Cortical tension, contact tension and tissue segregation: A clarification.

This article reports a study on the role of ephrin-Eph signalling in cell sorting. Ephrin-Eph signalling is one of the major molecular mechanism involved in tissue segregation (Fagotto, 2014; Fagotto, 2020; Fagotto et al., 2014). Kindberg and colleagues reproduce this ephrin-Eph-dependent sorting in an artificial system, namely the human HEK cell line. The authors confront two cell populations, one expressing an ephrin, the other a cognate Eph receptor. This simplified system roughly mimics the situation in embryonic tissues: Ephrin-Eph signalling in the latter is usually more complex, due to the expression of multiple ephrins and Eph receptors, but the general logic based on asymmetric expression of selective ligands and receptors is similar (Fagotto et al., 2014). Using virtually identical assays as those previously applied to embryonic cells by our team, Kindberg et al obtain results that are largely in agreement with our previous data and model (Canty et al., 2017). Yet, they also propose additional properties about which some clarification is needed.

To put this discussion in the right context, a reminder of the principles of tissue segregation is required: Cell sorting and cell positioning within a tissue are directed by contractile forces exerted on the cortex and cell-cell adhesion. A unifying model formulated by Brodland (Brodland and Chen, 2000), emphasizes the dominant role of actomyosin cortical contractility, which must be downregulated along cell-cell contacts to allow effective adhesion. Brodland defined a “cell to medium interfacial tension”, which corresponds to the “cortical tension” along free edges of the cell, and a “cell to cell interfacial tension”, also named “contact tension” (Winklbauer, 2015)(*). Stemming from this principle, the “Differential Interfacial Tension Hypothesis” (DITH) posed that cell sorting was driven by global differences in tissue stiffness (Brodland, 2002; Krieg et al., 2008). However, ample experimental evidence and theoretical considerations have brought quite a different view of the process.

Firstly, it is now clear that what dictates sorting is not a difference in the stiffness of the two cell types, but rather local enhanced contact tension at heterotypic contacts (which we coined high heterotypic interfacial tension, HIT (Canty et al., 2017; Fagotto, 2020)). This came as an unavoidable conclusion of our own experimental results on early embryonic boundaries in Xenopus, in particular on the dissection and reconstitution of the process of separation of dorsal mesoderm and ectoderm (and not endoderm, as incorrectly referred by Kindberg et al) (Canty et al., 2017; Rohani et al., 2011; Rohani et al., 2014). Importantly, our biophysical simulations showed that the principle of HIT could be generalized to any type of tissue boundary (Canty et al., 2017). This conclusion, confirmed independently by Manning and colleagues using a different model of simulation (Sussman et al., 2018), was in full agreement with experimental data in various systems. Furthermore, the fact that tension is highest at heterotypic contacts can be simply inferred from the straightness of tissue boundaries, as noted by the Dahmann and Sanson teams in the case of compartment boundaries in Drosophila (Aliee et al., 2012; Landsberg et al., 2009; Monier et al., 2010). Ephrin-Eph signalling is obviously an optimal mechanism to boost tension at a heterotypic contact, thus producing HIT, although various other types of mechanisms can produce the same effect (Fagotto, 2015; Luu et al., 2015; Sharrock and Sanson, 2020; Wang and Dahmann, 2020).

The second issue is the effect of contractility on relative positioning of the two cell populations. In this case, tension at cell surfaces exposed to the medium plays an important role, thus softer cells tend to engulf the stiffer cells. This has led to an apparent contradiction (Krieg et al., 2008), because, in embryos, the ectoderm is typically stiffer than mesoderm and endoderm, yet it takes a peripheral position. The solution to this conundrum, definitively solved by Ninoyima and Winklbauer (Ninomiya and Winklbauer, 2008), is simple: Under physiological conditions, most cells are not exposed to the “medium”, and tissues are largely “protected” by the non-adhesive apical domain of an epithelial layer. As such, this domain cannot participate to the minimization of contacts and by default it must occupy the peripheral position, and is therefore excluded from the game between adhesion and contractility. Thus, in practice, sorting WITHIN the tissue is controlled by the relative tension at different types of cell-cell contacts. Importantly, although global cell stiffness does impact the properties of cell contacts, this effect cannot be simply inferred from the tension present at the cell-medium interface. Indeed, complex reactions occur at contacts, which might enhance or dampen contact tension, and/or reinforce cell-cell adhesion. Thus, it is important to evaluate contact tension for each particular combination of cells. In any case, the key point here is that differences in cortical tension can directly influence cell positioning only in the immediate vicinity of a (non-coated) tissue surface.

With this is mind, one can now evaluate the observation by Kindberg et al of EphB2 expression increasing free cortical tension. The authors propose that this property significantly contributes to cell sorting. However, this model can be directly applied only under conditions where cells expose significant surface to the medium. This is true in the type of non-confluent in vitro cultures used in their study (although the interaction with the extracellular matrix is likely to impact the tension along these edges). What about actual tissues? As said, most tissues are covered by either an epithelial layer, or by other tissues. The degree of freedom is even lower in the case of purely epithelial tissues, such as Drosophila embryonic blastoderm or wing imaginal disc, as cells can only move in the plane of the epithelium. Could one conceive, however, a relevance for non-epithelial tissues, which are less tight and have interstitial spaces? This could be the case if tension along these spaces is comparable to that at a “free” edge as measured in vitro for isolated cell doublets. Xenopus ectodermal inner layer is so far the only model where a precise analysis was performed. Quite interestingly, Winklbauer and colleagues demonstrated that the tension is in fact quite low, very much in the range of contact tension (Barua et al., 2017; Parent et al., 2017). Thus, interstitial spaces cannot be compared to the simple medium used in cell culture: Cells appear to control their environment, probably through secretion of extracellular glycoproteins acting as “surfactants”, in order to avoid unwanted cortical tension to build inside the tissue. These considerations indicate that a model involving differences in cortical tension at free cell edges is likely to be of limited significance in the physiological context of real tissues.

The interesting observation by Kindberg et al of an effect of EphB2 ectopic expression on cortical tension of HEK cells should be included among the many interesting complications of ephrin-Eph systems (Pasquale, 2010). Similarly, ephrinB1 and EphB4 appear to have a proadhesive activity in the Xenopus ectoderm (Rohani et al., 2011), thus opposed to the stereotypical repulsive function of these molecules. The actual impact of these properties on cell sorting and tissue segregation will continue to be a fascinating issue, which will require further investigation.

A last note about the cadherin independence of the EphB2 phenotype reported by Kindberg et al. This conclusion was based on experiments in a medium with low calcium, a condition that certainly strongly decreases, but does not fully block cadherin adhesion. Furthermore, it has been proposed that non-specific surface proteins may significantly contribute to adhesion (Winklbauer, 2019). Finally, it is also conceivable that EphB2 may interact with low levels of endogenous ephrins, in a regime that could directly provide some adhesion to the cells (Poliakov et al., 2004).

*Note: The original term “interfacial tension” (Brodland and Chen, 2000), included in the definition of the “Differential Interfacial Tension Hypothesis” (DITH) (Brodland, 2002), directly came from the ANALOGY with liquid surface tension. It is used in the Kindberg et al. article. However, this terminology can be confusing when describing morphogenetic processes, and, for the sake of clarity, we rather prefer to use “contact tension”, from the terminology proposed by Winklbauer (Winklbauer, 2015), and keep the term “interface” to refer to supracellular structures, i.e. tissue and compartment interfaces.

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