Fagotto F , Winklbauer R , Rohani N .

	Figure 1. Models for boundary formation. (A) Classical models for cell sorting and tissue separation based on differences in cell-cell adhesion and cortical contractility. Sorting may be achieved (A) by cell-type specific expression of different cell adhesion molecules (CAMs) with stronger affinity for homotypic binding than for heterotypic interaction, or (A’) by differences in adhesive strength between cell types (differential adhesion hypothesis, or DAH), resulting for instance from different levels of adhesion molecules. (A”) Difference in cell cortex contractility can also lead to cell sorting. Recently, adhesion and contractility have been integrated into a single description of adhesive cell interactions, but for simplicity of depiction they are shown in separate panels here. B. Cell sorting/tissue separation may also be produced by the complementary expression of repellent cell surface cues, which would trigger local cortex contraction and cell repulsion at heterotypic contacts, a process termed contact inhibition.
	Figure 2. Ephrin and Eph receptor expression in the early Xenopus embryo. Transcripts of 7 ephrins and 11 Eph receptors are detected in the early Xenopus stages. The color code symbolizes relative mRNA levels, determined by real-time RT-PCR using tissues dissected from late blastula (stage 9.5), early gastrula (stage 10.5) mid gastrula (stage 11.5) and early neurula (stage 14). Gray color indicates that levels were too low and variable to be quantified.
	Figure 3. Ephrin and Eph receptor distribution at vertebrate embryonic boundaries. Each diagram represents the pattern observed in the species where most studies were performed: (A) Dorsal ectoderm-mesoderm boundary in the early Xenopus gastrula. (B) Ventral ectoderm-mesoderm boundary in the middle Xenopus gastrula. (C) Notochord-presomitic boundary in the late Xenopus gastrula. (D) Somitogenesis in zebrafish. (E) Hindbrain segmentation in zebrafish. (E’) Comparative diagram from available data in the 4 species, zebrafish (z), Xenopus (x), chicken (c) and mouse (m). Species abbreviated in black denotes expression, in red absence of (or low) expression in the corresponding segment. Absence of symbol indicates that the gene was not investigated in this species. Note that the ephrinB3 gene is altogether absent from chicken. (F) Eye field segregation in zebrafish. In (A–C), the font and box size represent relative enrichments. Comparative data on expression levels are not available for the other systems, but in a few instances lower expression was clearly observed in the hindbrain (ephrinB2 in r1,r3 and r6, EphA4 in r2, EphB4 in r4). Patterns were built based on expression data from:35,36,38,47 for (A–C), 32,42,48,52,59,122-125 for (D), 33,48,53-56,124,126-128 for (E), and 37 for (F), complemented with the Mouse Gene Expression Databases for zebrafish (http://zfin.org) and mo
	Figure 4. Ephrin-Eph signaling in embryonic tissues. (A) Multiple activities and cross-talks. Ephrin-Eph receptor interactions typically activate an intracellular signal that induces reorganization of the actin cytoskeleton, including increased actomyosin contraction, leading to retraction. This repulsive activity is thought to be triggered only at high levels of signal. It requires disruption of the ephrin-Eph bonds which connect the 2 cells. This can occur either by endocytosis, or by shedding of the ephrin/Eph extracellular domains by ADAMs proteinases (not shown). It is believed that, under certain condition, in particular under low ephrin-Eph signaling, ephrin-Eph interactions contribute on the contrary to cell-cell adhesion. Note that the exact mechanisms that control adhesive versus repulsive modes are not well understood. Like many other ligand-receptor systems, ephrin-Eph signaling influences other aspects of cell function, such as gene regulation or cell-matrix adhesion. Ephrins and Eph receptors have been found to directly associate with other cell surface components, such as tight junctions. The effect of these interactions is still poorly understood. When coexpressed in the same cell, ephrins and Eph receptors may also establish cross-talks, which are generally considered to lead to signal dampening. (B–E) Examples of ephrin-Eph configurations (top 2 cell rows) and of predicted consequences of loss-of-function on signaling and adhesion within and between tissues (lower rows). (
	Figure 4 . Ephrin-Eph signaling in embryonic tissues. (B’–E’) Resulting effect on tissue cohesion/separation. Sorting/separation is symbolized by a gap between the cells. (B) Embryonic tissues express multiple ephrins and Eph receptors. In the simplest cases, they are expressed in fully complementary patterns, such that signaling is exclusively triggered at the tissue interface, where repulsion antagonizes cadherin cell adhesion, resulting in low effective adhesion. Lowering ephrins or Ephs is predicted to decrease repulsion/increase adhesion at the tissue interface and cause their fusion, without major effect on intratissular contacts. Note that the systematic expression of several ephrins and/or several Ephs observed in all systems creates partial - and sometimes even full -redundancy. As a consequence, more than one of these components may need to be removed to inhibit separation. (C) In more complex cases, expression is only partially complementary. Here ephrins and Eph receptors can also interact in one or both tissues. These intracellular interactions can generate repulsion, which may confer to the tissue different physical properties, e.g., lower rigidity and higher migration. (D) Under certain circumstances, EphrinEph intratissular signaling may generate a pro-adhesive activity, effectively increasing tissue cohesion. In this case, ephrin/Eph depletion is predicted to decrease tissue cohesion, and could even in principle result in artificial sorting from the tissue of origin, while concomitant inhibition of repulsion across the boundary could lead the depleted cells to join the opposite tissue. (E) In a situation where intratissular signaling is weakly repulsive (C), partial lossof-function may switch from repulsive to adhesive mode. Depleted cells may sort into a compact independent population.
	Figure 5. Ephrin-Eph code at embryonic boundaries. The same code, based on selective ephrin-Eph pairs, is used in different configurations. In each case, repulsion results strongest at the tissue interface. (A–C) Simplified examples, taking into consideration a subset of ephrins and Eph receptors. A. Full complementary ephrin/Eph expression accounts for separation of the eye field and of the central hindbrain segments. (B) In the early gastrula, significant intracellular signaling occurs in the mesoderm, but strongest repulsion is restricted to the ectoderm-mesoderm contacts. EphrinB3 and EphB4, both enriched in the ectoderm, do not interact. (C) Hypothetical diagram of ephrin-Eph signaling in the somites. Segmentation may involve the combined activation of EphA4 by ephrinB2 and ephrinA1. EphA4 may be expressed in a gradient, with highest levels coinciding with the boundary. The distribution of ephrinB2 remains unclear, but may span the whole segment, thus activating some intratissular signal in the anterior half. (D) Complete description of ephrinB-Eph signaling at the ectoderm-mesoderm boundary. Individual contributions of each functional pair are semi-quantitatively symbolized by red lines of different thicknesses.

Aliee, Physical mechanisms shaping the Drosophila dorsoventral compartment boundary. 2012, Pubmed

Aliee, Physical mechanisms shaping the Drosophila dorsoventral compartment boundary. 2012, Pubmed
Arvanitis, Eph/ephrin signaling: networks. 2008, Pubmed
Astin, Competition amongst Eph receptors regulates contact inhibition of locomotion and invasiveness in prostate cancer cells. 2010, Pubmed
Aulehla, Oscillating signaling pathways during embryonic development. 2008, Pubmed
Baker, Ephs and ephrins during early stages of chick embryogenesis. 2003, Pubmed
Barrios, Eph/Ephrin signaling regulates the mesenchymal-to-epithelial transition of the paraxial mesoderm during somite morphogenesis. 2003, Pubmed
Batlle, Molecular mechanisms of cell segregation and boundary formation in development and tumorigenesis. 2012, Pubmed
Becker, Several receptor tyrosine kinase genes of the Eph family are segmentally expressed in the developing hindbrain. 1994, Pubmed
Bergemann, ELF-2, a new member of the Eph ligand family, is segmentally expressed in mouse embryos in the region of the hindbrain and newly forming somites. 1995, Pubmed
Blits-Huizinga, Ephrins and their receptors: binding versus biology. 2004, Pubmed
Böttger, Hydra, a model system to trace the emergence of boundaries in developing eumetazoans. 2012, Pubmed
Brodland, The Differential Interfacial Tension Hypothesis (DITH): a comprehensive theory for the self-rearrangement of embryonic cells and tissues. 2002, Pubmed
Brodland, Cellular interfacial and surface tensions determined from aggregate compression tests using a finite element model. 2009, Pubmed
Calzolari, Cell segregation in the vertebrate hindbrain relies on actomyosin cables located at the interhombomeric boundaries. 2014, Pubmed
Cavodeassi, Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis. 2013, Pubmed
Chan, Morphogenesis of prechordal plate and notochord requires intact Eph/ephrin B signaling. 2001, Pubmed
Chang, Differential adhesion and actomyosin cable collaborate to drive Echinoid-mediated cell sorting. 2011, Pubmed
Chen, A protocadherin-cadherin-FLRT3 complex controls cell adhesion and morphogenesis. 2009, Pubmed , Xenbase
Chen, An enhancer element in the EphA2 (Eck) gene sufficient for rhombomere-specific expression is activated by HOXA1 and HOXB1 homeobox proteins. 1998, Pubmed
Chen, Paraxial protocadherin mediates cell sorting and tissue morphogenesis by regulating C-cadherin adhesion activity. 2006, Pubmed , Xenbase
Chung, ANR5, an FGF target gene product, regulates gastrulation in Xenopus. 2007, Pubmed , Xenbase
Cooke, EphA4 is required for cell adhesion and rhombomere-boundary formation in the zebrafish. 2005, Pubmed
Cooke, Eph signalling functions downstream of Val to regulate cell sorting and boundary formation in the caudal hindbrain. 2001, Pubmed
Coulthard, Characterization of the Epha1 receptor tyrosine kinase: expression in epithelial tissues. 2001, Pubmed
Dahmann, Boundary formation and maintenance in tissue development. 2011, Pubmed
Davy, Ephrin-B2 forward signaling regulates somite patterning and neural crest cell development. 2007, Pubmed
Day, Three distinct molecular surfaces in ephrin-A5 are essential for a functional interaction with EphA3. 2005, Pubmed
Dottori, EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract. 1998, Pubmed
Dravis, Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development. 2004, Pubmed
Durbin, Anteroposterior patterning is required within segments for somite boundary formation in developing zebrafish. 2000, Pubmed
Durbin, Eph signaling is required for segmentation and differentiation of the somites. 1998, Pubmed
Edelman, Cell adhesion molecules in the regulation of animal form and tissue pattern. 1986, Pubmed
Fagotto, The cellular basis of tissue separation. 2014, Pubmed
Fagotto, A molecular base for cell sorting at embryonic boundaries: contact inhibition of cadherin adhesion by ephrin/ Eph-dependent contractility. 2013, Pubmed , Xenbase
Flanagan, The ephrins and Eph receptors in neural development. 1998, Pubmed
Foty, Cadherin-mediated cell-cell adhesion and tissue segregation in relation to malignancy. 2004, Pubmed
Foty, The differential adhesion hypothesis: a direct evaluation. 2005, Pubmed
Gale, Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. 1996, Pubmed
Glazier, Coordinated action of N-CAM, N-cadherin, EphA4, and ephrinB2 translates genetic prepatterns into structure during somitogenesis in chick. 2008, Pubmed
Halloran, Repulsion or adhesion: receptors make the call. 2006, Pubmed
Harris, Is Cell sorting caused by differences in the work of intercellular adhesion? A critique of the Steinberg hypothesis. 1976, Pubmed
Heasman, Morpholino oligos: making sense of antisense? 2002, Pubmed , Xenbase
Helbling, The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis. 2000, Pubmed , Xenbase
Helbling, Requirement for EphA receptor signaling in the segregation of Xenopus third and fourth arch neural crest cells. 1998, Pubmed , Xenbase
Helbling, Comparative analysis of embryonic gene expression defines potential interaction sites for Xenopus EphB4 receptors with ephrin-B ligands. 1999, Pubmed , Xenbase
Himanen, Ligand recognition by A-class Eph receptors: crystal structures of the EphA2 ligand-binding domain and the EphA2/ephrin-A1 complex. 2009, Pubmed
Himanen, Eph signaling: a structural view. 2003, Pubmed
Himanen, Crystal structure of an Eph receptor-ephrin complex. , Pubmed
Hukriede, Conserved requirement of Lim1 function for cell movements during gastrulation. 2003, Pubmed , Xenbase
Irie, Bidirectional signaling through ephrinA2-EphA2 enhances osteoclastogenesis and suppresses osteoblastogenesis. 2009, Pubmed
Janes, Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. 2005, Pubmed
Janis, Ephrin-A binding and EphA receptor expression delineate the matrix compartment of the striatum. 1999, Pubmed
Jülich, Control of extracellular matrix assembly along tissue boundaries via Integrin and Eph/Ephrin signaling. 2009, Pubmed
Jungbluth, Cell mixing between the embryonic midbrain and hindbrain. 2001, Pubmed
Kao, Ephrin-mediated cis-attenuation of Eph receptor signaling is essential for spinal motor axon guidance. 2011, Pubmed
Kemler, From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. 1993, Pubmed
Kemp, EphA4 and EfnB2a maintain rhombomere coherence by independently regulating intercalation of progenitor cells in the zebrafish neural keel. 2009, Pubmed
Kietzmann, Xenopus paraxial protocadherin inhibits Wnt/β-catenin signalling via casein kinase 2β. 2012, Pubmed , Xenbase
Kim, The role of paraxial protocadherin in selective adhesion and cell movements of the mesoderm during Xenopus gastrulation. 1998, Pubmed , Xenbase
Kim, The protocadherin PAPC establishes segmental boundaries during somitogenesis in xenopus embryos. 2000, Pubmed , Xenbase
Klein, Eph/ephrin signaling in morphogenesis, neural development and plasticity. 2004, Pubmed
Kraft, Wnt-11 and Fz7 reduce cell adhesion in convergent extension by sequestration of PAPC and C-cadherin. 2012, Pubmed , Xenbase
Krens, Cell sorting in development. 2011, Pubmed , Xenbase
Krieg, Tensile forces govern germ-layer organization in zebrafish. 2008, Pubmed
Landsberg, Increased cell bond tension governs cell sorting at the Drosophila anteroposterior compartment boundary. 2009, Pubmed
Laplante, Asymmetric distribution of Echinoid defines the epidermal leading edge during Drosophila dorsal closure. 2011, Pubmed
Lin, Cell adhesion molecule Echinoid associates with unconventional myosin VI/Jaguar motor to regulate cell morphology during dorsal closure in Drosophila. 2007, Pubmed
Luu, Large-scale mechanical properties of Xenopus embryonic epithelium. 2011, Pubmed , Xenbase
Maghzal, The tumor-associated EpCAM regulates morphogenetic movements through intracellular signaling. 2010, Pubmed , Xenbase
Maître, Adhesion functions in cell sorting by mechanically coupling the cortices of adhering cells. 2012, Pubmed
Manning, Differential regulation of the Kar3p kinesin-related protein by two associated proteins, Cik1p and Vik1p. 1999, Pubmed
Manzanares, The role of kreisler in segmentation during hindbrain development. 1999, Pubmed
Maroto, Somitogenesis. 2012, Pubmed
Marston, Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion. 2003, Pubmed
Medina, Xenopus paraxial protocadherin has signaling functions and is involved in tissue separation. 2004, Pubmed , Xenbase
Mellitzer, Eph receptors and ephrins restrict cell intermingling and communication. 1999, Pubmed
Mellott, The molecular phylogeny of eph receptors and ephrin ligands. 2008, Pubmed
Nakamoto, Eph receptors and ephrins. 2000, Pubmed
Niessen, Cadherin-mediated cell sorting not determined by binding or adhesion specificity. 2002, Pubmed , Xenbase
Nieto, A receptor protein tyrosine kinase implicated in the segmental patterning of the hindbrain and mesoderm. 1992, Pubmed
Nikolov, Crystal structure of the ephrin-B1 ectodomain: implications for receptor recognition and signaling. 2005, Pubmed
Ninomiya, Cadherin-dependent differential cell adhesion in Xenopus causes cell sorting in vitro but not in the embryo. 2012, Pubmed , Xenbase
Noberini, Profiling Eph receptor expression in cells and tissues: a targeted mass spectrometry approach. 2012, Pubmed
Pabbisetty, Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: correlation to function in a growth cone collapse assay. 2007, Pubmed
Park, The involvement of Eph-Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements. 2011, Pubmed , Xenbase
Pasquale, Eph receptors and ephrins in cancer: bidirectional signalling and beyond. 2010, Pubmed
Prin, Hox proteins drive cell segregation and non-autonomous apical remodelling during hindbrain segmentation. 2014, Pubmed
Reddy, Genome-wide screening reveals the emergence and divergence of RTK homologues in basal Metazoan Hydra magnipapillata. 2011, Pubmed
Reintsch, beta-Catenin controls cell sorting at the notochord-somite boundary independently of cadherin-mediated adhesion. 2005, Pubmed , Xenbase
Rhee, The protocadherin papc is involved in the organization of the epithelium along the segmental border during mouse somitogenesis. 2003, Pubmed , Xenbase
Richter, The genomic and cellular foundations of animal origins. 2013, Pubmed
Rohani, Variable combinations of specific ephrin ligand/Eph receptor pairs control embryonic tissue separation. 2014, Pubmed , Xenbase
Rohani, EphrinB/EphB signaling controls embryonic germ layer separation by contact-induced cell detachment. 2011, Pubmed
Sawada, Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites. 2000, Pubmed
Schaupp, The composition of EphB2 clusters determines the strength in the cellular repulsion response. 2014, Pubmed
Sentürk, Ephrin Bs are essential components of the Reelin pathway to regulate neuronal migration. 2011, Pubmed
Shi, Biophysical properties of cadherin bonds do not predict cell sorting. 2008, Pubmed , Xenbase
Smith, The EphA4 and EphB1 receptor tyrosine kinases and ephrin-B2 ligand regulate targeted migration of branchial neural crest cells. 1997, Pubmed , Xenbase
Smith, Dissecting the EphA3/Ephrin-A5 interactions using a novel functional mutagenesis screen. 2004, Pubmed
Stein, Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase. 1998, Pubmed
Steinberg, Townes and Holtfreter (1955): directed movements and selective adhesion of embryonic amphibian cells. 2004, Pubmed
Steinberg, Experimental specification of cell sorting, tissue spreading, and specific spatial patterning by quantitative differences in cadherin expression. 1994, Pubmed
Steinberg, Does differential adhesion govern self-assembly processes in histogenesis? Equilibrium configurations and the emergence of a hierarchy among populations of embryonic cells. 1970, Pubmed
STEINBERG, Reconstruction of tissues by dissociated cells. Some morphogenetic tissue movements and the sorting out of embryonic cells may have a common explanation. 1963, Pubmed
Surawska, The role of ephrins and Eph receptors in cancer. 2004, Pubmed
Takahashi, Somitogenesis as a model to study the formation of morphological boundaries and cell epithelialization. 2008, Pubmed
Takeichi, Morphogenetic roles of classic cadherins. 1995, Pubmed
Tepass, Cell sorting in animal development: signalling and adhesive mechanisms in the formation of tissue boundaries. 2002, Pubmed
Theil, Segmental expression of the EphA4 (Sek-1) receptor tyrosine kinase in the hindbrain is under direct transcriptional control of Krox-20. 1998, Pubmed
Unterseher, Paraxial protocadherin coordinates cell polarity during convergent extension via Rho A and JNK. 2004, Pubmed , Xenbase
Wacker, Development and control of tissue separation at gastrulation in Xenopus. 2000, Pubmed , Xenbase
Wang, Xenopus Paraxial Protocadherin regulates morphogenesis by antagonizing Sprouty. 2008, Pubmed , Xenbase
Watanabe, EphrinB2 coordinates the formation of a morphological boundary and cell epithelialization during somite segmentation. 2009, Pubmed
Wimmer-Kleikamp, Elevated protein tyrosine phosphatase activity provokes Eph/ephrin-facilitated adhesion of pre-B leukemia cells. 2008, Pubmed
Winklbauer, Frizzled-7 signalling controls tissue separation during Xenopus gastrulation. 2001, Pubmed , Xenbase
Winklbauer, Cell adhesion in amphibian gastrulation. 2009, Pubmed
Winklbauer, Frizzled-7-dependent tissue separation in the Xenopus gastrula. 2008, Pubmed , Xenbase
Xu, Eph-related receptors and their ligands: mediators of contact dependent cell interactions. 1997, Pubmed
Xu, In vivo cell sorting in complementary segmental domains mediated by Eph receptors and ephrins. 1999, Pubmed
Xu, Boundary formation in the development of the vertebrate hindbrain. 2013, Pubmed
Xu, Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain. 1995, Pubmed , Xenbase
Yamamoto, Mouse paraxial protocadherin is expressed in trunk mesoderm and is not essential for mouse development. 2000, Pubmed , Xenbase
Yamamoto, Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm. 1998, Pubmed
Youssef, Quantification of the forces driving self-assembly of three-dimensional microtissues. 2011, Pubmed
Zimmer, EphB-ephrinB bi-directional endocytosis terminates adhesion allowing contact mediated repulsion. 2003, Pubmed