During embryonic development, cells or groups of cells migrate from their locations of origin to assume their correct anatomical positions. Intercellular adhesion plays an active and instructive role in orchestrating this process. Precisely how adhesion provides spatial positioning information is a subject of intense interest. In the 1960s, Steinberg proposed the differential adhesion hypothesis (DAH) to explain how differences in the intensity of cell adhesion could give rise to predictable spatial interactions between different cell types. The DAH is grounded in the same set of physical principles governing the interaction of immiscible fluids and thus provides a rigorous conceptual framework connecting the chemistry of cell adhesion to the physics underlying cell and tissue segregation. Testing the DAH required the development of methods to measure intercellular cohesion and of assays to accurately assess relative spatial position between cells. The DAH has been experimentally verified and computationally simulated. Moreover, evidence concerning the role of differential adhesion in a number of morphodynamic events is accumulating. It is clear that differential adhesion is a major driving force in various aspects of embryonic development, but recent studies have also advanced the concept that other factors, such as cortical tension and elasticity, may also be involved in fine tuning, or even driving the process. It is likely that an interplay between adhesion and these other factors co-operate to generate the forces required for tissue self-organization. (c) 2013 Wiley Periodicals, Inc.

In their letter, Krens et al. (1) question whether our tissue surface tension model can be applied to zebrafish aggregates and explants. They argue that the stretched-out surface cells shown in our manuscript (2) are differentiated squamous epithelial enveloping-layer (EVL) cells, which implies that these zebrafish explants consist of two tissue types and that our model does not apply to these explants. Their data neither contradict our results nor support their claim that “the model as such has very little predictive value for zebrafish explants” (1).

In the course of animal morphogenesis, large-scale cell movements occur, which involve the rearrangement, mutual spreading, and compartmentalization of cell populations in specific configurations. Morphogenetic cell rearrangements such as cell sorting and mutual tissue spreading have been compared with the behaviors of immiscible liquids, which they closely resemble. Based on this similarity, it has been proposed that tissues behave as liquids and possess a characteristic surface tension, which arises as a collective, macroscopic property of groups of mobile, cohering cells. But how are tissue surface tensions generated? Different theories have been proposed to explain how mesoscopic cell properties such as cell-cell adhesion and contractility of cell interfaces may underlie tissue surface tensions. Although recent work suggests that both may be contributors, an explicit model for the dependence of tissue surface tension on these mesoscopic parameters has been missing. Here we show explicitly that the ratio of adhesion to cortical tension determines tissue surface tension. Our minimal model successfully explains the available experimental data and makes predictions, based on the feedback between mechanical energy and geometry, about the shapes of aggregate surface cells, which we verify experimentally. This model indicates that there is a crossover from adhesion dominated to cortical-tension dominated behavior as a function of the ratio between these two quantities.

This study provides direct functional evidence that differential adhesion, measurable as quantitative differences in tissue surface tension, influences spatial positioning between zebrafish germ layer tissues. We show that embryonic ectodermal and mesendodermal tissues generated by mRNA-overexpression behave on long-time scales like immiscible fluids. When mixed in hanging drop culture, their cells segregate into discrete phases with ectoderm adopting an internal position relative to the mesendoderm. The position adopted directly correlates with differences in tissue surface tension. We also show that germ layer tissues from untreated embryos, when extirpated and placed in culture, adopt a configuration similar to those of their mRNA-overexpressing counterparts. Down-regulating E-cadherin expression in the ectoderm leads to reduced surface tension and results in phase reversal with E-cadherin-depleted ectoderm cells now adopting an external position relative to the mesendoderm. These results show that in vitro cell sorting of zebrafish mesendoderm and ectoderm tissues is specified by tissue interfacial tensions. We perform a mathematical analysis indicating that tissue interfacial tension between actively motile cells contributes to the spatial organization and dynamics of these zebrafish germ layers in vivo.

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Self-organizing behavior is one of the most remarkable properties of regulative animal embryos. The reorganization of disarranged embryonic primordia to form an approximation of the "correct" structure by any number of abnormal pathways constitutes a form of goal-directed behavior. One might suppose that such anatomical "goals" are specified by genetic programs evolved through mutation and natural selection to produce useful structures. However, one might also ask the following questions: how can genes direct morphogenesis without specifying the pathways to be followed? How can genetic systems have evolved to specify the organization of the never-before-assembled structures reproducibly generated by abnormal tissue combinations? Experiments have shown that the layered structures generated in such experiments belong to the category of "inherently precise" machines, in which a specific pattern is generated with great precision by the constant repetition of a simple local behavior throughout the pattern-forming system. The organization characteristic of the chordate body plan—the "goal" of early development—also arises by very different developmental pathways in the various members of the phylum. Yet divergent evolution can hardly have altered the mechanisms governing gastrulation and neurulation while holding the end results essentially constant. Evidence suggests that the striking differences in these pathways may be understood less as fundamental alterations of morphogenetic mechanisms than as the physical consequences arising from heterochrony—differences in the times at which a shared set of underlying cellular changes are initiated.

We have developed rapid and efficient methods for the isolation of desmosomes and the fractionation of their components. These methods involve the use of 6 M guanidine HCl to isolate the desmosomes from bovine epidermis, followed by hydroxyapatite column chromatography in the presence of SDS to fractionate the desmosomal components. All of the desmoplakins and desmogleins were purified at least partially by these procedures, and desmoglein II was purified to apparent homogeneity. We expect these procedures to facilitate a detailed biochemical analysis of the molecular components of desmosomes. In addition, these methods may be applicable to the purification of other plasma membrane domains involved in cell adhesion.

A panel of monoclonal antibodies and conventional antisera directed against desmosomal proteins from bovine muzzle epidermis was used to identify immunologically related proteins from two other bovine stratified squamous epithelia, cornea and esophagus. Desmosome-enriched tissue fractions were prepared from epidermis, cornea, and esophagus. These tissue extracts were electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels, blotted onto nitrocellulose paper, and labeled using an indirect immunoperoxidase technique. Labeling with the conventional antisera demonstrates that each of the previously characterized epidermal desmosomal proteins or protein families has an immunologically cross-reacting counterpart in cornea and esophagus. However, chemical differences between homologous desmosomal proteins in these three tissues have also been detected. The corresponding proteins in the different tissues have similar but not always identical apparent molecular weights. Moreover, tissue-restricted antigenic determinants were detected in two of the desmosomal proteins families using four monoclonal antibodies, each of which recognizes a distinct antigenic determinant.

A sensitive method for assaying aggregation of dissociated cells has been developed which allows the determination of the mean number of cells per aggregate of a cell population. We have demonstrated that exposure of dissociated 6- or 7-day chick embryo neural retinal cells to trypsin in calcium-free solution renders them unable to aggregate for a half hour in stirred cell suspensions. Aggregation was noticeable first at 30 to 40 minutes and, progressed to the formation of massive compact aggregates. Because the half-hour aggregation lag occurred both in the absence of serum and in medium reclaimed from aggregated preparations, the possibilities were excluded that it was due either to an inhibitor of aggregation in the serum, or was the time required for release into the medium of soluble aggregation-promoting materials emanating from the cells themselves. Cells dissociated by divalent cation withdrawal (Ca++, Mg++-free saline with EDTA) aggregated without a lag. The trypsin-induced lag does not appear to be the result of trypsin adsorbed to the, surfaces of dissociated cells, as the lag is not abolished by addition of trypsin inhibitors to the aggregation medium. Microelectrophoresis of dissociated cells did not reveal changes in surface charge density during recovery from trypsinization. A variety of proteins and calcium ion, if present during trypsinization, protect the cells against the trypsin-induced aggregation lag. If the temperature was reduced from 37 to 6°C, aggregation of fully adhesive cell populations came to a complete halt within 2 to 3 minutes. Aggregation resumed with a 5 to 10 minute delay when the temperature was returned to 37°C. The rapidity of onset and reversal of inhibition of aggregation by low temperature treatment militates against the hypothesis that the low-temperature inhibition of aggregation acts by suppressing the synthesis of cell surface components necessary for adhesion. The abolition of the aggregation lag in trypsinized cells was also shown to be temperature-dependent; a 20-minute cold, pulse administered in the middle of the lag period extended the length of the lag by exactly 20 minutes.

AbstractThe differential adhesion hypothesis attributes sorting‐out behavior of embryonic cells to differences in the strengths of their intercellular adhesions. It suggests that cells in mixed populations rearrange themselves to minimize their total adhesive free energy. The final configuration adopted is viewed as approaching a moststable or equilibrium configuration, with the less cohesive cell/population tending to envelop the more cohesive one.The hypothesis enables two predictions to be made. First, if the final configuration adopted by a mixed cell population indeed approximates an equilibrium configuration, the same final configuration should be approached irrespective of the starting configuration. Second, comparison of the equilibrium configurations for all possible pair‐combinations of a series of cell populations of different types should reveal a hierarchy of “preference” for the internal position, reflecting a hierarchy of cellular cohesiveness.These predictions have been tested and confirmed. First, any particular pair of tissues approaches the same final configuration, irrepective of whether the cells are initially randomly intermixed or tissue fragments are laterally joined. Second, the predicted hierarchy of preference for the internal position has been found, consistent with a hierarchy of cellular cohesiveness.An effort has been made to clarify confusions that have arisen concerning the differential adhesion hypothesis. It is particularly stressed that the hypothesis offers an explanation for cell population behavior in terms of relative strengths of cell adhesions and makes no proposals concernnig the chemistry of adhesion. However, the value of equilibrium configurations in guiding biochemical investigations of cell surface adhesives is pointed out, as is the relevance of the present observations to natural morphogenetic events.