Ephrin signalling in the developing nervous system.

Available online at www.sciencedirect.com

ScienceDirect Ephrin signalling in the developing nervous system§ Ru¨diger Klein1,2 and Artur Kania3,4,5 Ephrin ligands and their Eph receptors hold our attention since their link to axon guidance almost twenty years ago. Since then, they have been shown to be critical for short distance cell–cell interactions in the nervous system. The interest in their function has not abated, leading to ever-more sophisticated studies generating as many surprising answers about their function as new questions. We discuss recent insights into their functions in the developing nervous system, including neuronal progenitor sorting, stochastic cell migration, guidance of neuronal growth cones, topographic map formation, as well as synaptic plasticity. Addresses 1 Department of Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany 2 Munich Cluster for Systems Neurology (Synergy), Munich, Germany 3 Institut de Recherches Cliniques de Montreál (IRCM), Montreál, QC, Canada H2W 1R7 4 De´partement de Me´decine, Universite´ de Montreál, Montreál, QC, Canada H3T 1J4 5 Division of Experimental Medicine, Departments of Biology, and, Anatomy and Cell Biology and Integrated Program in Neurosciences, McGill University, Montreál, QC, Canada H3A 1A3 Corresponding authors: Klein, Ru¨diger ([email protected]) and Kania, Artur ([email protected])

Current Opinion in Neurobiology 2014, 27:16–24 This review comes from a themed issue on Development and regeneration 2014 Edited by Oscar O Marin and Frank F Bradke

0959-4388/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.conb.2014.02.006

Introduction Since the discovery of their function in establishing topographic organisation, ephrins and Ephs have shaped our thinking about the developing nervous system. Because they reside in the cytoplasmic membrane, Ephs and ephrins act in processes occurring over short distances such as cell–cell contact, cell migration and the synaptic dialogue between neurons. Ephs are tyrosine kinase transmem-

§ We dedicate this review to Tony Pawson whose insights into signal transduction and contributions to our understanding of Eph signalling continue to shine.

Current Opinion in Neurobiology 2014, 27:16–24

brane receptors, while ephrin ligands are either transmembrane (ephrin-Bs) or membrane-tethered via a GPI tail (ephrin-As; Figure 1). The ‘classic’ mode of signalling or forward signalling is from ephrins to Ephs (ephrin:Eph), and frequently results in repulsion. Pervasive evidence of reverse signalling from Ephs to ephrins (Eph:ephrin), which generally promotes adhesion, has blurred the line between ligand and receptor labels. The roles of Eph and ephrin structural domains and the identity of their signalling intermediates are known, but what is still unclear is the nature of higher order interactions, for example those between clusters of Ephs and ephrins expressed in the same or separate neurons. Other pressing questions are related to the balance of forces of cell–cell repulsion and adhesion, neuronal morphology, migration, and axonal growth cone behaviour. Our picture of short distance signals between synaptic partners also remains sketchy, as does modelling of Eph/ephrin-driven biological phenomena. Here, we discuss the recent studies aiming to answer these questions, and apologise in advance to those whose work did not fit in the limited space allotted to discuss this vast field.

Structure–function and signalling Eph/ephrin structure–function studies suggest that Eph cluster formation is crucial to Eph/ephrin signalling in many different cell types. Xu and colleagues describe the structure of EphA4 extracellular (EC) domain alone and bound to ephrin-A5, and uncover interactions between the ligand-binding domain and the fibronectin domain. These suggest the formation of pre-clustering complexes, possibly enabling faster recruitment of unbound receptors into ligand-bound clusters [1]. Different affinities of Eph receptors for cognate class ephrins [2] raise the question of whether, for example, different EphA receptors might respond differentially to a particular ephrin-A. Starting with the observation that when exposed to ephrin-A5, EphA4 collapses growth cones, whereas EphA2 increases adhesion, a new study shows that the structural basis for this functional differentiation resides mainly in the EC domain [3]. The ligand-bound EphA4 EC domain forms oligomeric clusters in contrast to the previously characterised EphA2 EC domain forming continuous arrays [4,5], mirroring the observation that the clusters formed by EphA4 are smaller than those containing EphA2 [3]. In parallel, a study from the Klein lab [6] uses chemical dimerizers to generate different EphB2 cluster sizes and shows that the relative abundance of inactive monomers/ dimers and active higher-order clusters determines the www.sciencedirect.com

Ephrin signalling in the developing nervous system Klein and Kania 17

Figure 1

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Structure–function and signalling. (A) Eph and ephrin structure and their interactions. Domain composition and tyrosine phosphorylation sites are indicated. LBD: ligand-binding; RBD: receptor-binding; Sushi/EGF: Sushi and Epidermal Growth Factor-like; Fn: fibronectin-like; SAM: sterile alpha motif; PDZ: Psd-95, Dlg and ZO1-binding motif. (B) Specific ephrin-B3 functional domain requirements for different dendritic development processes. Deletion of the PDZ domain, which interacts with Pick1 and Syntenin, leads to exuberant dendrite growth but sparse spines, while mutation of the Grb4-interacting phosphorylation sites leads to exuberant dendritic growth, with normal spine density. (C) C-Ret acts as a detector of EphA4 receptor www.sciencedirect.com


18 Development and regeneration 2014

strength in the repulsive response. Interestingly, these data also argue that Eph intracellular modules such as the SAM domain have negative regulatory effects on ephrininduced clustering. A genetic and biochemical study in Caenorhabditis elegans brought new insights into the mechanism of Ephmediated inhibition of axonal growth [7]. Here, the Eph receptor stops axon growth by having two effects on actin dynamics: inhibition of actin polymerization and formation of an extensive network of short, branched actin filaments.

in retinal ganglion cell (RGC) neurons and their tectal or collicular targets keeps producing important insights into the development of topographic maps, and remains an inspiration for the study of nervous system organisation. Topographic map compression results in all RGCs innervating the available tectum [15]. One interpretation of this was that the tectal pattern of the chemoaffinity labels changes in response to the deletion. A recent quantitative study demonstrated that, indeed, such manipulations make the ephrin-A expression gradient in the superior colliculus steeper, paralleling the shift in retino-collicular targeting [16]; thus topographic map target size intrinsically regulates ephrin gradient steepness.

Differentiation and plasticity The links between Ephs and ephrins and specific intracellular cascades regulating differentiation and synaptic plasticity remain obscure. A new connection to transcriptional pathways emerges from the finding that ephrinB2:EphB4 signalling from astrocytes to neural stem cells promotes differentiation during adult hippocampal neurogenesis [8]. Surprisingly EphB signalling can activate bcatenin independently of Wnt signalling, leading to the upregulation of proneural gene expression, providing a way for Ephs to influence transcriptional control of cell fate. A contribution of Eph signalling to experience-dependent dendritic spine pruning in cortical neurons is revealed in ephrin-A2 knockout mice [9] where glutamate-dependent spine elimination is accelerated and the glial glutamate transporter, which normally co-localizes with ephrin-A2, is downregulated. Previously, ephrin-A3 loss was found to disrupt hippocampal dendritic plasticity through the opposite mechanism: in contrast to loss of ephrin-A2, ephrin-A3 mutation led to higher levels of glutamate transporter expression [10,11]. Additionally, ephrin-B3 reverse signalling is also a postsynaptic effector of dendritic plasticity in the developing hippocampus [12]. Tyrosine phosphorylation and PDZ interactions of the ephrin-B3 cytoplasmic domain lead to inhibition of dendrite branching, and only PDZ binding is essential for the development of spines and excitatory synaptic function. Further work in cultured neurons identifies GRB4, PICK1 and syntenin as downstream mediators that recruit additional proteins to form dendritic spines, prune exuberant processes, and establish functional synapses. (Figure 1)

Topography and modelling Sperry’s chemoaffinity theory [13] was molecularly validated by the in vitro stripe assays of Bonhoeffer and colleagues [14]. The manipulation of ephrins and Ephs

The olfactory system contains a topographic map where olfactory receptor neuron (ORN) axons meet the dendrites of projection neurons (PNs) in specific glomeruli. In the Drosophila mutant Meigo, glomerular targeting is disrupted in both ORN axons and PN dendrites [17]. Meigo encodes an endoplasmic reticulum protein that promotes N-glycosylation, which is required for the normal cell surface expression of ephrin. Indeed, ephrin loss of function causes Meigo-like defects, supporting the first in vivo evidence of the requirement of N-glycosylation for ephrin function. The study of topographic maps also brought forth the idea that ephrins co-expressed with Ephs can either bind them in cis and attenuate their function [18], or signal in parallel from separate membrane compartments [19]. Kao and Kania provide evidence for the first scenario, where decreasing motor neuron ephrin levels increase their sensitivity to ephrins [20]. Mouse genetics provides evidence of parallel signalling in motor axon guidance: a knockout of EphA4 from motor neurons and/or their limb target tissues leads to different incidences of axon guidance errors reflecting changes in in vitro motor axon responses to EphA4 [21] (Figure 1). Elaborating further this idea, another motor neuron study reports the interaction between EphA:ephrin-A reverse and GDNFGFRalpha1/c-Ret signalling pathways. It emerges that GDNF receptor c-Ret interacts with ephrin-A5 and promotes its reverse signalling, acting as a detector of coincidence of cell membrane-bound EphA and diffusible GDNF [22] (Figure 1). Eph/ephrin function in populations of neurons has also been subjected to mathematical modelling, germinating from Gierer’s prescient ideas on topographic maps [23]. However, a model that explains all experimental data remains elusive. Three new studies bring us closer to a unified model of topographic mapping. The Gebhard et al.

( Figure 1 Legend Continued ) and GDNF co-incidence, through its interactions with ephrin-A5 and GFRa1 proteins. (D) Parallel forward and reverse signalling in response to ephrin-A and EphA is achieved by sequestration of EphAs and ephrin-As to different cell membrane domains. Ephrin-A interaction with EphA in cis results in the attenuation of its signalling ability. (E) The silencing of neuronal activity does not disrupt EphA signallingmediated early guidance of motor or retinal axons. Current Opinion in Neurobiology 2014, 27:16–24

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Figure 2

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New models of retinotopic/retinocollicular topographic map development. (A) The Gebhard et al. model based on ligand and receptor gradients and counter-gradients, a balance between forward and reverse signalling, and axon–axon interactions. (B) The Steratt model in which tectal gradient:counter-gradient-driven targetting is refined by ephrin gradient-driven modulation of target-area branching. (C) The Grimbert and Cang model also includes molecular gradients and counter-gradients but emphasises axon branching driven by RGC spontaneous activity and neurotrophin signalling.

model is based on ligand and receptor gradients and counter-gradients, a balance between forward and reverse signalling, and axon–axon interactions (Figure 2) [24]. Its predictions are used to set up in vitro experiments monitoring RGC axon responses to Eph/ephrin stripes. The results argue that signalling balance and axon–axon interactions are sufficient to describe some of the observed effects of in vitro manipulations and genetic experiments, surprisingly, without invoking cis-attenuation. The Steratt model features gradient:counter-gradient interactions, but targeting is refined by ephrin gradient-driven modulation of target-area branching (Figure 2) [25]. This model fares well against the Math5 knockout where decreased RGC numbers result in a compressed map [26], and against EphA3 overexpression in RGCs, which results in a double map [27]. However, this model does not describe accurately what is observed when one Eph receptor is deleted and another overexpressed. www.sciencedirect.com

In the third model, molecular gradients and countergradient function is strongly influenced by axon branching driven by RGC spontaneous activity and neurotrophin signalling [28], challenging the view that hard-wired molecular pathways drive topographic map development (Figure 2). This model accurately predicts several knockout phenotypes [29,30,27]; however, the predicted severe disruption of topography when neuronal activity is blocked is contradicted by a study using the Kir 2.1 channel to block activity in RGCs and motor neurons [31]. Under such conditions, the early establishment of topographic order through ephrin-A:EphA signalling is normal, emphasizing the importance of hard-wired molecular pathways for initial target mapping (Figure 1).

Cell segregation and dispersion Contact repulsion between ephrin/Eph-expressing cells leads to cell segregation into domains with matching ephrin/Eph expression, and together with adhesive interactions between kindred cells, underlies tissue boundary Current Opinion in Neurobiology 2014, 27:16–24


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Tissue boundary formation and cell dispersion. (A) Formation of stable boundaries occurs when ephrin-expressing and Eph-expressing cells segregate and establish a balanced degree of adhesion/repulsion at the tissue boundary. (B) Formation of dynamic boundaries that allow the sliding of cells along the boundary require cell blebbing induced by Eph/ephrin signalling. (C) Eph/ephrin-mediated contact repulsion contributes to the dispersion of migrating Cajal-Retzius cells that are born in distinct regions of the embryonic pallium. (D) The limited lateral dispersion of pyramidal neurons during radial migration is controlled by ephrinB1 reverse signalling.

formation (Figure 3; [32]). Examples of Eph/ephrinmediated boundary formation include hindbrain and somite segmentation, and the positioning of intestinal epithelium cells [33]. In the zebrafish forebrain, Eph/ ephrin signalling also segregates eye precursors from telencephalon and diencephalon precursors [34], but the relationship of this activity with neuronal identity is unclear [35]. A recent study [36] revisited this question by demonstrating that complementary ephrin/Eph expression in adjacent forebrain domains is downstream of regional fate acquisition. Thus, Eph/ephrin signalling prevents cell mixing, allowing adjacent tissues to deploy their distinct morphogenetic programmes. While some boundaries evolve to become permanent and stable barriers, others remain flexible. In vertebrates, the dorsal mesoderm partitions into the notochord and presomitic mesoderm, and during convergent extension [37], adhesion at their boundaries decreases dramatically allowing them to ‘slide’ against each other. The decreased adhesion is not a consequence of downregulation of adhesion molecules, but derives from Eph/ephrinCurrent Opinion in Neurobiology 2014, 27:16–24

induced changes in actomyosin-driven contractility that generates membrane blebbing-like behaviour along the boundary [38], inhibiting cadherin clustering (Figure 3). Eph/ephrin-mediated contact repulsion also drives the dispersal of Cajal-Retzius (CR) cells in the cerebral cortex. These are born in discrete regions of the pallium, from which they disperse tangentially to colonize the entire cortex evenly (Figure 3). CR cells express multiple Ephs and ephrins, and their disruption affects both homotypic and heterotypic CR interactions leading to more frequent CR cell–cell contact and a reduction of their dispersal [39]. Once CR cells reach their cortical marginal zone destination, they secrete reelin which coordinates the inside-out migration of cortical neurons [40]. New evidence shows that besides binding its receptors from the LDL protein family, reelin also interacts with Ephs and ephrins. For example, it is able to induce ephrin-B clustering and phosphorylation of Dab1, an essential mediator of Reelin signalling [41], and to bind the ectodomain and activate EphB receptors [42]. Mice deficient in ephrin-Bs or EphBs display reeler mutant www.sciencedirect.com


phenotypes in cerebral cortex and hippocampus, respectively [41,42]. Radially migrating cortical pyramidal neurons also undergo dispersion in response to ephrinA:EphA forward signalling which, through an unclear mechanism, promotes their intermingling during the tangential phase of migration [43]. Contrasting the above ideas, other new work implicates ephrin-B1 in cell adhesion. Apical progenitors of the cor-

tical neuroepithelium show strong adhesion requiring ephrin-B1-mediated localization of integrin-b1 [44]. During their multipolar stage, ephrin-B1 reverse signalling also activates Rac3 through the P-Rex1 guanylate exchange factor, inhibiting neurite dynamics and effectively restricting tangential migration independently of radial migration (Figure 3) [45]. Hence, ephrin-B1 signalling blocks lateral dispersion probably as a consequence of reduced neurite dynamics and exploratory behaviour.

Figure 4

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Regulation of EphA4 expression during axon fasciculation and guidance. (A) Pou3f4 regulates EphA4 expression in otic mesenchyme (OM). EphA4 promotes fasciculation of spiral ganglion axons via binding to ephrin-B2. CE: cochlear epithelium. (B) Zic2 regulates EphA4 expression in dorsal spinal neurons that form an ipsilateral ascending tract. EphA4+ corticospinal (CST) axons descend in the same spinal tract. Both neuron populations are guided by repulsive ephrinB3/EphA4 forward signalling. (C) Axon–axon interactions involve forward ephrin-A:EphA signalling-mediated repulsion, and a balance between EphA:ephrin-A reverse signalling attraction and an unknown repulsion signal leads to axon tracking. www.sciencedirect.com



Axon guidance and fasciculation Ephrin-B reverse signalling-mediated adhesion has been implicated in the developing auditory system by a study examining the transcriptional regulation of ephrins and Ephs [46,47]. Auditory spiral ganglion neuron (SGN) axons form radial fascicles that project through the otic mesenchyme to synapse onto cochlear hair cells (Figure 4). The POU-domain (Pit1-Oct1/2-unc86) protein Pou3f4 was shown to be expressed in otic mesenchymal cells and regulate EphA4 expression, promoting fasciculation of SGN axons via ephrin-B2 [46]. This posits the surprising idea that EphA4:ephrin-B2 reverse signalling promotes axon fasciculation. The zinc finger transcription factor Zic2 promotes EphB1 expression in RGCs, instructing their axons to follow an ipsilateral trajectory [48]. In the dorsal spinal cord, Zic2 also promotes ipsilateral axon trajectories, through the expression of EphA4, as recently shown in [49]. In both cases, Eph receptors function to repel axons from a midline ephrin-B domain, suggesting an evolutionarily conserved role for Zic2 in midline avoidance. A parallel study suggests that the Zic2+EphA4+ spinal neurons relay mechanosensory information [50], and their developing axons ascend ipsilaterally in the dorsal funiculus, in the same location as the later-descending corticospinal (CST) axons (Figure 4). Surprisingly, ephrin-B3:EphA4 forward signalling operates in both ascending and descending axon tracts raising the question of whether the ascending tracts are pioneering a tract followed by CST axons. Similarly, sensory axons were found to use ephrin-As to track along motor axons that express EphA3/4 receptors (Figure 4), whose deletion results in sensory axon repulsion from motor axons, hinting that a balance between repulsive and adhesive interactions underlies axon tracking [51]. Eph receptor forward signalling requires its intrinsic tyrosine kinase activity [52] according to genetic experiments in which kinase-dead EphA4 receptors phenocopy EphA4 lacking the entire cytoplasmic domain ([53] and R.K., unpublished observations). However, Eph kinaseindependent forward signalling events have also been described [54,55]. To more rigorously investigate them, Greenberg and colleagues used chemical genetics to generate mice in which EphB kinase activity could be specifically and acutely blocked [56]. They found that the tyrosine kinase activity of EphBs was required for axon guidance but not for synaptogenesis, suggesting that repulsive Eph/ephrin interactions require kinase activity whereas adhesive interactions may not.

ephrin/Eph signalling is important for neuronal connectivity led to many ideas about ephrin/Eph function but some original observations remain without a molecular explanation. The biological significance of specificity of ephrin–Eph interactions is emerging [3], but we are still far from understanding whether same-class ligands are interchangeable or the significance of ligand dissociation constant differences for a particular receptor [2]. Could some of these be explained by post-translational modifications [17]? Is a combinatorial deployment of ephrin ligands a solution to the problem of synaptic diversity? Another pressing question is the relationship between forward and reverse signalling. Reverse Eph:ephrin signalling results in attractive interactions [46], and concomitantly, ephrin binding to Eph receptor on the opposing cell should also lead to forward repulsive signalling. The evidence of two neighbouring cells receiving such conflicting signals is lacking, raising the question of how the repulsive effects of forward signalling are blocked. Since Eph/ephrin signalling occurs within a certain cellular context, it interacts with specific signalling cascades [22,41,42]. What is the cellular and molecular consequence of these interactions? Which signalling pathways are activated by the co-incident activity of Ephs and other receptors? How would such interactions impact topographic map formation models? The development of biosensors and tools allowing biochemical investigation of live neurons will certainly answer some of the above questions. However, even a rudimentary understanding of the spatial relationship between receptor activation and cytoskeletal dynamics would be a very exciting next step. One such experiment might be visualising the temporal and spatial dynamics of activated Eph receptors within a growth cone. With the advent of new tools, combined with imaging advances allowing us to see more clearly, this field is on the verge of many exciting discoveries.

Acknowledgements We are grateful to He´le`ne Lambin for figure preparation, Daniel Morales and Chris Law for critical comments on the manuscript. R.K. is supported by the Max-Planck Society, and by grants from the Deutsche Forschungsgemeinschaft [SFB870] and the European Union. A.K. is supported by the Canadian Institutes of Health Research, National Sciences and Engineering Research Council of Canada, Que´bec Pain Research Network, Brain Canada and the W. Garfield Weston Foundation.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest

Conclusions The past two years have been packed with many exciting studies ranging from reductionist approaches exploring Eph/ephrin signalling in its pure form, to those extending such studies in vivo, to highly specialised events peculiar to the nervous system. The original observation that Current Opinion in Neurobiology 2014, 27:16–24

1.

Xu K, Tzvetkova-Robev D, Xu Y, Goldgur Y, Chan Y-P, Himanen JP, Nikolov DB: Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5. Proc Natl Acad Sci U S A 2013, 110:14634-14639. www.sciencedirect.com


2.

Gale NW, Holland SJ, Valenzuela DM, Flenniken A, Pan L, Ryan TE, Henkemeyer M, Strebhardt K, Hirai H, Wilkinson DG et al.: Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Neuron 1996, 17:9-19.

3.

Seiradake E, Schaupp A, del Toro Ruiz D, Kaufmann R, Mitakidis N, Harlos K, Aricescu AR, Klein R, Jones EY: Structurally encoded intraclass differences in EphA clusters drive distinct cell responses. Nat Struct Mol Biol 2013, 20:958-964. Study of contrasting cellular and biochemical effects of ephrin-A5 binding to EphA2 and EphA4 and mapping of their origin to the structure of the extracellular domain.

4.

Himanen JP, Yermekbayeva L, Janes PW, Walker JR, Xu K, Atapattu L, Rajashankar KR, Mensinga A, Lackmann M, Nikolov DB et al.: Architecture of Eph receptor clusters. Proc Natl Acad Sci U S A 2010, 107:10860-10865.

5.

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6.

Schaupp A, Sabet O, Dudanova I, Ponserre M, Bastiaens P, Klein R: The composition of EphB2 clusters determines the strength in the cellular repulsion response. J Cell Biol 2013. (in press). By using chemical dimerizers to generate specific EphB2 cluster sizes of various abundance in living cells, this study provides evidence that the strength of the cellular response is determined by the relative abundance of inactive monomers/dimers and active higher-order clusters.

7.

Mohamed AM, Boudreau JR, Yu FPS, Liu J, Chin-Sang ID: The Caenorhabditis elegans Eph receptor activates NCK and NWASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance. PLoS Genet 2012, 8:e1002513.

8.

Ashton RS, Conway A, Pangarkar C, Bergen J, Lim K-I, Shah P, Bissell M, Schaffer DV: Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling. Nat Neurosci 2012, 15:1399-1406. Ephrin-B expression by astrocytes influences hippocampal neurogenesis, and signalling through EphB receptor leads to activation of betacatenin, and transcriptional responses.

9.

Yu X, Wang G, Gilmore A, Yee AX, Li X, Xu T, Smith SJ, Chen L, Zuo Y: Accelerated experience-dependent pruning of cortical synapses in ephrin-A2 knockout mice. Neuron 2013, 80:64-71. A genetic analysis combined with high resolution live imaging showing that Ephrin-A2 is important for sensory experience-dependent dendritic spine pruning and apparently associates with glial glutamate transporter. 10. Carmona MA, Murai KK, Wang L, Roberts AJ, Pasquale EB: Glial ephrin-A3 regulates hippocampal dendritic spine morphology and glutamate transport. Proc Natl Acad Sci U S A 2009, 106:12524-12529.

11. Filosa A, Paixao S, Honsek SD, Carmona MA, Becker L, Feddersen B, Gaitanos L, Rudhard Y, Schoepfer R, Klopstock T et al.: Neuron–glia communication via EphA4/ephrin-A3 modulates LTP through glial glutamate transport. Nat Neurosci 2009, 12:1285-1292. 12. Xu N-J, Sun S, Gibson JR, Henkemeyer M: A dual shaping mechanism for postsynaptic ephrin-B3 as a receptor that sculpts dendrites and synapses. Nat Neurosci 2011, 14:14211429. A complex genetic analysis of ephrin-B3 mutations deleting specific intracellular domains showing that reverse signalling through ephrin-B3 shapes dendrite and synapse function. 13. Sperry RW: Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc Natl Acad Sci U S A 1963, 50:703-710. 14. Drescher U, Kremoser C, Handwerker C, Loschinger 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. 15. Yoon MG: Progress of topographic regulation of the visual projection in the halved optic tectum of adult goldfish. J Physiol 1976, 257:621-643. www.sciencedirect.com

16. Tadesse T, Cheng Q, Xu M, Baro DJ, Young LJ, Pallas SL: Regulation of ephrin-A expression in compressed retinocollicular maps. Dev Neurobiol 2013, 73:274-296. An elegant study providing a molecular answer to the cellular observation of retinotectal maps compression caused by tectal target deletion. In this paper, ephrin expression gradients are shown to be shifted in animals with superior colliculus deletions, suggesting global ephrin regulation in topographic map targets. 17. Sekine SU, Haraguchi S, Chao K, Kato T, Luo L, Miura M, Chihara T: Meigo governs dendrite targeting specificity by modulating ephrin level and N-glycosylation. Nat Neurosci 2013, 16:683-691. Mutations in Meigo, a Drosophila gene encoding an endoplasmic reticulum protein, uncover a role for Ephrin signalling in olfactory circuit connectivity, and provide first evidence that N-glycosylation is required for normal ephrin function. 18. Carvalho RF, Beutler M, Marler KJ, Knoll B, Becker-Barroso E, Heintzmann R, Ng T, Drescher U: Silencing of EphA3 through a cis interaction with ephrinA5. Nat Neurosci 2006, 9:322-330. 19. 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. 20. Kao T-J, Kania A: Ephrin-mediated cis-attenuation of Eph receptor signaling is essential for spinal motor axon guidance. Neuron 2011, 71:76-91. In vivo and in vitro motor axon manipulation showing the importance of ligand–receptor cis interaction in axon guidance. 21. Dudanova I, Kao TJ, Herrmann JE, Zheng B, Kania A, Klein R: Genetic evidence for a contribution of EphA:EphrinA reverse signaling to motor axon guidance. J Neurosci 2012, 32:52095215. 22. Bonanomi D, Chivatakarn O, Bai G, Abdesselem H, Lettieri K, Marquardt T, Pierchala BA, Pfaff SL: Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals. Cell 2012, 148:568-582. A multi-faceted study of the role of EphA reverse signalling through ephrin-A2/5, and its association with c-Ret, a GDNF receptor, in motor axon guidance. Here, c-Ret functions as a coincidence detector, with distinct signalling responses to either ligand separately, and to their concomitance. 23. Gierer A: Development of projections between areas of the nervous system. Biol Cybern 1981, 42:69-78. 24. Gebhardt C, Bastmeyer M, Weth F: Balancing of ephrin/Eph forward and reverse signaling as the driving force of adaptive topographic mapping. Cambridge, England: Development; 2011, . A very thorough modeling analysis including in vitro experimental validation of the development of retino-topic topographic maps arguing for an elemental role of the balance between forward and reverse ephrin:Eph signalling between the tectum and RGC neurons, as well as in axon–axon interactions. 25. Sterratt DC: On the importance of counter gradients for the development of retinotopy: insights from a generalised gierer model. PLOS ONE 2013, 8:e67096. A theoretical model paper, describing the importance of ligand and receptor gradients and counter-gradients, as well as local axon branching near the target in the development of retinotopic topography. 26. Triplett JW, Pfeiffenberger C, Yamada J, Stafford BK, Sweeney NT, Litke AM, Sher A, Koulakov AA, Feldheim DA: Competition is a driving force in topographic mapping. Proc Natl Acad Sci U S A 2011, 108:19060-19065. 27. Brown A, Yates PA, Burrola P, Ortuno D, Vaidya A, Jessell TM, Pfaff SL, O’Leary DD, 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. 28. Grimbert F, Cang J: New model of retinocollicular mapping predicts the mechanisms of axonal competition and explains the role of reverse molecular signaling during development. J Neurosci 2012, 32:9755-9768. This theoretical paper proposes a new model of retinotopic mapping that emphasizes neuronal activity-driven processes as playing a more prominent role in RGC targeting than hard-wired Eph–ephrin interactions. Current Opinion in Neurobiology 2014, 27:16–24


29. Rashid T, Upton AL, Blentic A, Ciossek T, Knoll B, Thompson ID, Drescher U: Opposing gradients of ephrin-As and EphA7 in the superior colliculus are essential for topographic mapping in the mammalian visual system. Neuron 2005, 47:57-69. 30. Lim YS, McLaughlin T, Sung TC, Santiago A, Lee KF, O’Leary DD: p75(NTR) mediates ephrin-A reverse signaling required for axon repulsion and mapping. Neuron 2008, 59:746-758. 31. Benjumeda I, Escalante A, Law C, Morales D, Chauvin G, Muça G, Coca Y, Ma´rquez J, Lo´pez-Bendito G, Kania A et al.: Uncoupling of EphA/ephrinA signaling and spontaneous activity in neural circuit wiring. J Neurosci 2013, 33:18208-18218. A two-pronged approach in chicken and mouse, to tackle the question of the role of neuronal activity in ephrin/Eph signalling. Blocking activity in developing motor neurons or RGCs through overexpression of the Kir2.1 channel does not result in ephrin/Eph-dependent axon trajectory mistargeting, arguing against a prominent role of activity, at these early stages of axon pathfinding. 32. Batlle E, Wilkinson DG: Molecular mechanisms of cell segregation and boundary formation in development and tumorigenesis. Cold Spring Harb Perspect Biol 2012, 4: a008227. 33. Klein R: Eph/ephrin signalling during development. Development 2012, 139:4105-4109. 34. Xu Q, Alldus G, Macdonald R, Wilkinson DG, Holder N: Function of the Eph-related kinase rtk1 in patterning of the zebrafish forebrain. Nature 1996, 381:319-322. 35. Moore KB, Mood K, Daar IO, Moody SA: Morphogenetic movements underlying eye field formation require interactions between the FGF and ephrinB1 signaling pathways. Dev Cell 2004, 6:55-67. 36. Cavodeassi F, Ivanovitch K, Wilson SW: Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis. Development 2013, 140:4193-4202. 37. Wallingford JB, Fraser SE, Harland RM: Convergent extension: the molecular control of polarized cell movement during embryonic development. Dev Cell 2002, 2:695-706. 38. Fagotto F, Rohani N, Touret A-S, Li R: A molecular base for cell sorting at embryonic boundaries: contact inhibition of cadherin adhesion by Ephrin/Eph-dependent contractility. Dev Cell 2013. An interesting study which shows that membrane blebbing at flexible boundaries allows cells to ‘slide’ against each other. Blebbing is caused by actomyosin-driven contractility that inhibits cadherin clustering and is initiated by Eph/ephrin signalling. 39. Villar-Cervinõ V, Molano-Mazoń M, Catchpole T, Valdeolmillos M, Henkemeyer M, Martıńez LM, Borrell V, Marıń O: Contact repulsion controls the dispersion and final distribution of cajal-retzius cells. Neuron 2013, 77:457-471. A new mechanism based on Eph:ephrin-mediated contact repulsion underlies the tangential dispersion of Cajal-Retzius cells in the developing neocortex. 40. Tissir F, Goffinet AM: Reelin and brain development. Nat Rev Neurosci 2003, 4:496-505. 41. Sentu¨rk A, Pfennig S, Weiss A, Burk K, Acker-Palmer A: Ephrin Bs are essential components of the Reelin pathway to regulate neuronal migration. Nature 2011. 42. Bouche´ E, Romero-Ortega MI, Henkemeyer M, Catchpole T, Leemhuis J, Frotscher M, May P, Herz J, Bock HH: Reelin induces EphB activation. Cell Res 2013. 43. Torii M, Hashimoto-Torii K, Levitt P, Rakic P: Integration of neuronal clones in the radial cortical columns by EphA and ephrin-A signalling. Nature 2009, 461:524-528. 44. Arvanitis DN, Behar A, Tryoen-Toth P, Bush JO, Jungas T, Vitale N, Davy A: Ephrin B1 maintains apical adhesion of neural progenitors. Development 2013, 140:2082-2092.


A study that implicates ephrin-B1-mediated localization of integrin-b1 in cell adhesion in apical progenitors of the developing cortex. 45. Dimidschstein J, Passante L, Dufour A, van den Ameele J, Tiberi L, Hrechdakian T, Adams R, Klein R, Lie DC, Jossin Y et al.: Ephrinb1 controls the columnar distribution of cortical pyramidal neurons by restricting their tangential migration. Neuron 2013, 79:1123-1135. This study shows that ephrin-B1 reverse signalling reduces neurite dynamics in radially migrating cortical neurons thereby restricting their ability to disperse in the tangential direction. 46. Coate TM, Raft S, Zhao X, Ryan AK, Crenshaw EB III, Kelley MW: Otic mesenchyme cells regulate spiral ganglion axon fasciculation through a Pou3f4/EphA4 signaling pathway. Neuron 2012, 73:49-63. A study that suggests that ephrin-B2 reverse signalling promotes axon fasciculation. The transcription factor Pou3f4, expressed in otic mesenchymal cells, regulates EphA4 expression which promotes fasciculation of SGN axons via binding to ephrinB2. 47. Arvanitis D, Davy A: Eph/ephrin signaling: networks. Genes Dev 2008, 22:416-429. 48. Garcia-Frigola C, Carreres MI, Vegar C, Mason C, Herrera E: Zic2 promotes axonal divergence at the optic chiasm midline by EphB1-dependent and -independent mechanisms. Development 2008, 135:1833-1841. 49. Escalante A, Murillo B, Morenilla C, Klar A, Herrera E: Zic2 controls the formation of major ipsilateral tracts in the CNS through active midline avoidance. Neuron 2013, 80:1392-1406. In this study, the zinc finger transcription factor Zic2 is shown to determine ipsilateral axon wiring by controling midline avoidance in the CNS across species and signaling pathways. One mechanism by which Zic2 operates is by regulating EphA4 expression. 50. Paixao S, Balijepalli A, Serradj N, Niu J, Luo W, Martin JH, Klein R: EphrinB3/EphA4-mediated guidance of ascending and descending spinal tracts. Neuron 2013, 80:1407-1420. This work shows that ephrin-B3:EphA4 forward signalling operates in both ascending and descending spinal axon tracts and therefore represents an example of the economical usage of the limited vocabulary of guidance cues with which neural development creates axon projections. 51. Wang L, Klein R, Zheng B, Marquardt T: Anatomical coupling of sensory and motor nerve trajectory via axon tracking. Neuron 2011, 71:263-277. A sophisticated combination of genetic observations with in vitro reconstitution studies. The authors show that forward and reverse ephrinA:EphA signalling is important for sensory axons tracking along preexisting motor axons with EphA mutant analysis revealing a balance of attractive and repulsive forces. 52. Lisabeth EM, Falivelli G, Pasquale EB: Eph receptor signaling and ephrins. Cold Spring Harb Perspect Biol 2013:5. 53. Dufour A, Egea J, Kullander K, Klein R, Vanderhaeghen P: Genetic analysis of EphA-dependent signaling mechanisms controlling topographic mapping in vivo. Development 2006, 133:4415-4420. 54. Dalva MB, Takasu MA, Lin MZ, Shamah SM, Hu L, Gale NW, Greenberg ME: EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell 2000, 103:945-956. 55. Kullander K, Klein R: Mechanisms and functions of Eph and ephrin signalling. Nat Rev Mol Cell Biol 2002, 3:475-486. 56. Soskis MJ, Ho HY, Bloodgood BL, Robichaux MA, Malik AN, Ataman B, Rubin AA, Zieg J, Zhang C, Shokat KM et al.: A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development. Nat Neurosci 2012, 15:1645-1654. An elegant chemical genetic approach to generate mice in which EphB1/ B2/B3 kinase activity can be specifically and acutely blocked. It was found that EphBs’ tyrosine kinase activity was required for axon guidance, but not for synaptogenesis.

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