Regulation of cell adhesion and cell sorting at embryonic boundaries.

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Regulation of Cell Adhesion and Cell Sorting at Embryonic Boundaries François Fagotto1 Department of Biology, McGill University, Montre´al, Que´bec, Canada 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. A Short History of Tissue Separation 2.1 Cell sorting and “affinities” 2.2 Compartments 2.3 The differential adhesion hypothesis 2.4 Differential CAM expression 2.5 Contact inhibition 2.6 Differential interfacial tension 2.7 Direct investigation of basic properties: Embryonic boundaries are not stable physical structures, but the dynamic product of cell–cell interactions 3. Adhesion and Contractility of Embryonic Tissues 3.1 Methodology 3.2 Germ layers 3.3 Notochord–presomitic mesoderm boundary 3.4 Somite and hindbrain segmentation 3.5 Drosophila tissues 3.6 Boundaries reflect abrupt discontinuities in tissue properties 4. Molecular Base of Separation in Vertebrates: Ephrins–Eph Signaling 5. Homophilic Contact Molecules at Embryonic Boundaries 5.1 Immunoglobulin CAMs, Echinoid 5.2 Leucine-rich repeat proteins, FLRT3 5.3 Protocadherins, PAPC 5.4 EpCAM, inducer of tissue mixing 6. Regulation of Tension and Adhesion by Contact Cues 6.1 The action of homophilic regulators 6.2 Putting pro- and antiadhesive activities together 7. Conclusions Acknowledgments References

Current Topics in Developmental Biology ISSN 0070-2153 http://dx.doi.org/10.1016/bs.ctdb.2014.11.026

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2015 Elsevier Inc. All rights reserved.

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Abstract Embryonic boundaries are sharp delimitations that prevent intermingling between different cell populations. They are essential for the development of well-organized structures and ultimately a functional organism. It has been long believed that this process was driven by global differences in cell adhesion strength, or expression of different types of adhesion molecules. The actual picture turns out to be quite different: Boundaries should be viewed as abrupt discontinuities, where cortical contractility is acutely upregulated in response to specific cell surface contact receptors which act as repulsive cues. Cell adhesion is also modulated along the interface, in different ways depending on the type of boundary, but in all cases the process is subordinated to the function of the cortical actomyosin cytoskeleton.

1. INTRODUCTION Development proceeds by subdivision of a single mass of cells into progressively smaller regions, which will eventually give rise to the tissues and organs of the adult organism. The position and size of these regions are determined by the interplay between patterning signals and gene regulatory networks, which have been characterized in detail and show amazing degrees of precision and sophistication. It is perhaps less widely appreciated that the newly determined regions become rapidly physically separated by embryonic boundaries, which impede any future exchange of cells. We will see that the property to separate from an adjacent population is acquired as an inheritable, cell-autonomous property. Without this physical separation, embryonic cells, which divide frequently and are generally highly motile, would be constantly at risk of ending up in the wrong territory. Short term, patterning signals are capable of reprogramming misplaced cells. To constantly maintain a developing structure based on patterning information, however, would be a challenging task, in particular for regions undergoing intense proliferation, such as the insect imaginal discs. It would certainly be close to impossible during large scale movements such as gastrulation. Boundaries largely relieve development from these constraints and allow each separate region to further evolve into complex structures. Consistently, experimental interference with boundary formation causes distortion of the insect wings (e.g., Janody, Martirosyan, Benlali, & Treisman, 2003) and catastrophic defects in the general body plan when it targets the initial vertebrate ectoderm–mesoderm separation (e.g., Rohani, Canty, Luu, Fagotto, & Winklbauer, 2011; Winklbauer, Medina, Swain,

ARTICLE IN PRESS Regulation of Cell Adhesion and Cell Sorting at Embryonic Boundaries

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& Steinbeisser, 2001). The capacity of cells to sort into different populations is thus a fundamental property indissociable with the multicellularity of metazoans, which can be traced to the deepest roots of animal evolution (Wilson, 1907). The phenomenon has fascinated scientists for decades, and despite recent significant advances in understanding its cellular basis, it continues to withhold many mysteries. Regulation of cell–cell adhesion is clearly at the core of the problem, but different views have been opposed as to whether it constituted the main force that caused cell sorting and tissue separation, or whether other parameters were driving the process, such as the contractility of the cell cortex, or regulated repulsive reactions. One of the goals of this review is to provide an updated compilation of the scattered information gathered over the past years on tissue and boundary properties and to confront them to the theoretical models. This exercise will highlight the paucity of evidence for global differences between separating tissues, opposed to a strong case for local high tension as hallmark of all boundaries. I will then focus on the role of ephrin–Eph signaling in building this tension at vertebrate boundaries, discussing the different possible mechanisms through which these repulsive cues may control the tension and adhesive properties of the boundaries. A significant part of this essay will be devoted to the many remaining open issues in the field. One should note in particular that the prototypic compartment boundaries in Drosophila are still in want of upstream cues, which so far have remained inexplicably elusive. As for vertebrates, tissue separation clearly depends on an ephrin–Eph-mediated reaction that resembles classical contact inhibition, but a description of the process only based on this mechanism is likely to be a coarse oversimplification. We will see in particular that ephrin–Eph signaling may have multiple effects beyond simple repulsion. We will also see that even its main target, i.e., stimulation of actomyosin contractility, can have different effects on cell adhesion and boundary properties. Furthermore, several other molecules have been implicated in separation, which bear no obvious direct connection to ephrins and Eph receptors, except for having myosin as common target. Although far less understood, these other components must be taken into account, and I will propose some ideas for their integration in a general model. The most exciting next challenge in my opinion is to move from a coarse description of a generic boundary to the more subtle regulations that likely provide each boundary with the right properties required for each different morphogenetic process.

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2. A SHORT HISTORY OF TISSUE SEPARATION I summarize here the original discoveries of “tissue affinities” and compartment boundaries, and briefly review the major hypotheses that were proposed to explain these phenomena.

2.1. Cell sorting and “affinities” The field was founded by the discovery of the phenomenon of cell sorting: When cells dissociated from different embryonic regions are mixed and left to reaggregate, they initially form a mixed aggregate, but then gradually sort into distinct populations. Remarkably, these cell clusters develop into organized structures that bear the histological signatures of the tissues that would normally derive from the regions from which the cells originated. First observed in sponges (Wilson, 1907), the phenomenon was systematically analyzed in frog embryos (Holtfreter, 1939; Townes & Holtfreter, 1955), and its generality was confirmed in chicken embryos (Moscona & Moscona, 1952). The key characteristics conceptualized by Holtfreter constitute fundamental principles of metazoan organization and continue to bear deep implications for our understanding of the process: First, the fact that mixed aggregates can be produced implies that all cells of an embryos share a common adhesive mechanism. We now know that the main actors are cadherin adhesion molecules. Second, each cell, once determined, acquires an autonomous tissue identity, which can be maintained after cell dissociation and isolation, and even when the cell finds itself surrounded by cells of another type. This identity translates into the capacity to discriminate between neighbors and react by adopting a specific cell behavior, i.e., to group with cells of the same type. Holtfreter named this property “tissue affinity” (Holtfreter, 1939). The nature of the mechanisms that mediate the recognition of self (homotypic contacts) and nonself (heterotypic contacts) and drive the appropriate response remains the central question in the field.

2.2. Compartments A second key discovery was made by Drosophila geneticists, who observed that the expansion of proliferating clones in the embryo blastoderm and in the larval imaginal discs was restricted by invisible yet sharp and fully impermeable partitions. Thus, these epithelial sheets were subdivided into “compartments,” which were delimited by stably inherited “boundaries”


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(Garcia-Bellido, Ripoll, & Morata, 1973; Lawrence & Green, 1975; Lawrence, Green, & Johnston, 1978; Morata & Lawrence, 1978; Martinez-Arias & Lawrence, 1985; reviewed in Dahmann & Basler, 1999). Similar compartment boundaries were subsequently found in the vertebrate embryo, e.g., in the brain and limb buds (Altabef, Clarke, & Tickle, 1997; Dahmann, Oates, & Brand, 2011; Fraser, Keynes, & Lumsden, 1990; Zervas, Millet, Ahn, & Joyner, 2004).

2.3. The differential adhesion hypothesis Steinberg had the revolutionary idea to consider cell–cell adhesion from a physical point of view. He noticed that tissue explants behaved very much like liquids, from which he conceived the following analogy: Individual cells would correspond to the molecules of a liquid and cell adhesion to the cohesive bonds between these molecules. The principle of liquid surface tension predicted with astonishing accuracy many of the configurations adopted by cells and tissues. For instance, single cells and pieces of tissues in isolation invariably round up, thus minimizing the surface exposed to the medium, just as a drop of oil in water. When placed against an adhesive surface, whether matrix or cells, they spread, or in biophysical terms they “wet” the surface. When two groups of cells are put into contact, they either coalesce or, on the contrary, they remain fully separated, again similar to the behavior of immiscible liquids. Based on this analogy, Steinberg proposed that quantitative differences in cell adhesion were sufficient to explain cell sorting and thus tissue separation, a model that was named the differential adhesion hypothesis (DAH) (Davis, Phillips, & Steinberg, 1997; Steinberg, 1970, 1978). Sorting in mixed aggregates based on DAH was demonstrated in vitro, using cells expressing different cadherin levels (Foty & Steinberg, 2004, 2005). We will see that DAH in its original form does not seem to explain tissue separation in the embryo. However, the concept of representing morphogenesis based on a simple combination of adhesive and tensile forces exerted on the cell surface (i.e., cell membrane and its actin cortex) arguably constituted the most influential ideas in the field of morphogenesis. The analogy to liquid surface tension remains a very successful way to simulate the behavior of cells and tissues (Foty & Steinberg, 2004; Lecuit & Lenne, 2007; Manning, Foty, Steinberg, & Schoetz, 2010).

2.4. Differential CAM expression As one entered the cloning era, and cellular functions could finally start to be assigned to particular gene products, biophysical considerations on cell

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sorting were temporarily left aside, and attention shifted naturally to a resolutely molecular perspective. The two first cell adhesion molecules (CAMs), N-CAM and E-cadherin (Edelman, 1986), soon joined by many other CAMs, were found to preferentially bind to themselves (Inuzuka, Miyatani, & Takeichi, 1991; Matsuzaki et al., 1990; Nose, Nagafuchi, & Takeichi, 1988; Steinberg & Takeichi, 1994). This homophilic adhesive property and the striking tissue-specific expression of most CAMs seemed to provide a perfect explanation for tissue segregation (Takeichi, 1995). According to this model, individualization of each tissue would rely on the expression of a particular kit of CAMs (reviewed in Oda & Takeichi, 2011). This hypothesis received a broad acceptance among developmental biologists, entered the textbooks, and reigned almost undisputed until recently. Evidence supporting this hypothesis however remained scarce (Inoue et al., 2001; Price, De Marco Garcia, Ranscht, & Jessell, 2002). More accurate methods led to a reevaluation of the concept of homophilic binding. Notably, two studies showed that cells which expressed two different cadherins—at similar levels—failed to sort (Duguay, Foty, & Steinberg, 2003; Niessen & Gumbiner, 2002). In vitro measurements confirmed that binding is not strictly homophilic (Katsamba et al., 2009; Ounkomol, Yamada, & Heinrich, 2010; Prakasam, Maruthamuthu, & Leckband, 2006; Shi, Chien, & Leckband, 2008; Shimoyama, Tsujimoto, Kitajima, & Natori, 2000), and functional N-/E-cadherin heterotypic interactions were detected in vivo (Straub et al., 2011). A role for differential expression of homophilic CAMs in separation remains uncertain. Note that several studies hint at specific functions for different cadherin cytoplasmic tails in regulating signaling pathways (e.g., Schafer, Narasimha, Vogelsang, & Leptin, 2014; Seidel, Braeg, Adler, Wedlich, & Menke, 2004; Wheelock, Shintani, Maeda, Fukumoto, & Johnson, 2008), which could provide an alternative explanation for the multiplicity of cadherins and for their mosaic expression.

2.5. Contact inhibition Contemporarily to the discovery of CAMs, the identification of ephrins and Eph receptors as cell surface repulsive cues (Flanagan & Vanderhaeghen, 1998) pointed to a completely different model for tissue separation, based on contact inhibition of migration, a phenomenon that had been originally


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proposed based on observations on cell lines (Abercrombie, 1967). Although ephrins and Ephs have mostly been studied for their role in development of neuronal networks and in angiogenesis, they are also widely expressed in early embryos (see Fagotto, Winklbauer, & Rohani, 2014 for review). The striking complementarity of ephrin/Eph expression patterns in the developing hindbrain suggested a role in segmentation, which was supported by functional data (Xu, Alldus, Holder, & Wilkinson, 1995). Similar observations were then made for segmentation of the somites (Durbin et al., 1998). This model, although rather straightforward, remained less popular than the cadherin-based models, perhaps because it was until recently limited to the two vertebrate segmentation processes. However, ephrins and Ephs are now known to control other types of boundaries, including between germ layers (Fagotto, Rohani, Touret, & Li, 2013; Park, Cho, Kim, Choi, & Han, 2011; Rohani et al., 2011), and emerge as major regulators of separation in vertebrates.

2.6. Differential interfacial tension The turn of the century witnessed strong revival of biophysical approaches to developmental processes. Myosin II-mediated contractility was known to be involved in most aspects of cell adhesion and motility, and it became apparent that this parameter could explain quite a few aspects of morphogenesis. The importance of cell cortex contractility had been already highlighted in a seminal critical analysis of DAH by Harris (1976). Adhesion and contractility were formally integrated into a broader theory, named differential interfacial tension hypothesis (DITH) (Brodland, 2002; Brodland, Yang, & Sweny, 2009). DITH was used to interpret sorting of zebrafish germ layers, and it was concluded that the system was dominated by differences in cortical tension (Krieg et al., 2008; Maitre et al., 2012). Note that an old puzzle remains: When tested in reaggregation experiments, ectoderm cells always sorted toward the center, endoderm to the periphery, and mesoderm in between. This configuration fitted perfectly with DITH, but was opposite to the normal organization of embryos. This discrepancy may at least partly be due to the fact that the dissected explants are artificially exposed to the medium, which causes a strong surface tension, whereas in the embryo, the tissues are wrapped in an outer polarized epithelial layer (outer ectoderm in Xenopus, enveloping layer in zebrafish), which alleviates internal tensions (Ninomiya & Winklbauer, 2008).

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2.7. Direct investigation of basic properties: Embryonic boundaries are not stable physical structures, but the dynamic product of cell–cell interactions What about the extracellular matrix (ECM)? It is a robust isolator of adult organs. Would not it be an obvious candidate for the separation of embryonic tissues? ECM is indeed deposited soon after separation and contributes to consolidate the boundaries ( Julich, Mould, Koper, & Holley, 2009; Koshida et al., 2005), but it does not appear to participate to the initial boundaries, which are clearly not physical fences. A boundary is in fact only impermeable insofar as it keeps cells of the two populations within their respective territories. Missorted cells, on the contrary, can freely cross it to reintegrate the proper tissue. This has been unambiguously demonstrated by following migration of single cells in mosaic notochords (Fagotto et al., 2013; Reintsch, Habring-Mueller, Wang, Schohl, & Fagotto, 2005) and rhombomeres (Calzolari, Terriente, & Pujades, 2014). Further support came from in vitro reconstitution of the ectoderm– mesoderm boundary. This assay, based on the simple juxtaposition of tissue explants (Wacker, Grimm, Joos, & Winklbauer, 2000), played a significant role in the recent progress in uncovering the mechanism of tissue separation (Ibrahim & Winklbauer, 2001; Medina, Swain, Kuerner, & Steinbeisser, 2004; Rohani et al., 2011; Rohani, Parmeggiani, Winklbauer, & Fagotto, 2014; Wacker et al., 2000; Winklbauer et al., 2001). It thus became clear that cells react almost instantaneously to contacts with a tissue of the same or of the other cell type, “melting” selectively in the former within minutes, while remaining stably separated from the latter. This separation behavior (Wacker et al., 2000) can be observed for single dissociated cells laid on an explant (Wacker et al., 2000) and even between two single dissociated cells (Rohani et al., 2014). These observations are important because they firmly validate Holtfreter’s original interpretation that tissue separation relies on cellautonomous properties. A key experimental consequence of this property is the possibility to study the mechanisms underlying tissue separation in vitro using isolated cells (Rohani et al., 2014). What is then the mechanism that allows single cells to find their way and eventually gather with cells of the same type? Classical DAH and DITH state that boundaries result from the juxtaposition of two cell populations displaying global differences in cell–cell adhesion, cortical contractility, or both. Alternatively, cell populations may express different types of adhesion molecules. Contact inhibition does not presume of such differences, but


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predicts local effects at heterotypic contacts. One would thus expect that the analysis of the tissue properties would provide support for one or the other model.

3. ADHESION AND CONTRACTILITY OF EMBRYONIC TISSUES There are four main models of boundaries in vertebrates: The mesoderm is first separated from the ectoderm (Fig. 1A). Its most axial portion then splits from the paraxial (or presomitic) mesoderm (PSM) to form the notochord (Fig. 1B). The PSM eventually segments into somites (Fig. 1D). For the developing neural tissue, the best studied process is the segmentation of the hindbrain into seven rhombomeres (Fig. 1C). In Drosophila, the two main models are the parasegment boundaries and the compartment boundaries of imaginal discs (Fig. 1F). I have also included the particular case of the egg appendages, which has been studied for the function of the Echinoid protein (Fig. 1E, see below). I have compiled in this section the available information on adhesion and contractility, which is fragmentary and heteroclite, making comparison between models still rather rash. A brief overview of these methods will be useful to define what has been actually measured.

3.1. Methodology Cell adhesion can be estimated in vitro by determining the degree of reaggregation of dissociated cells, resistance of tissues to mechanical dissociation or adhesion of single cells plated on immobilized recombinant CAMs (e.g., Yap, Niessen, & Gumbiner, 1998). Adhesion can also be directly measured as the force necessary to pull apart a pair of cells using AFM or dual aspiration pipette (Krieg et al., 2008; Maitre et al., 2012). These two methods can also be used to measure cortical stiffness of single cells (Krieg et al., 2008; Maitre et al., 2012). The advantage of in vitro measurements is a better controlled environment and reduced parameter complexity. A caveat of these single cell measurements is the fact that cells are bound to actively react to the artificial environment, including the large free surface exposed to the medium, and in the case of AFM, the type of substrate used to hold the cells (inert or adherent, chemical or biological). Thus, it is important to validate these measurements in vivo. Cortical tension can be estimated by laser ablation (e.g., Landsberg et al., 2009). Biosensors are being developed for direct force

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A

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Germ layers

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Dorsal endoderm dorsal endoderm

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Figure 1 Embryonic boundary models. The enlarged view of each boundary provides information about cadherin distribution (C-cad, N-cad, and DE-Cad), actomyosin structures the cell surface, and the identity and localization of cell surface cues functionally implicated in separation (Ephrins, Eph receptors, PAPC, and Echinoid). The boundary is represented as a dashed line. (A) Separation of the dorsal ectoderm and mesoderm in the early Xenopus gastrula. The process depends on a complex network of partially selective ephrin–Eph pairs setting bidirectional signals across the boundary. PAPC also participate in a parallel and less understood pathway. Differences in C-cadherin levels and actomyosin activity are indicated. Gaps between the two tissues represent the occurrence of dynamic cycles of detachments and reattachments. (B) Separation of the most axial mesoderm (notochord) from the paraxial or presomitic mesoderm (PSM). A similar ephrin–Eph network controls separation. PAPC is restricted to the


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measurement in live tissues (Borghi et al., 2012; Kuriyama et al., 2014) but they have not yet been used in the context of tissue separation. Indirect information can also be obtained from the myosin distribution (e.g., Calzolari et al., 2014; Landsberg et al., 2009; Rohani et al., 2014) and from the relative cadherin levels and their degree clustering (Fagotto et al., 2013). Another simple but quite informative criterion is the cell geometry, which is predicted to directly reflect the strength and direction of forces exerted on the cell: At the two extremes, cells with high adhesion tend to maximize their contacts and adopt a hexagonal shape, while highly contractile/low adhering cells are close to round. In principle, a rather precise map of local tensions may be drawn using refined morphological criteria, including membrane curvature, angles at cell vertices, or cell elongation (e.g., Brodland et al., 2014, 2009; Lynch, Veldhuis, Brodland, & Hutson, 2014; Manning et al., 2010). Finally, cohesion is a general physical property integrating adhesion and tension, which can be measured for whole tissue explants (David et al., 2014; David, Ninomiya, Winklbauer, & Neumann, 2009; Kalantarian et al., 2009; Luu, David, Ninomiya, & Winklbauer, 2011; Ninomiya & Winklbauer, 2008). PSM, but a role in formation of this boundary has not yet been demonstrated. Myosin activation and C-cadherin levels are similar on both sides. Myosin is hyperactivated along the boundary and cadherin adhesion is inhibited, which is indicated by the space separating the two tissues. Eventually, the gap is permanently stabilized by secretion of a thick layer of extracellular matrix. (C) Hindbrain segmentation. The hindbrain becomes segmented in seven rhombomeres r1–7. The process depends on several ephrins and Eph receptors. The central segments r3–5 presented here express complementary sets of multiple ligands and receptors. Expression in the other segments is more complex. All segments express N-cadherin homogenously. Actin and myosin are enriched along the boundaries. (D) Somitogenesis. The PSM becomes progressively segmented, starting anteriorly, under the control of ephrinB2, expressed in the posterior half of the newly forming somite, and EphA4, complementary expressed in the most anterior portion of the unsegmented PSM. PAPC, also expressed anteriorly, contributes to the process. Eventually, somites become completely isolated from each other (empty space), through deposition of extracellular matrix. (E) Drosophila dorsal appendages. These two cuticular structures of the Drosophila egg are secreted by extensions of the follicular epithelium. Their primordia are delimited as two regions devoid of Echinoid. The juxtaposition of Echinoid-positive and -negative cells produces a smooth interface with high actomyosin and low DE-cadherin levels. (F and F0 ) Drosophila compartment boundaries. The embryonic blastoderm (F) and the wing imaginal disc epithelium (F0 ) are two examples of epithelia partitioned by sharp compartment boundaries. Increased cortical tension is consistent with accumulation of actomyosin fibers along the interface, but upstream cues have not yet been identified (?).

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3.2. Germ layers The best studied systems are the germ layers of lower vertebrates, fish and amphibians. There are discrepancies between studies, which partly reflect intrinsic specificities of the two models, but mostly differences in the type of approach and in the interpretation of the data. Thus, there is the need to discuss the two cases in some detail. In zebrafish, adhesion, measured by AFM, was found to be lowest for ectoderm, intermediate for endoderm, and highest for mesoderm cells (Krieg et al., 2008). Consistently, E-cadherin levels were reported to be higher in the mesoderm (Montero et al., 2005; Ulrich et al., 2005). For cortical stiffness, AFM measurements gave the highest value for ectoderm, intermediate for mesoderm, and lowest for endoderm (Krieg et al., 2008). Inference of the adhesive and tensile components of interfacial tension highlighted the predominant influence of cortical tension (Maitre et al., 2012). Results from cell sorting experiments also yielded configurations consistent with cortical tension serving as the main driving force (Krieg et al., 2008). The caveat of these conclusions is that the interfacial properties of heterotypic contacts were estimated based on DITH, which assumes that these properties should be somewhat intermediate between those of the two tissues. Yet heterotypic adhesion had been in fact measured in the original report, although not commented, and values were lower than for homotypic adhesion (Krieg et al., 2008), a result that did not fit with DAH/DITH. Other parameters of the boundary interface have not yet been explicitly studied, but some information can be extracted from published images: Cadherin staining showed no particularity at the boundary (Krieg et al., 2008), but there seems to be some myosin enrichment (Maitre et al., 2012). The angles formed by the cell edges appear close to 90° along the boundary (Fig. 1F in Krieg et al., 2008), which is a typical configuration that reflects high interfacial tension. Based on these various criteria, interfacial tension at heterotypic contacts must be significantly higher than in each of the two tissues. Although this conclusion awaits confirmation, it would be fully consistent with the properties of the other boundaries, including the Xenopus ectoderm–mesoderm boundary. Cortical properties seem to be shared between zebrafish and Xenopus embryos: Xenopus ectoderm cells are also stiffer than mesoderm cells (AFM unpublished data, Canty and Fagotto), consistent with significantly higher levels of activated myosin (Rohani et al., 2014). The parallel with zebrafish does not hold for adhesion, though cadherin levels were found


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to be higher in the ectoderm (Angres, M€ uller, Kellermann, & Hausen, 1991; Ogata et al., 2007), and ectoderm performed better than mesoderm in all adhesion assays, reaggregation (Brieher & Gumbiner, 1994), adhesion to immobilized recombinant cadherin extracellular domain (Zhong, Brieher, & Gumbiner, 1999), and dissociation assay (Canty and Fagotto, unpublished data). However, a more detailed analysis of different mesoderm subregions showed that this tissue was not homogenous, and that the presumed lower adhesion of this layer only applied to the anterior region, while the posterior chordomesoderm appeared similar to the ectoderm in all these aspects (Chen, Koh, Yoder, & Gumbiner, 2009; Fagotto, unpublished observations; Winklbauer, 2009). This was corroborated by measurements of surface tension of ectoderm, anterior and posterior mesoderm explants (Kalantarian et al., 2009; Ninomiya & Winklbauer, 2008; Winklbauer, 2009). The lack of correlation between tissue properties and separation behavior is incompatible with a role of DAH/DITH. The distinct characteristics of each region of the embryo probably reflect specific requirement for other aspects of gastrulation, which also explains differences between zebrafish and Xenopus. As for the boundary, it showed unique properties: heterotypic contacts across the interface were constantly disrupted by transient but dramatic repulsive reactions followed by a phase of relaxation and reattachment (Rohani et al., 2011). This behavior, which was never observed within the tissues, suggested a mechanism of contact inhibition. We demonstrated that it was indeed directly controlled by ephrins and Eph receptors interacting across the boundary (Rohani et al., 2011). Detachments were found to correlate with bursts of Rho activation at heterotypic contacts (Rohani et al., 2011), consistent with myosin accumulation along the boundary (Rohani et al., 2014).

3.3. Notochord–presomitic mesoderm boundary Both tissues showed identical cadherin and myosin staining and similar cell shapes (Fagotto et al., 2013). The boundary, however, was characterized by strong accumulation of actomyosin structures and intense membrane blebbing, indicative of extreme cortical tension, and most interestingly, almost complete lack of cadherin clusters (see below). All these particularities depended on myosin activity, which, similar to the ectoderm–mesoderm case, was activated downstream of ephrin–Eph signaling (Fagotto et al., 2013).

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3.4. Somite and hindbrain segmentation Somitogenesis is a particularly complex process, where segmentation is accompanied by other morphogenetic movements, including compaction and epithelization, a 90° rotation of cell alignment in the lower vertebrates, and differentiation of somite subregions. This complexity makes it difficult to distinguish those parameters that are directly involved in separation sensus stricto from those reflecting other events. N-cadherin and N-CAM have distinct distributions in the forming somites (Duband et al., 1987), but these patterns appear to relate to epithelization. Another classical cadherin, cadherin 11 is specifically expressed in somites, starting first in the posterior half of the newly formed somite (Kimura et al., 1995), but its appearance is also probably more relevant for subsequent somite cohesion, not for the initial segmentation. Loss of N-cadherin in mice did not impair segmentation, but on the contrary led to somite fragmentation, a phenotype that was enhanced in double N-cadherin/cadherin 11 knockouts (loss of cadherin 11 alone had no effect) (Horikawa, Radice, Takeichi, & Chisaka, 1999). Somite segmentation is actually resistant to general interference with type I cad (Giacomello et al., 2002). Actin and myosin distribution was examined in zebrafish. Levels were higher along the somatic boundaries, but homogenous within the forming somites ( Julich et al., 2009). Less is known about the properties of the rhombomeres. The hindbrain expresses N-cadherin homogenously. Cadherin 6, however, is expressed specifically in rhombomeres 6 and 7, but its function is not known (Inoue, Asami, & Inoue, 2008; Inoue, Chisaka, Matsunami, & Takeichi, 1997). Similar to the somites, actin and myosin are enriched at the boundaries but homogeneous within the rhombomeres (Calzolari et al., 2014). As already mentioned, both segmentation processes are known to depend on ephrin–Eph signaling, which readily explains the accumulation of actomyosin along the boundary (Calzolari et al., 2014).

3.5. Drosophila tissues In Drosophila, all criteria examined so far argue that tissue properties are similar on both sides of compartment boundaries, whereas cortical tension is higher along the boundaries: Cells have identical polygonal shapes on both sides, but vertex angles approach 90° along the smooth boundary interface (Aliee et al., 2012). Myosin levels are indistinguishable between the tissues, but significantly higher at the boundary (Aliee et al., 2012; Landsberg et al., 2009; Laplante & Nilson, 2006; Major & Irvine, 2006; Monier,


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Pelissier-Monier, Brand, & Sanson, 2010). Laser ablation in the wing imaginal disc confirmed that cortical tension is identical in anterior and posterior compartments, but higher along the boundary (Landsberg et al., 2009). Cell adhesion has not yet been examined.

3.6. Boundaries reflect abrupt discontinuities in tissue properties We have seen that, in most cases, the cell populations show no obvious differences in terms of adhesive or tensile properties. The only exception are the ectoderm and the mesoderm, but even this case hardly supports DAH/DITH, since there is no consistency between fish and frog tissues and high heterogeneity between mesoderm subregions. According to DAH/DITH, tissue interfaces should represent some kind of middle point (average) between tissue properties, which certainly does not predict for the remarkable characteristics of the actual boundaries. In fact, simply considering the stereotypical cell alignment and smooth interface of all boundaries from a biophysical point of view leads to the inescapable conclusion that boundaries must be sharp discontinuities. Although direct evidence for high interfacial tension so far has only been obtained on the wing compartment boundary (Landsberg et al., 2009), we have seen numerous indications that this is a general property of embryonic boundaries, including actomyosin enrichment (all boundaries), repulsive behavior between ectoderm and mesoderm cells in Xenopus (Rohani et al., 2011, 2014), and blebbing along the notochord–PSM boundary (Fagotto et al., 2013) (see below and Fig. 5). Functional evidence for the importance of contractility has been demonstrated in all cases, either by biochemical inhibition of myosin function (Drosophila imaginal discs, Landsberg et al., 2009; vertebrate ectoderm– mesoderm, Rohani et al., 2011, 2014; notochord, Fagotto et al., 2013; hindbrain, Calzolari et al., 2014) or by myosin optical inactivation (Drosophila parasegments, Monier et al., 2010). Retrospectively, it makes sense that boundary formation must be driven by a local and robust mechanism. In a DAH/DITH situation, sorting would rely on probing differences between different neighbors, which, in order to produce fast and sharp separation, would need to be extreme. In other words, such a mechanism would work only if one of the tissues is extremely compact and the other one extremely loose. These conditions would be cripplingly limiting for embryonic development. They would be incompatible, for instance, with morphogenesis of the axial mesoderm, since both the

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notochord and the paraxial mesoderm undergo simultaneously convergent extension, for which they need essentially identical biophysical properties. Creation of a local discontinuity uncouples separation from other morphogenetic requirements. The next exciting question is about the mechanisms which create this discontinuity. We have seen that boundaries form as a result of local reactions at heterotypic contacts, they rely on cell-autonomous components, and myosin is systematically overactivated. This is highly reminiscent of the established repulsive mechanisms active in neuronal guidance. The potential cues responsible for setting Drosophila compartment boundaries have still not been identified. The case is however largely elucidated in vertebrates, where ephrin–Eph receptors play a major role. These molecules are well known to activate myosin through Rho, but we will see that the consequences of this activity on cell adhesion are potentially complex. Before discussing these reactions, it may be useful to remember some of the general factors that are thought to regulate adhesion, in particular those related to the actin cytoskeleton. 3.6.1 Principles of regulation of cell adhesion This brief overview is restricted to classical cadherins, which account for most adhesion in all metazoans and are far better understood than any other CAM. The simplest and most common experimental strategy to modify cell–cell adhesion is to change cadherin levels (e.g., Dahmann & Basler, 2000; Foty & Steinberg, 2005). Yet, it is not clear that expression levels constitute a major mode of regulation in vivo. In the embryo, manipulation of cadherin levels has surprisingly mild effects on gastrulation movements, cell sorting being particularly resilient (Fagotto et al., 2013; Ninomiya et al., 2012; Reintsch et al., 2005). Similar to integrins, cell–cell (or “trans”) interactions between cadherins are weak, and efficient adhesion involves clustering (Brasch, Harrison, Honig, & Shapiro, 2012). Clustering is due to the intrinsic property of the cadherin extracellular domains to form both cis and trans interactions, with additional contributions from the transmembrane domain and from the cytoplasmic tail. The size of the clusters is considered to relate to adhesive strength, and may be regulatable, for instance, through p120catenin (Yap et al., 1998). Cadherin stability at the cell surface is an important determinant of adhesion, which has been implicated in a variety of morphogenetic processes (e.g., Kuriyama et al., 2014; Levayer, Pelissier-Monier, & Lecuit, 2011).


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Several factors have been identified that control the rate of cadherin internalization: Cadherin endocytosis is antagonized by p120catenin binding to cadherin cytoplasmic tail (Ishiyama et al., 2010; Nanes et al., 2012). Surface localization is also strongly stabilized by trans cell–cell adhesive interactions. However, the largest clusters are also the favored targets for internalization (Truong Quang, Mani, Markova, Lecuit, & Lenne, 2013). Cadherins can also be inactivated by shedding of the extracellular domain, performed by ADAM metalloproteases (Maretzky et al., 2005; Reiss et al., 2005). The actin cytoskeleton has a predominant influence on adhesion. Its effects are diverse and complex, and here I will only touch upon a few aspects, focusing on actomyosin contractility: Mechanical coupling between cadherin adhesions and actin structures is considered essential to cell adhesion, similar to the link between focal contacts and stress fibers for cellmatrix adhesion. Like integrin-matrix adhesion, cadherin adhesion involves reinforcement of the actin connection, which can occur via the mechanosensing molecules α-catenin and vinculin (Huveneers et al., 2012; Ladoux et al., 2010; le Duc et al., 2010; Yonemura, Wada, Watanabe, Nagafuchi, & Shibata, 2010), or by myosin II-dependent regulation of actin dynamics (Engl, Arasi, Yap, Thiery, & Viasnoff, 2014). On the other hand, the establishment of new adhesive bonds requires cell protrusive activity, a process that is countered by Rho. More generally, contractility of the actomyosin cell cortex represents a force that works toward minimizing the cell contact surface, thus antagonizing cell adhesion (Fig. 2A). There are two ways to consider these two antagonistic activities, which I will call here proadhesive and antiadhesive (Fig. 2). In one model, two distinct actomyosin pools would be in charge of the tension on the cell cortex and of cadherin anchoring. Optimal adhesion would result from the balance of these two opposite activities (Fig. 2B). There is experimental evidence supporting the existence of these two pools (e.g., Cavey, Rauzi, Lenne, & Lecuit, 2008) (Fig. 2A and B), but details on their composition, distribution, and interplay remains unclear. In the second model, the cadherin–actomyosin structures are integrated with the cortical cytoskeleton in a single tensile network (Fig. 2A). The existence of such continuum between various actin structures was recently visualized by high-resolution electron microscopy (Hoelzle & Svitkina, 2012). The pro- and antiadhesive roles could then correspond to two different regimes of a single, intrinsically bimodal, system (Fig. 2B0 ). The mechanosensing α-catenin–vinculin interaction could provide an underlying molecular mechanism: Weak tension is needed to expose the vinculin binding site of α-catenin, but too high tension

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Figure 2 Models for integration of actomyosin contractile structures and cell–cell adhesion. (A) Independent versus integrated systems. Actomyosin may form two independent types of tensile structures responsible for contractility of the cell cortex (1) or reinforcement of adhesions (2). The former may be locally regulated, for instance, along nonadhesive membrane domains (1), or on the contrary in the proximity of adhesive sites (10 ). Alternatively, mechanical connections between the adhesive sites and the cytoskeleton may be integrated with the cell cortex (3). Global changes in actomyosin contractility may propagate to multiple pools (4). (B) Bimodal dependence of adhesion on tension. Available data argue for an optimal tension, beyond which increasing tension leads to decreased adhesion. This optimum may be defined by the overlap of the two opposite activities of cortical contractility (1) and adhesion reinforcement (2) (B). Alternatively, a single contractile system (3) may be intrinsically bimodal (B0 ).

unfolds it (Yao et al., 2014). The actual situation is likely to be a combination of these two models. We will see below that both pro- and antiadhesive effects must be taken into account to explain the properties of embryonic boundaries. Cell–cell adhesion also feeds back on cortical tension: Indeed, cadherin engagement induces local downregulation of Rho activity and decreased contractility along the cell contacts (Yamada & Nelson, 2007). This


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regulation is essential, as the tension of the cortex along a free cell surface would be too high to be overcome by the strength of cadherin–cadherin bonds (David et al., 2014; Maitre et al., 2012). Cortical actin can also influence adhesion more indirectly, for instance, by restricting cadherin lateral diffusion by the process of corralling or by cadherin tethering, which, even in the absence of cadherin–cadherin engagement, contribute to cadherin stabilization and clustering (Hong, Troyanovsky, & Troyanovsky, 2013; Sako, Nagafuchi, Tsukita, Takeichi, & Kusumi, 1998). Furthermore, actin dynamics are also involved in the process of endocytosis, and thus can also control cadherin cell surface distribution. The complexity of these multiple functions explains why interference with general cytoskeleton regulators such as RhoGTPases has often yielded inconsistent results. Note that most of what we know about cadherin adhesion comes from studies on epithelial cells. The fact that these cells adhere through highly specialized adherens junctions adds extra layers of complexity. The study of simpler adhesion clusters (spot junctions or puncta) present in nonepithelial cells, including embryonic mesoderm, should help clarify the basic regulatory mechanisms of cadherin-mediated adhesion.

4. MOLECULAR BASE OF SEPARATION IN VERTEBRATES: EPHRINS–EPH SIGNALING Ephrins and Eph receptors are widely expressed in all embryonic tissues, including early germ layers. The fact that highly localized patterns delineated specific structures pointed to a role in segmentation (Xu et al., 1995; Xu & Wilkinson, 1997). It turns out that ephrin–Eph signaling plays a major role for all vertebrate boundaries examined so far, ectoderm– mesoderm (Park et al., 2011; Rohani et al., 2011, 2014), notochord–paraxial mesoderm (Fagotto et al., 2013), somites (Davy & Soriano, 2007; Durbin et al., 1998; Watanabe, Sato, Saito, Tadokoro, & Takahashi, 2009), hindbrain rhombomeres (Calzolari et al., 2014; Xu et al., 1995), and eye field (Cavodeassi, Ivanovitch, & Wilson, 2013) (see Batlle & Wilkinson, 2012; Fagotto, 2014; Fagotto et al., 2014 for reviews). The identity, distribution, and function of various ephrins and Eph receptors were recently reviewed in detail (Fagotto et al., 2014). I summarize here the key points and focus the discussion on the effect of ephrin–Eph signaling on the adhesive properties of the boundary. Gain- and loss-of-function experiments have solidly established a requirement for these molecules in all of the above-mentioned models.

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Their interaction across the boundary is expected to trigger contraction of the actomyosin cortices, leading to mutual cell retraction/repulsion. There is evidence that these events do occur at the boundary (e.g., Calzolari et al., 2014; Rohani et al., 2011), and this simple mechanism explains satisfactorily how cell mixing is inhibited. However, there is far more complexity in these systems, which must be addressed in order to understand how separation may truly work and, moving one step further, start to distinguish mechanistic similarities and differences between different types of boundaries. Ephrins and Eph receptors represent large families, subdivided into A and B subfamilies. It was long assumed that A ligands bind promiscuously to A receptors and B ligands to B receptors. There are, however, strong differences in affinity between individual pairs, even within subfamilies (BlitsHuizinga, Nelersa, Malhotra, & Liebl, 2004; Pabbisetty et al., 2007). Thus, only some of the possible ephrin–Eph combinations may pair efficiently enough to generate physiologically significant signals. We have recently confirmed such partial selectivity in the context of embryonic tissues (Rohani et al., 2014). Ephrin and Eph expression patterns in early embryos show an unexpected sophistication, comparable to what is found at later stages in the developing brain. The simplest case, where one tissue expresses one ephrin and the adjacent tissue a cognate Eph receptor, is rare. Instead, most tissues express more than one ephrin and/or one Eph, creating various degree of complexity: Segregation of the eye field is a relatively simple example, where the eye field itself expresses several ephrins and the surrounding neural tissue expresses a set of complementary to Eph receptors, all likely to contribute to strong repulsion at the interface (Cavodeassi et al., 2013). In most other cases, however, both ligands and receptors are coexpressed (reviewed in Fagotto et al., 2014). The output is then difficult to predict, since functional ephrin–Eph interactions can occur both within the tissues and across the boundaries. The logic of these networks was elucidated for the Xenopus dorsal ectoderm–mesoderm boundary (Rohani et al., 2014). Here, the system could be satisfactorily explained based on two parameters, partial selectivity of ephrin–Eph pairing and partial complementary of expression (Fig. 3): Despite widespread ephrin and Eph expression, more ephrin–Eph interactions could form at the tissue interface than within each tissue, due to the asymmetric expression of key pairs, such as ephrinB3 and EphA4. The resulting global signal is low in the ectoderm, moderate in the mesoderm, but strongest across the boundary, where it overcomes cell–cell adhesion


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EphB2 EphB4 EphA4

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Figure 3 Partial selectivity in ephrin–Eph pairing serves as an identity code for vertebrate embryonic tissues. (A) Selective interactions between ephrinB ligands and cognate receptors. (B) Ephrin and Eph network in the Xenopus dorsal ectoderm and mesoderm. Tissues are represented by single boxes. Each cell of a tissue expresses the full set of ligands and receptors. The relative abundance of each component is represented by the font size. The lines represent ephrin–Eph interactions between cells of the same tissue and across the boundary. The thickness of the lines corresponds to the relative signal intensity, which depends on the ligand–receptor binding affinity and of the abundance of the two partners. Multiple interactions can form in the mesoderm. On the contrary, few combinations are available in the ectoderm, despite high expression of several components. The strongest global output is generated at the tissue boundary, and only there it is sufficient to cause overt cell–cell deadhesion.

and results in overt repulsion. The occurrence of weaker repulsive reactions in the mesoderm was revealed when adhesion was experimentally weakened by partial cadherin depletion (Rohani et al., 2014). Similar principles appear to account for ventral ectoderm–mesoderm and notochord–paraxial mesoderm separation (Rohani et al., 2014) and are likely to apply to other boundaries, such as somites and rhombomeres, which also show some partial coexpression of ligands and receptors. Ephrin–Eph signaling is usually defined as repulsive, based on the typical reaction of neurites contacting a negative cue. Ephrin–Eph signaling is however complex and its effects on cell behavior diverse, which will be here briefly discussed in the context of tissue separation (Fig. 4). The classical reaction, often defined as “collapse” for neuron growth cones, typically involves RhoA activation as the major target. The process of collapse is still relatively poorly characterized. It may involve increased

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Figure 4 Regulation of cell–cell adhesion by ephrin–Eph signaling. (A) Myosindependent antiadhesive mechanisms. Ephrin–Eph signaling generates high cortical tension through activation of the Rho pathway. Contraction of the cell cortex pulls cells apart, exerting a physical force antagonistic to cell–cell adhesion. Exacerbated cortical tension can further lead to transient rupture of the cortex–membrane connections,


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actomyosin contractility and/or stimulation of actin depolymerization in the lamellipodium. Within a compact tissue, a simple action of ephrin–Ephdependent boost in cortical actomyosin contractility is to pull on the adhesive contacts, eventually disrupting them (Fig. 4A). Time lapses of the Xenopus ectoderm–mesoderm boundary are consistent with this mechanism (Rohani et al., 2011). The observed cycles of alternating detachments– reattachments may be described as follows (Fig. 5B): Tight cadherin contacts favor ephrin–Eph interactions. Rho activation builds up, and eventually triggers repulsion and deadhesion. Once cells apart, the repulsive signal decays, contractility decreases, and protrusions are reemitted until adhesive contacts are reestablished. This system is well suited for the ectoderm– mesoderm boundary, which must accommodate tissue separation with the need of the mesoderm to migrate using the ectoderm as a cell substrate. Note that the details of the process may be more sophisticated than described here: Ephrin–Eph signaling may contribute to cell detachment not only by increasing cortical tension, but could also involve cointernalization of cadherin clusters with ephrin–Eph complexes or ephrin–Eph-induced shedding of cadherin extracellular domains by ADAM metalloproteases (Fig. 4B) (Solanas, Cortina, Sevillano, & Batlle, 2011). In other contexts, ephrin–Eph-dependent Rho and myosin activation can create boundary interfaces with different properties. In the case of the notochord boundary, contractility seems to be particularly strong. It causes rupture of the links between the actomyosin cortical structures and the plasma membrane, leading to membrane blebbing (Fagotto et al., 2013) (Fig. 4A). Under this peculiar situation, cadherins fail to cluster, even though the two membranes remain closely apposed due to the compaction of the tissues (Fagotto et al., 2013). The mechanism is not understood, but may depend on the composition and mechanical properties of the blebbing

leading to membrane blebbing. Under these conditions, cadherin clustering appears to be inhibited through an unknown mechanism. (B) Other potential antiadhesive mechanisms. Repulsion requires removal of ephrin–Eph clusters, which can occur either by endocytosis or by proteolytic cleavage by ADAM proteinases. Cadherins could conceivably be internalized together with ephrin–Eph clusters. Ephrin–Eph-dependent ADAM activation can also cause cadherin shedding. (C). Ephrins and Eph receptors are also known to stimulate cell-cell adhesion. It has been proposed that under certain conditions, ephrin-Eph clusters may fail to be internalized and that their trans-interactions may then contribute to adhesion (?). Another potential mechanism could rely on mild myosin activation, which could reinforce cell adhesion.

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Adhesive boundary Actin “cable”

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Figure 5 Potential impact of cortical tension on boundary properties. (A) Adhesive boundary. Mild cortical contractility reinforces lateral cadherin adhesions. The mechanical coupling between adjacent cells through “actin cables” connected by cadherin adhesion can be viewed as a physical seal that prevents incursion of cells from the other tissue, while maintaining stable adhesion between the tissues. This mechanism may apply to insect compartment boundaries. (B) Dynamic boundary. Ephrin–Ephdependent myosin activation along the ectoderm–mesoderm boundary in Xenopus leads to transient cell detachment. The repulsive signal then decays, cells extend protrusions, and cadherin bonds are reestablished. Cell–cell contact stimulates a new burst of ephrin–Eph-dependent contraction, initiating a new cycle of detachment– reattachment (symbolized by the two curved arrows). (C) Nonadhesive boundary. Maximal tension can produce a fully not adhesive boundary interface by inhibiting cadherin adhesion (see Fig. 3A).

membrane and/or of the associated actin layer. An important future question will be to determine whether inhibition of cadherin clustering is a reaction that only occurs when tension reaches extreme intensities, or if it may constitute a more pervasive effect, the importance of which would progressively increase as tension increases. Paradoxically, ephrins and Eph receptors are also known to promote cell adhesion under some circumstances (Halloran & Wolman, 2006) (Fig. 4C). The decrease in tissue cohesion observed upon ephrin/Eph loss-of-function in zebrafish rhombomeres (Cooke, Kemp, & Moens, 2005) and in the Xenopus ectoderm (Rohani et al., 2011) suggests that this phenomenon is relevant for tissue separation. Ephrins and Eph receptors could then control separation by two parallel mechanisms, simultaneously decreasing adhesion at the boundary and increasing it within the tissues. The molecular mechanism responsible for this proadhesive activity is unclear. In one model, ephrin–Eph complexes would directly function as adhesive bonds. In fact, it is thought that these complexes must be removed from the cell surface


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to allow repulsion, which can occur via endocytosis or proteolytic cleavage ( Janes et al., 2005; Marston, Dickinson, & Nobes, 2003; Zimmer, Palmer, Kohler, & Klein, 2003). Although other types of regulations could be involved, one possible parameter could simply be intensity of receptor activation, which may need to reach a given threshold to trigger internalization/ cleavage of these molecules, thus shifting from an adhesive to a repulsive mode (Halloran & Wolman, 2006). I would argue that the ability of mild actomyosin contractility to reinforce cell adhesion could constitute another explanation for the ephrin–Eph proadhesive activity (Fig. 4C). This simple model is generally not discussed in the context of ephrin–Eph signaling, but would be compatible with observations of the formation of the notochord boundary, where transient strong cadherin clusters are observed across the boundary, likely reflecting cell–cell contact attempting to resist the increasing ephrin–Eph-dependent tension (Fagotto et al., 2013). Extrapolating these observations to different separation processes, one may postulate that different types of boundaries could be built simply based on different intensities of contractile reactions along the interface (Fig. 5) (Fagotto, 2014). Positive feedback between cadherins and moderate tension could explain the presence of the so-called “actin cables” along many boundaries. Under these conditions, all cells would remain adherent, but cell mixing would be prevented by a lateral compaction of the row of cells lining the boundary (Fig. 5A). This scenario would correspond to the situation of Drosophila compartments. Higher tension will disrupt cell contacts, producing either a “dynamic” or a “nonadhesive” boundary (Fig. 5B and C). The Xenopus ectoderm–mesoderm boundary is the prototype of the former type, where local bursts of repulsion temporarily disrupt adhesion (Fig. 5B) (Rohani et al., 2011). The notochord represents a nonadhesive boundary, seemingly maintained under a stronger interfacial tension (Fig. 5C) (Fagotto et al., 2013). This tentative classification is undoubtedly an oversimplification, as we are only at the very beginning of the exploration of boundary properties. An important piece of the puzzle will be to determine the strength and orientation of the various forces exerted on lateral and interfacial contacts, and how adhesion bonds are affected by local manipulation of these forces. An ephrin–Eph-based model is valid for vertebrates, but the determinants setting interfacial tension at Drosophila boundaries are not known. Even in vertebrates, the effects of ephrin–Eph signaling are likely to be complex and diverse, and additional mechanisms are certainly involved. The next section gives a glimpse of other interesting surface molecules which were shown to induce separation.

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5. HOMOPHILIC CONTACT MOLECULES AT EMBRYONIC BOUNDARIES The identification of additional cell surface cues came essentially from studies in Drosophila and in Xenopus. These proteins can all form direct homophilic or heterophilic cell to cell interactions, consistent with the importance of direct cell–cell contact at tissue interfaces. Most of them were originally classified as CAMs, but a bona fide direct adhesive function has generally not been validated, and their effect on tissue separation seems to be rather attributable to intracellular activities. The downstream pathways remain generally poorly understood, but, quite strikingly, they all seem to target myosin (Fig. 6). The four best studied examples, Echinoid in Drosophila, FLRT3, paraxial protocadherin (PAPC), and EpCAM in Xenopus, belong to four different families of membrane proteins and illustrate well the diversity of strategies of regulation of cell contractility and adhesion.

5.1. Immunoglobulin CAMs, Echinoid The immunoglobulin superfamily includes classical CAMs such as N-CAM and L1CAM, as well as a variety of other cell surface proteins that can undergo homophilic or heterophilic interactions (Shimono, Rikitake, Mandai, Mori, & Takai, 2012). In vertebrate models, most of them have been studied in the context of the nervous system or other specialized tissues. One pair, Nephrin and of its heterophilic partner Neph1 is responsible for forming the filtering slits of the kidney glomerula (Heikkila et al., 2011). In Drosophila, Nephrin-like Hibris and Sticks-and-Stones (Sns), and Neph-like Kirre and Roughest control the organization of the omnatidia, which can be considered a cell sorting process (Bao & Cagan, 2005; Bao, Fischbach, Corbin, & Cagan, 2010; Shimono et al., 2012). The best studied member is Echinoid: Natural and experimental juxtaposition of Echinoid-expressing and nonexpressing cells create smooth boundaries in the ovarian follicular epithelium and in the wing imaginal disc of the larvae, marked by an accumulation of actomyosin cortical structures along the heterotypic contacts (Chang et al., 2011; Laplante & Nilson, 2006, 2011). A similar structure also forms at the edge of the ventral epidermis as it closes over the amnioserosa in the embryo. This actin “cable” requires the presence of Echinoid in the epidermis and its disappearance from the amnioserosa (Laplante & Nilson, 2011). Based on sequence alignment of the extracellular domain, Echinoid is most similar to Drosophila Sns, to

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Regulation of Cell Adhesion and Cell Sorting at Embryonic Boundaries

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Figure 6 Models of regulation of separation by repulsive and proadhesive cues. (A) Contact cues involved in Xenopus ectoderm–mesoderm separation. The diagram represents the cell surface cues, their direct molecular interactions, and their major downstream targets, which all regulate myosin: PAPC activates Rho via ANR5. FLRT3 (together via Unc5B) inhibits it via Rnd1. EpCAM represses Erk activity through direct inhibition of novel PKCs. PAPC and FLRT3 also directly interact, which inhibits FLRT3– Rnd1 interaction. Additional reported interactions include direct binding to cadherins (PAPC and FLRT3) or indirect cadherin regulation by competition with the Wnt receptor Frizzled 7 (PAPC). The function of these heterophilic interactions remains unclear. Note (Continued)

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vertebrate ECM proteins hemicentrin and fibulin, and to nephrin. The intracellular domain of Ed does not show any conservation with other sequences. Functionally, Echinoid appears quite comparable to another subfamily of vertebrate IgG CAMs, the Nectin and nectin-like molecules, which in vertebrates establish the initial contacts between epithelial cells, prior to recruitment of cadherins and various other components that build mature adherens and tight junctions (Takai, Miyoshi, Ikeda, & Ogita, 2008). Both Nectin and Echinoid recruit the junctional soluble proteins Afadin/ Canoe and Par3/Bazooka (Takai et al., 2008; Wei et al., 2005). Echinoid is known to be localized to homotypic contacts, presumably through homophilic binding but absent (or less stable) from heterotypic contacts. As a consequence, Bazooka is also asymmetrically distributed, and its absence from heterotypic contacts leads to the accumulation of actomyosin along the boundary (Laplante & Nilson, 2011). Whether sequestration of Bazooka is sufficient to account for Echinoid function is unclear: From data on the process of convergence extension, where Bazooka and myosin II are also complementarily distributed, myosin localization does not depend on Bazooka (Simoes Sde et al., 2010). It is then likely that another pathway is responsible for myosin II regulation downstream of Echinoid. An alternative mechanism based on differential adhesion has also been proposed (Chang et al., 2011). Figure 6—Cont'd that Frizzled 7 was reported to inhibit cadherin dimerization, but the data did not discriminate between dimerization and higher order clustering. The effect of cis and trans homophilic interactions is still unknown. (B–E) Models for regulation of boundary by homophilic cues. Homophilic cell surface proteins such as PAPC and Echinoid are differentially expressed on one side of the boundary. Trans interactions stabilize them at homotypic contacts. Various mechanisms may explain their ability to induce separation: (B) sequestration of a negative regulator of cortical tension away from the heterotypic contact. (C) Strengthening of cell adhesion on one side of the boundary. (D) Global increase in tension, indirectly boosting tension at the interface. (E) A free pool could also directly activate tension (and/or inhibit adhesion) at the boundary interface, in the absence of trans interactions. (F) Putative range of regulation of tension and adhesion by ephrins/Eph receptors, EpCAM, PAPC, and FLRT3. The top curve represents the dependence of adhesion on increasing myosin activation (tension), as in Fig. 2B0 . For each surface cue, the corresponding triangles symbolize levels/activity, aligned to the hypothetical effect on myosin activity and adhesion. Ephrin–Eph signaling typically produces cell retraction and deadhesion, but a proadhesive function is also observed, perhaps restricted to the lowest range of activity. EpCAM was found to function exclusively as a proadhesive cue. PAPC and FLRT3 seem to be antiadhesive when expressed alone, but proadhesive when coexpressed, but the actual relationship to tension levels is unknown.


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5.2. Leucine-rich repeat proteins, FLRT3 Several leucine-rich repeat proteins function as guidance cues for neurons, either as ligands (Slit is one example) or receptors (Trks; de Wit, Hong, Luo, & Ghosh, 2011). Two members, Tartan and Capricious, play a role at the dorsoventral boundary of the Drosophila wing imaginal disc and at the leg segment boundaries (Milan, Weihe, Perez, & Cohen, 2001; Milan, Perez, & Cohen, 2005; Sakurai, Kojima, Aigaki, & Hayashi, 2007; Tepass, Godt, & Winklbauer, 2002). Note that they are not absolutely required for boundary formation, and may rather contribute to its refinement (Sakurai et al., 2007). Neither the cellular mechanism involved nor the identity of their partners in adjacent cells is known. Vertebrate “fibronectin and leucine-rich repeat” proteins FLRT1–3 are potent regulators of cell–cell adhesion, and, although a role for endogenous boundary formation has not yet been demonstrated, they are quite remarkable for their ability to induce cell sorting in the early Xenopus embryo (Chen et al., 2009; Karaulanov et al., 2009). The most studied is FLRT3, which is particularly strongly expressed in the early Xenopus mesoderm and plays a role in gastrulation movements (Ogata et al., 2007). FLRT3 can interact with several transmembrane proteins, including itself (Karaulanov, Bottcher, & Niehrs, 2006), the FGF receptor (Bottcher, Pollet, Delius, & Niehrs, 2004), the Netrin receptor Unc5B (Karaulanov et al., 2009), PAPC (Chen et al., 2009), and classical cadherins (Chen et al., 2009) (Fig. 6A). FLRT3 and Unc5B were originally proposed to bind in cis on the same membrane, but trans interactions should also be considered (Yamagishi et al., 2011). Data aiming at determining the function of FLRT proteins gave inconsistent results: FLRT3 homophilic binding was originally proposed to mediate cell–cell adhesion (Karaulanov et al., 2006), but since this effect was observed in cells expressing endogenous cadherins and was calcium-dependent, it could be due to indirect positive regulation of cadherin-mediated adhesion. On the contrary, gain and lossof-function experiments in Xenopus indicated that FLRT3 regulated cadherin adhesion negatively (Karaulanov et al., 2009; Ogata et al., 2007). The effect appeared to involve FLRT3 interaction with Rnd1 (Ogata et al., 2007) (Fig. 6A), an atypical RhoGTPase that is thought to antagonize Rho. The deadhesive activity of FLRT3 and Rnd1 was originally attributed to stimulation of cadherin endocytosis and consequently decrease in cadherin levels at the cell surface (Ogata et al., 2007). However, Chen et al. (2009) showed evidence that the inhibition of adhesion preceded and was probably the cause for the drop in cadherin levels.

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A similar conclusion was obtained for PAPC (see below). These data are again consistent with an indirect regulation of adhesion through the actin cytoskeleton.

5.3. Protocadherins, PAPC The discovery of the large protocadherin subfamily had originally brought the promise of definitive explanation of cell sorting based on differential CAM expression. However, protocadherins appear to be unable to promote efficient cell–cell adhesion (Chen & Gumbiner, 2006), most likely due to the absence of a β-catenin binding site. Although a potential direct role in cell– cell adhesion cannot be excluded, the current consensus is that they rather function as cell contact receptors. Consistently, their cytoplasmic tails, which are quite diverse in sequence, regulate a variety of intracellular pathways (e.g., Nap1/WAVE for protocadherin 10, Wnt for protocadherin 11Y, Rho for protocadherin 8, Kim, Yasuda, Tanaka, Yamagata, & Kim, 2011). Protocadherin 8, also called paraxial protocadherin (PAPC) has been implicated in early vertebrate morphogenesis, including in tissue separation. It induces efficient cell sorting, producing exceptionally sharp boundaries when ectopically expressed (Chen & Gumbiner, 2006; Chen et al., 2009). During early embryonic development it is systematically expressed on one side of a forming boundary (Fig. 1A, B, and D): It first appears in the dorsal mesoderm as this layer separates from the ectoderm, later it disappears from the axial mesoderm (future notochord), and remains restricted to the paraxial mesoderm when both structures split. The same process of restriction occurs once more during somitogenesis, with PAPC remaining in the anterior half of the forming somites (Kim, Jen, De Robertis, & Kintner, 2000; Kim, Yamamoto, Bouwmeester, Agius, & Robertis, 1998; Rhee, Takahashi, Saga, Wilson-Rawls, & Rawls, 2003). PAPC loss-offunction impairs ectoderm–mesoderm separation in Xenopus (Medina et al., 2004) and somite segmentation in Xenopus and the mouse (Kim et al., 2000; Rhee et al., 2003). A role for formation of the notochord boundary has not yet been confirmed. Chen and Gumbiner showed that PAPC was unable to provide cell–cell adhesion on its own. On the contrary, it negatively regulates the function of classical cadherins (Chen & Gumbiner, 2006). PAPC was found to upregulate Rho activity (Medina et al., 2004) through an unknown mechanism that involves the ankyrin repeat protein ANR5 (Chung, Yamamoto, & Ueno, 2007) (Fig. 6A). As explained above, Rho


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overactivation and cortical hypercontractility could be sufficient to explain the observed decrease in adhesion. The actual function of PAPC is however more complicated. First, PAPC cytoplasmic domain, and thus presumably its interaction with ANR5, is dispensable for the capacity to induce sorting (Chen & Gumbiner, 2006; Chen et al., 2009). PAPC was also reported to laterally interact with FLRT3 (Chen et al., 2009), with classical cadherins (Chen et al., 2009) (also observed for another protocadherin, Pcdh19, Biswas et al., 2014), and with the Wnt receptor Fz7 (Kraft, Berger, Wallkamm, Steinbeisser, & Wedlich, 2012) (Fig. 6A). The consequences of these interactions are incompletely understood. In one model, PAPC mitigates the antiadhesive activity of FLRT3 by preventing recruitment of Rnd1 (Chen et al., 2009). The interaction with Fz7 seems to favor PAPC stabilization at the cell membrane (Kraft et al., 2012). Note that Fz7 also forms a complex with classical cadherins that inhibits cadherin dimerization (Kraft et al., 2012). These authors failed to detect a PAPC–cadherin interaction. There is clearly a need to reconcile and integrate these various data into a coherent picture. Note that another protocadherin, axial protocadherin (AxPC of Pcadh1) replaces PAPC in the notochord at the time of its separation from the paraxial mesoderm (Kuroda, Inui, Sugimoto, Hayata, & Asashima, 2002). AxPC depletion disrupted the corresponding boundary (Yoder & Gumbiner, 2011). The underlying mechanism has not yet been investigated.

5.4. EpCAM, inducer of tissue mixing Although its loss-of-function in early Xenopus and zebrafish does not lead to any obvious separation phenotype, EpCAM is included here as prototypic example of positive regulator of cell migration and adhesion, which must be taken into account to understand how tissue properties influence tissue boundaries. Its case is quite informative about the relationship between regulation of cortical actomyosin, cell adhesion, and cell motility. EpCAM was mostly known as a tumor-associated protein highly expressed in human carcinomas, but very little is known about its potential role in metastasis. EpCAM can bind homophilically (Balzar et al., 1999), but its classification as CAM remains to be validated. It turns out that EpCAM is also abundantly expressed in early vertebrate embryos. We originally identified Xenopus EpCAM in a gain-of-function screen through its remarkable capacity to induce mixing between ectoderm and mesoderm (Maghzal, Vogt, Reintsch, Fraser, & Fagotto, 2010). As mentioned, EpCAM depletion

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does not affect ectoderm–mesoderm separation, but has other effects on morphogenesis: it first impairs the thinning and spreading of the ectoderm during gastrulation, a process called epiboly (Maghzal et al., 2010; Slanchev et al., 2009). After gastrulation, EpCAM loss yields a dramatic phenotype, with cells losing adhesion, leading to complete tissue disaggregation and embryo death (Maghzal, Kayali, Rohani, Kajava, & Fagotto, 2013). We will see that, although diverse in appearance, all these phenotypes have a single common molecular cause. Tissue mixing upon EpCAM overexpression and epiboly defect upon EpCAM depletions are two opposite results of modulating intercellular motility, i.e., the capacity of cells to move among other cells. We found that EpCAM controls this process by repressing myosin activation (Maghzal et al., 2010). When EpCAM levels are lowered, high myosin activity makes the cells poorly motile and the tissue stiff, when EpCAM is high, low myosin activity allows cells to be more motile and tissues more “fluid.” Cell motility is boosted enough to bypass boundary repulsion and cause tissue mixing. EpCAM-mediated repression of myosin activity has also a positive effect on cadherin adhesion. Note that unlike commonly assumed, decreasing myosin activation stimulates both adhesion and migration. We will discuss below possible ways to reconcile this phenomenon with the assumed requirement for myosin in cell–cell adhesion. Cell dissociation in EpCAM depleted postgastrula embryos is an extreme result of myosin overactivation (Maghzal et al., 2013). High contractility prevents maintaining cell–cell adhesion. This in turn leads to cadherin internalization and degradation, which further feeds back into accelerating loss of adhesion. At the molecular level, EpCAM acts by directly binding and inhibiting novel PKCs, thus blocking the downstream PKD–Raf–Erk cascade (Maghzal et al., 2013). Although several targets are certainly affected, all the developmental defects result from myosin deregulation (Maghzal et al., 2013).

6. REGULATION OF TENSION AND ADHESION BY CONTACT CUES The identification of these various cell surface molecules has given us some idea of the type of regulations that control tissue and boundary properties, and has highlighted the central role of myosin. Yet the preliminary study of these molecules, far from solving the problem, has brought additional puzzles. I discuss here general conceptual issues raised by the


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implication of homophilic molecules in regulating heterotypic contacts and by the coexistence of pro- and antiadhesive activities.

6.1. The action of homophilic regulators PAPC and Echinoid share the ability to bind homophilically and the particularity of being expressed only on one side of the boundary. As a consequence, they both accumulate at homotypic contacts and are low or absent from the membranes abutting the boundary (Laplante & Nilson, 2006 and unpublished observations), which, by analogy with classical cadherins, is probably determined by internalization of the nonengaged molecules and stabilization by homophilic interactions. PAPC and Echinoid can be thus considered as “homophilic cues,” by opposition to “heterophilic cues” such as ephrins and Eph receptors. How can then homophilic cues control the properties of an interface from which they are precisely absent? Among several possible scenarios, the one favored for Echinoid is that it sequesters some regulator of myosin contractility, resulting in its depletion from the boundary interface (Laplante & Nilson, 2011) (Fig. 6B). Alternatively, PAPC and Echinoid may favor cell–cell adhesion within the tissue, either functioning directly as CAMs, or indirectly, via regulation of cadherins or of contractility. Separation would then be stimulated by the building of differential adhesion (and/or tension) between homotypic and heterotypic contacts (Fig. 6C). Changes in adhesion or contractility along homophilic contacts may also affect global cellular forces, which could propagate to the heterotypic contact (Fig. 6D). These possible scenarios are based on the reasonable assumption that the functional pool is the one engaged in homophilic interactions. This, however, has not yet been demonstrated, and it remains formally possible that what counts is the free pool, which, despite its presumed instability, may directly affect the boundary properties (Fig. 6E). A better characterization of homophilic and heterophilic interactions, and of their impact on intracellular activities should clarify the mode of action of these interesting molecules.

6.2. Putting pro- and antiadhesive activities together So far, the function of the various cell surface cues can be largely explained by their action on actomyosin contractility. Although our knowledge is still too incomplete to draw more specific conclusions, studies on the best known molecules, ephrins/Eph receptors and EpCAM (Fig. 6F) give us

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some general ideas about how multiple pathways may be integrated. Ephrin–Eph signaling and EpCAM work essentially antagonistically: the former stimulates myosin activation, contractility, and decreases adhesion. EpCAM has the exact inverse action. Since both are simultaneously active in the early embryo, one first conclusion is that the local properties must be influenced by their balance. This explains, for instance, why EpCAM overexpression causes ectoderm to mix with mesoderm (Maghzal et al., 2010): by inhibiting myosin, it decreases basal cortical tension, such that the bursts induced by ephrin–Eph signaling are no longer sufficient to overcome cell– cell adhesion. Assuming this simple scenario is however not necessarily sufficient to predict the combined outcome of multiple cues. It is indeed important to remember that myosin has multiple functions, which can be pro- or antiadhesive, which could be differently regulated by different cues, either because a specific pool may be preferentially targeted or because the regulation would be restricted to a given range of tensile strength. The case of EpCAM seems relatively straightforward: when EpCAM levels were manipulated, myosin activation and of adhesion always consistently responded in opposite directions. Taking a simple linear model (Fig. 6F), we may conclude that EpCAM activity spans essentially the “antiadhesive” range of contractility. The case of ephrins and Ephs is complicated by the ill-defined proadhesive activity, which may occur within a weak tensile mode, even though the system is probably antiadhesive over a broad range of ephrin–Eph signaling (Fig. 6F). Predictions are currently more risky for PAPC and FLRT3 (Fig. 6F), due to outstanding inconsistencies in the reported effects on adhesion and cell sorting and their mutual regulations (Chen et al., 2009). It remains that PAPC and FLRT proteins are remarkably potent regulators of adhesion and inducers of cell sorting, and one should consider them as potential heavy weights in setting the adhesive and tensile conditions. Defining their mode and range of regulation should be a priority if one wants to build a coherent picture of separation in vertebrates. 6.2.1 Note about complexity and redundancy Studies in fish and Xenopus have implicated several components in tissue separation, including multiple ephrins and Eph receptors, PAPC, and FLRT3. Quite strikingly, depletion of a single component, including one out of several ephrins/Ephs, is sufficient to affect separation rather severely (Cooke et al., 2005; Fagotto et al., 2013; Rohani et al., 2011, 2014; Watanabe


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et al., 2009). On the contrary, the corresponding knockouts in mice gave no obvious phenotype (e.g., Dottori et al., 1998; Yamamoto, Kemp, Bachiller, Geissert, & De Robertis, 2000). An extreme case is the removal of all ephrinBs, which did not impair any of the major early developmental processes (Senturk, Pfennig, Weiss, Burk, & Acker-Palmer, 2011). Yet the expression patterns of all these molecules is very conserved throughout vertebrates, which makes it difficult to imagine that the function would not be similarly conserved. The lack of phenotype in mammals is probably due to a higher degree of redundancy, particularly between the ephrins and PAPC, two systems that are active in parallel for at least three boundaries. This apparent redundancy is a serious obstacle for studying boundaries and probably other morphogenetic processes in mammals. Note that a certain degree of redundancy must also occur in fish and frogs, suggested by the fact that none of the depletions completely abolished separation. Complexity could then serve as buffer for small variations in levels/activity of individual components. The existence of multiple pathways may also be related to additional functions in other morphogenetic processes. This is clearly the case for PAPC, which is involved in mesoderm convergence extension (Medina et al., 2004; Unterseher et al., 2004). Redundancy could similarly explain the current lack of strong candidates for setting high contractility at Drosophila compartment boundaries. Mutants for Tartan and Capricious, for instance, do yield a wiggling boundary (Milan et al., 2005), suggesting that they do play a role in separation. Using a sensitized mutant background could reveal a second parallel pathway. 6.2.2 After separation: Relationship between separation and epithelization Formation of Drosophila parasegments, imaginal disc boundaries and vertebrate hindbrain segmentation, all correspond to the transverse partition of an epithelial layer, with little direct impact on the tissue structure. In other cases, however, the separating tissues undergo important reorganization. A well-known example is somitogenesis, where segmentation is immediately followed by compaction of the new somite into a closed epithelial layer that constitutes an independent structure. The de novo appearance of a polarized organization in a mesoderm tissue is a particularly intriguing phenomenon, which is not restricted to somitogenesis. By this stage, it has already occurred twice during mesoderm morphogenesis, each time as a consequence of a separation event. This first happens after the separation of the mesoderm from the ectoderm (and from the endoderm, an event that has

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not yet been studied): Mesoderm organizes then as two parallel layers, which correspond to the somatic mesoderm (facing the ectoderm) and the splanchnic mesoderm (facing the endoderm), two key elements of the coelomate body structure. When the dorsal section of this mesoderm splits into the notochord and the presomitic mesoderm, a similar reorganization takes place along the new boundary. The alignment and polarization of the cells abutting the boundary results in connecting the dorsal and ventral mesoderm layers on both sides of the boundary into two continuous monolayers, as summarized in Fig. 7. When somites segment out of the PSM, the process is repeated, virtually identical except for the 90° rotation of the plane of separation. In the case of somitogenesis, epithelization had been sometimes considered as a step toward separation (e.g., Duband et al., 1987). However, a boundary interface is unambiguously detected before any morphological sign of epithelization (e.g., Youn & Malacinski, 1981), although this latter process contributes to the appearance of a visible gap (Fig. 7D). Comparison of the three consecutive separations suggests a common mechanism, initiated by local ephrin–Eph-mediated repulsion (we will ignore for simplicity the poorly understood contribution from PAPC). The resulting decrease or loss in cell adhesion creates a nonadhesive interface, which is interpreted by the cells as a “basal” side, where extracellular matrix is deposited. It is likely that the matrix serves then as a basal cue to reorganize and polarize the cells along the boundary.

7. CONCLUSIONS High actomyosin cortical tension appears to be a fundamental property of embryonic boundaries, and ephrin–Eph-dependent repulsion provides a satisfactory mechanism to set this local tension, at least in vertebrates. With the generalization of these concepts, our view of morphogenesis has been radically modified: Rather than selecting their neighbors based on shared properties, cells appear to stick together by default and to specifically detect those other cell types that they should repulse. In other words, development does not proceed by increasing “affinities,” but by creating “identities.” This is one more example where behind a phenomenon that seemed to be explainable based on simple physical principles, is actually controlled by active cellular signals. How much did we solve so far of this fascinating process? We have then learnt how embryonic tissues are being cut into parts, which answers an old

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A

Apical adherens junctions

Basal surface, adhesion to matrix

B Ephrin/Eph repulsion Ephrin–Eph-mediated separation Local loss of adhesion

C

Reorganization of apical–basal polarity

D

Completed “epithelization”

Figure 7 Local epithelization during separation of vertebrate mesoderm tissues. Once separated from the ectoderm and the mesoderm, the mesoderm layer becomes organized as two parallel layers of polarized cells. The basal domain faces the outside, and adherens junction-like structures are in the center (A). During the two successive separations that first produce the notochord and the presomitic mesoderm, then the somites, a similar pattern of changes in tissue organization is observed: A boundary forms due to ephrin–Eph-dependent increase in cortical tension and a corresponding drop in cell–cell adhesion (B). The boundary interface becomes equivalent to a basal domain, where extracellular matrix is deposited (C). Cells are reoriented, the continuity of the polarized layer is reestablished on each side of the boundary, and the two new regions become permanently isolated by extracellular matrix (D).

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and fundamental question. We are however still far from being able to build a realistic picture of tissue separation. We have in particular only vague hints for what confers different boundaries with specific properties, such that tissues can at times slide relative to each other, crawl, or remain tightly connected. There is certainly a whole set of regulations to uncover, a mine of new excitement for cell biologists.

ACKNOWLEDGMENTS F. F. research is supported by the Canadian Institute for Health Research, the Canadian Cancer Society Research Institute, and the Cancer Research Society.

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Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain.

Cell Sorting and Noise-Induced Cell Plasticity Coordinate to Sharpen Boundaries between Gene Expression Domains.

Differential effects of the cytoplasmic domains of cell adhesion molecules on cell aggregation and sorting-out.

Cell adhesion and sorting in embryoid bodies derived from N- or E-cadherin deficient murine embryonic stem cells.

Wnt Signaling Gradients Enriched at Adult Intestinal Compartment Boundaries.

Modelling cell movement, cell differentiation, cell sorting and proportion regulation in Dictyostelium discoideum aggregations.

Role of cell-cell adhesion complexes in embryonic stem cell biology.

Expression of adhesion molecules and the establishment of boundaries during embryonic and neural development.

Differential regulation of thyroid cell-cell and cell-substrate adhesion by thyrotropin.

Resolving Heterogeneity: Fluorescence-Activated Cell Sorting of Dynamic Cell Populations from Feeder-Free Mouse Embryonic Stem Cell Culture.

Regulation of human B-cell activation and adhesion.

Embryonic cell-cell adhesion: a key player in collective neural crest migration.

Beyond cell-cell adhesion: Plakoglobin and the regulation of tumorigenesis and metastasis.

Cell cycle regulation of embryonic stem cells and mouse embryonic fibroblasts lacking functional Pax7.

Clusters of neural cell adhesion molecule at sites of cell-cell contact.

Regulation of LFA-1-mediated T cell adhesion by CD4.

Cell adhesion in the stromal regulation of haemopoiesis.

Down regulation of T-cell adhesion by CD4.

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution.

Transmembrane interactions at cell adhesion and invasion sites.

Talin: role at sites of cell-substratum adhesion.

MicroBubble activated acoustic cell sorting.

Cell-cell recognition in Saccharomyces cerevisiae: regulation of mating-specific adhesion.

Retinoids and cell adhesion.