within individual maps probably plays a key role in their ability to organize both incoming and outgoing projections, and thus integrate the topography of multiple maps interconnected by complex neural pathways. the human genome contains only a few tens of thousands of protein-coding genes. This means that there must be highly efficient mechanisms for a limited amount of genetic information to encode the complex pattern of neural wiring [35]. The use of gradients is one way to achieve efficiency, because a small set of signaling molecules can specify a range of positional values, patterning many connections over an extended area. Another mechanism to enhance efficiency seems to be the repeated use of the same labels in multiple maps. Ephrin gradients are found in multiple maps formed by retinal axons [4] (Figure 3a). They can also function at multiple stages of a neural pathway, mapping complex serial or parallel interconnections. This is illustrated by emerging work on pathways leading to and from the cortex (Figure 3c), where ephrins map not only projections up to thalamic nuclei such as the LGN but also higher projections from thalamic nuclei to individual cortical areas [33,34,36], and reciprocal projections from the cortex to the thalamus [37]. In addition, they seem to function earlier in development at an intermediate target in the ventral telencephalon to map the broader distribution of thalamic axons across multiple areas of the cortex [33] (Figure 3c). Gradients of ephrins also function in mapping other sensory modalities, including somatosensory [33,34] and auditory [38] maps, in addition to nonsensory projections such as the hippocamposeptal map [39]. Ephrins also function in olfactory [40] and motor [41 43] maps, although the patterns generally seem to be discrete rather than graded in those systems. The idea that different parts of the nervous system rely on the same mapping mechanisms is directly addressed by a recent study using a brain rewiring paradigm. Visual axons can form topographic maps in auditory targets when surgically rewired during development [44], and similar to normal maps in the visual system, these rewired projections were recently shown to depend on ephrin gradients for map specification (Figure 3b) [45]. Taken together, these studies indicate that topographic maps are discrete developmental modules, in which the same mapping molecules can be used repeatedly, provided that axons are routed to the correct target by distinct pathway selection cues. This modular organization is an efficient use of genetic information in development, and presumably enables efficient evolution of new neural systems by duplication of existing modules. www.sciencedirect.com

64 Development Other positional mapping labels Although the ephrins remain the best characterized mapping labels, other candidates have been identified. Repulsive guidance molecule (RGM) is a lipid-anchored protein in a gradient in the chick tectum. RGM repels temporal axons in vitro with topographic specificity [46], and functions through a receptor, neogenin, which is graded in the retina [47](Figure 1). Although no mapping phenotype was seen following gene targeting of the mouse homolog RGMa [48], redundancy with other cues might have masked a function in mapping. Recently, two studies have identified additional graded retinal axon guidance cues in novel classes. Wnt proteins are known primarily as extracellular factors that regulate patterning of cell fate, and can function as graded morphogens, although recent studies have revealed that they can also function as axon guidance cues [49]. Zou and coworkers [10 ] now show that Wnt3 is expressed in a gradient in the tectum (Figure 1). Misexpression studies in vivo and axon guidance assays in vitro show a topographically specific effect on retinal axon mapping. Similar to the ephrin-as, Wnt3 shows a concentrationdependent transition from positive to negative effects (Figure 2). Moreover, these actions can be attributed to different receptors, with Ryk mediating repulsion, whereas the attraction at lower concentrations is mediated by Frizzled receptors [10 ](Figure 1). Engrailed proteins have long been known as homeoboxcontaining nuclear transcription factors that regulate cell fate, including A P patterning of the tectum [50]. Consistent with this patterning function, tectal En-2 misexpression causes mapping errors [51 53], and this clearly involves cell fate changes because ephrin-a2 and -A5 are upregulated [52,53]. Remarkably, Holt, Prochiantz and co-workers [9 ] now report that soluble En-2 can also function directly on retinal axons as a guidance factor. A background to this study is provided by extensive work by the Prochiantz group showing that homeobox proteins added to the extracellular medium of cultured cells can readily penetrate across the membrane [54]. The new work shows that when a soluble En-2 gradient is presented to cultured retinal axons, it causes attraction of nasal axons and repulsion of temporal axons [9 ] (Figure 1). The requirement for Wnt3 and En2 as guidance factors remains to be fully tested by loss-of-function in vivo, which presents experimental challenges in view of their actions in cell fate determination. Nevertheless, these exciting studies provide new examples of the emerging principle that positional information gradients provide a coordinate system that can be interpreted to regulate essentially any cell function, depending on the receptor systems used to decode this positional information [49]. This principle now extends not only to factors such as Wnt proteins, extracellular signals that can regulate both cell fate and guidance, but even to factors such as En-2, which can function both within the nucleus as transcription factors and extracellularly as guidance cues. Conclusions The identification of ephrins as mapping labels during the 1990s brought the study of mapping to a molecular level. The general molecular principles to specify map position have continued to emerge over the past few years. Ephrins and other candidate mapping labels show concentration-dependent positive and negative effects on guidance, which can explain the specification of a unique termination point within the target gradient. Meanwhile, axon axon competition functions to ensure the target is filled. Ephrins function in maps throughout the nervous system, providing an efficient modular organization for map development and evolution. The recent identification of Wnt3 and En-2 as graded guidance cues broadens our concepts of the molecular nature of the available positional information, and raises new questions for the future about the function and integration of different guidance cues in mapping. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Sperry RW: Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc Natl Acad Sci USA 1963, 50:703-710. 2. Cheng H-J, Nakamoto M, Bergemann AD, Flanagan JG: Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell 1995, 82:371-381. 3. Drescher U, Kremoser C, Handwerker C, Löschinger J, Noda M, Bonhoeffer F: In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kda tectal protein related to ligands for Eph receptor tyrosine kinases. Cell 1995, 82:359-370. 4. Feldheim DA, Vanderhaeghen P, Hansen MJ, Frisen J, Lu Q, Barbacid M, Flanagan JG: Topographic guidance labels in a sensory projection to the forebrain. Neuron 1998, 21:1303-1313. 5. Frisén J, Yates PA, McLaughlin T, Friedman GC, O Leary DDM, Barbacid M: Ephrin-A5 (AL-1/RAGS) is essential for proper retinal axon guidance and topographic mapping in the mammalian visual system. Neuron 1998, 20:235-243. 6. Nakamoto M, Cheng H-J, Friedman GC, McLaughlin T, Hansen MJ, Yoon C, O Leary DDM, Flanagan JG: Topographically specific effects of ELF-1 on retinal axon guidance in vitro and retinal axon mapping in vivo. Cell 1996, 86:755-766. 7. Hindges R, McLaughlin T, Genoud N, Henkemeyer M, O Leary DD: EphB forward signaling controls directional branch extension and arborization required for dorsal-ventral retinotopic mapping. Neuron 2002, 35:475-487. 8. Mann F, Ray S, Harris W, Holt C: Topographic mapping in dorsoventral axis of the Xenopus retinotectal system depends on signaling through ephrin-b ligands. Neuron 2002, 35:461-473. 9. Brunet I, Weinl C, Piper M, Trembleau A, Volovitch M, Harris W, Prochiantz A, Holt C: The transcription factor Engrailed-2 guides retinal axons. Nature 2005, 438:94-98. www.sciencedirect.com

Gradients in neural mapping Flanagan 65 En-2 is well known as a homeobox nuclear factor, which is expressed in a tectal gradient and controls cell fate. Earlier work by Prochiantz and coworkers [54] showed that homeobox proteins can cross cell membranes. Here, En-2 is shown to function as an extracellular guidance factor for retinal axons in vitro, with topographic specificity, repelling temporal axons and attracting nasal axons. 10. Schmitt AM, Shi J, Wolf AM, Lu CC, King LA, Zou Y: Wnt-Ryk signalling mediates medial-lateral retinotectal topographic mapping. Nature 2005, epub ahead of print. Wnt proteins have long been known as extracellular regulators of cell fate, with recent studies also identifying effects on axon guidance. Here, Wnt3 is shown to be expressed in a dorsoventral gradient in the tectum. It has concentration-dependent positive and negative effects on retinal axons in vitro, with topographic specificity for nasal versus temporal axons. In the retina, the Ryk receptor mediates repulsion, whereas Frizzled receptors mediate attraction. 11. Gierer A: Model for the retino-tectal projection. Proc R Soc Lond B Biol Sci 1983, 218:77-93. 12. Brown A, Yates PA, Burrola P, Ortuno D, Vaidya A, Jessell TM, Pfaff SL, O Leary DDM, Lemke G: Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling. Cell 2000, 102:77-88. 13. O Leary DD, Yates PA, McLaughlin T: Molecular development of sensory maps: representing sights and smells in the brain. Cell 1999, 96:255-269. 14. Feldheim DA, Kim YI, Bergemann AD, Frisen J, Barbacid M, Flanagan JG: Genetic analysis of ephrin-a2 and ephrin-a5 shows their requirement in multiple aspects of retinocollicular mapping. Neuron 2000, 25:563-574. 15. Feldheim DA, Nakamoto M, Osterfield M, Gale NW, DeChiara TM, Rohatgi R, Yancopoulos GD, Flanagan JG: Loss-of-function analysis of EphA receptors in retinotectal mapping. 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Dev Biol 1997, 191:14-28. 18. McLaughlin T, Hindges R, Yates PA, O Leary DD: Bifunctional action of ephrin-b1 as a repellent and attractant to control bidirectional branch extension in dorsal-ventral retinotopic mapping. Development 2003, 130:2407-2418. 19. Huynh-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 alpha(v)beta(3) and alpha(5)beta(1) integrins. EMBO J 1999, 18:2165-2173. 20. Honda H: Competitive interactions between retinal ganglion axons for tectal targets explain plasticity of retinotectal projection in the servomechanism model of retinotectal mapping. Dev Growth Differ 2004, 46:425-437. 21. Matsuoka H, Obama H, Kelly ML, Matsui T, Nakamoto M: Biphasic functions of the kinase-defective Ephb6 receptor in cell adhesion and migration. J Biol Chem 2005, 280:29355-29363. Ephrin-B2 was found to attract migrating cells at low concentrations and repel them at high concentrations. These studies support other evidence for a biphasic concentration-dependent response to ephrin-bs, and indicate that in the context of a guidance assay in vitro they can either promote or inhibit migration. 22. Fraser SE, Hunt RK: Retinotectal specificity: models and experiments in search of a mapping function. Annu Rev Neurosci 1980, 3:319-352. 23. Prestige MC, Willshaw DJ: On a role for competition in the formation of patterned neural connexions. Proc R Soc Lond B Biol Sci 1975, 190:77-98. 24. Goodhill GJ, Richards LJ: Retinotectal maps: molecules, models and misplaced data. Trends Neurosci 1999, 22:529-534. 25. Reber M, Burrola P, Lemke G: A relative signalling model for the formation of a topographic neural map. Nature 2004, 431:847-853. EphA3 was overexpressed under control of the Islet2 promoter in a scattered subset of projecting retinal ganglion cells. Here, and in a previous study [12], the overexpressing axons were found to shift anteriorly in the map, whereas axons with normal EphA levels shifted posteriorly. Qualitative analysis and quantitative modeling of the map shows that ephrins specify relative and not absolute position in the map, and that gradients function together with axon axon competition in mapping. 26. Cohen-Cory S, Fraser SE: Effects of brain-derived neurotrophic factor on optic axon branching and remodelling in vivo. Nature 1995, 378:192-196. 27. Demyanenko GP, Maness PF: The L1 cell adhesion molecule is essential for topographic mapping of retinal axons. J Neurosci 2003, 23:530-538. 28. Hornberger MR, Dutting D, Ciossek T, Yamada T, Handwerker C, Lang S, Weth F, Huf J, Wessel R, Logan C et al.: Modulation of EphA receptor function by coexpressed ephrina ligands on retinal ganglion cell axons. Neuron 1999, 22:731-742. 29. 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EphA7 is expressed in an anterior > posterior gradient in the tectum, opposite to the ephrin-a gradients. Here, gene targeting of EphA7 results in abnormally located retinotectal termination zones, and EphA7 repels retinal axons in vitro. These results lead to a model in which EphA7 reverse signaling suppresses termination zones anterior to the topographically correct location. 32. Marquardt T, Shirasaki R, Ghosh S, Andrews SE, Carter N, Hunter T, Pfaff SL: Coexpressed EphA receptors and ephrin-a ligands mediate opposing actions on growth cone navigation from distinct membrane domains. Cell 2005, 121:127-139. Motor axon growth cones co-express both ephrin-as and EphA receptors. Here, they are shown to have opposite responses to ephrin-a (forward) signaling, which causes axon growth or attraction, and EphA (reverse) signaling, which causes growth cone collapse or repulsion. 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