Malcolm S. Steinberg

Viscoelastic Properties of Living Embryonic Tissues: a Quantitative Study

A number of properties of certain living embryonic tissues can be explained by considering them as liquids. Tissue fragments left in a shaker bath round up to form spherical aggregates, as do liquid drops. When cells comprising two distinct embryonic tissues are mixed, typically a nucleation-like process takes place, and one tissue sorts out from the other. The equilibrium configurations at the end of such sorting out phenomena have been interpreted in terms of tissue surface tensions arising from the adhesive interactions between individual cells. In the present study we go beyond these equilibrium properties and study the viscoelastic behavior of a number of living embryonic tissues. Using a specifically designed apparatus, spherical cell aggregates are mechanically compressed and their viscoelastic response is followed. A generalized Kelvin model of viscoelasticity accurately describes the measured relaxation curves for each of the four tissues studied. Quantitative results are obtained for the characteristic relaxation times and elastic and viscous parameters. Our analysis demonstrates that the cell aggregates studied here, when subjected to mechanical deformations, relax as elastic materials on short time scales and as viscous liquids on long time scales.

Cadherins and their connections: adhesion junctions have broader functions.

Cadherins - a family of cell-cell adhesion molecules - are linked to the actin cytoskeleton via intervening proteins. Recent results address molecular explanations for observed cadherin behavior, point to signals that regulate adhesion by modulating elements of the cadherin-associated complex, challenge the belief that different cadherins generally cannot cross-adhere, and highlight instructive roles for cadherins in cell signaling and differentiation.

Tissue spreading on implantable substrates is a competitive outcome of cell–cell vs. cell–substratum adhesivity

While the interactions of cells with polymeric substrata are widely studied, the influence of cell–cell cohesivity on tissue spreading has not been rigorously investigated. Here we demonstrate that the rate of tissue spreading over a two-dimensional substratum reflects a competition or “tug-of-war” between cell–cell and cell–substratum adhesions. We have generated both a “library” of structurally related copolymeric substrata varying in their adhesivity to cells and a library of genetically engineered cell populations varying only in cohesivity. Cell–substratum adhesivity was varied through the poly(ethylene glycol) content of a series of copolymeric substrata, whereas cell–cell cohesivity was varied through the expression of the homophilic cohesion molecules N- and R-cadherin by otherwise noncohesive L929 cells. In the key experiment, multicellular aggregates containing about 600 cells were allowed to spread onto copolymeric surfaces. We compared the spreading behavior of aggregates having different levels of cell–cell cohesivity on a series of copolymeric substrata having different levels of cell–substratum adhesivity. In these experiments, cell–cell cohesivity was measured by tissue surface tensiometry, and cell–substratum adhesivity was assessed by a distractive method. Tissue spreading was assayed by confocal microscopy as the rate of cell emigration from similar-sized, fluorescence-labeled, multicellular aggregates deposited on each of the substrata. We demonstrate that either decreasing substratum adhesivity or increasing cell–cell cohesivity dramatically slowed the spreading rate of cell aggregates.

Coaction of intercellular adhesion and cortical tension specifies tissue surface tension

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.

Mechanism of Tissue Reconstruction by Dissociated Cells, II: Time-Course of Events

The details of the process by which cells sort out and reconstruct tissues within aggregates containing two kinds of tissue cells have been correctly predicted from considerations of the kinetic and adhesive properties of such cells. The requisite properties are discreteness, motility, and differential mutual adhesiveness among the types of cells present.

A rheological mechanism sufficient to explain the kinetics of cell sorting

Abstract When two different vertebrate embryonic tissues are dissociated into individual cells, which are then recombined into mixed aggregates, the differing cells sort out within the aggregates to form a characteristic structure. The kinetics of cell sorting closely resembles the kinetics of breaking of an unstable emulsion of two immiscible liquids. We investigate the consequences of the postulate that cell rearrangement in such a system is driven by the tension at the interfaces between the two cell populations and resisted by “tissue viscosities”, the latter being a newly recognized parameter of cell sorting. Using preliminary experimental data on cell population interfacial tensions and on the time for fusion of two identical spherical aggregates, the viscous liquid model leads to estimates for tissue viscosities in the range of 0.4 × 10 6 to 0.7 × 10 8 poise. Also, using two other independent sets of data, one on the time for breaking of a roughly cylindrical cell aggregate into a few clusters, and the other on the time for the rounding-up of an approximately ellipsoidal tissue mass into a roughly spherical mass, tissue viscosities are again estimated to be in the range of 10 6 to 1.5 × 10 8 poise. In attempting to find a possible basis for such high effective viscosities, we propose that: (i) tissue viscosities would most likely result from sliding friction between the cell membranes; (ii) the cell membranes would have to possess protrusions of molecular or macromolecular dimensions; and (iii) the ratio of the surface tension to the logarithm of viscosity should, if this model is correct, be approximately constant, independent of the tissue.

Quantitative differences in tissue surface tension influence zebrafish germ layer positioning

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.

On the recovery of adhesiveness by trypsin-dissociated cells

SummaryA 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.

Two distinct adhesion mechanisms in embryonic neural retina cells. I. A kinetic analysis.

The reaggregation kinetics of embryonic chick neural retina cells prepared using several different dissociation procedures were monitored through decreases in the small-angle light scattering of aggregating samples. Two distinct modes of aggregation were revealed, one Ca2+ independent, the other Ca2+ dependent, suggesting the existence of two separate adhesion mechanisms. By varying the concentrations of Ca2+ and trypsin in the dissociation medium, we obtained cells which exhibited both, either, or neither mode of aggregation. The Ca2+-independent adhesiveness is active in the absence of proteolysis, is resistant to low levels of trypsin (0.001%), but is readily inactivated at higher trypsin concentrations in either the presence or absence of Ca2+. It is relatively temperature independent. By contrast, the Ca2+-dependent adhesiveness is not detected before exposure of the cells to proteolysis. It is expressed after tryptic proteolysis in the presence of Ca2+ and is then highly temperature dependent. It is resistant to further digestion by trypsin in the continued presence of Ca2+ but is lost when Ca2+ is subsequently removed, apparently through the expression of tryptic cleavage incurred earlier. We suggest that its increased activity may result at least in part from the clustering of surface components into adhesive patches. A provisional model is presented correlating these data.

Adhesion-guided multicellular assembly: a commentary upon the postulates, real and imagined, of the differential adhesion hypothesis, with special attention to computer simulations of cell sorting*

The differential adhesion hypothesis (DAH) explains cell sorting and related cell rearrangements as progressions of motile and mutually adhesive cell populations toward configurations of minimal interfacial (adhesive) free energy. Many behavioral predictions based upon this hypothesis have been confirmed. However, elements of the hypothesis itself have been misunderstood by some authors, who have consequently erred in their expectations of the behavior it would predict. One commonly held misconception is that this hypothesis entails an assumption that all cells adhere to one another through a common chemical mechanism. Other errors have been introduced in connection with the interpretation of the results of computer simulations of cell sorting. In general, when a computer simulation based upon the DAH has failed to generate all of the expected behavior, there has been a tendency to attribute this to shortcomings of the DAH itself. It is here shown, however, that the difficulty, in each instance of which I am aware, has lain either in faulty understanding of the behavior actually displayed by living cell populations as they sort out, or in some inadequacy of the simplified rules fed to the computer. When these deficiences are corrected, DAH-based computer simulations of cell sorting, although awkward, nevertheless produce fairly lifelike approximations of cell-sorting behavior.