Differential Adhesion Hypothesis Research Articles

Preferential Adhesion Mediated by Hibris and Roughest Regulates Morphogenesis and Patterning in the Drosophila Eye

Cell adhesion is essential for morphogenesis; however, the mechanisms by which cell adhesion coordinates precisely regulated morphogenesis are poorly understood. Here we analyze the morphogenetic processes that organize the interommatidial precursor cells (IPCs) of the Drosophila pupal eye. We demonstrate that the Drosophila immunoglobulin superfamily members Hibris and Roughest are essential for IPC morphogenesis in the eye. The two loci are expressed in complementary cell types, and Hibris and Roughest proteins bind directly in vivo. Primary pigment cells employ Hibris to function as organizers in this process; IPCs minimize contacts with neighboring IPCs and utilize Roughest to maximize contacts with primaries. In addition, we provide evidence that interactions between Hibris and Roughest promote junction formation and that levels of Roughest in individual cells determine their capacity for competition. Our results demonstrate that preferential adhesion mediated by heterophilic interacting cell-adhesion molecules can create a precise pattern by minimizing surface free energy.

The differential adhesion hypothesis: a direct evaluation

The differential adhesion hypothesis (DAH), advanced in the 1960s, proposed that the liquid-like tissue-spreading and cell segregation phenomena of development arise from tissue surface tensions that in turn arise from differences in intercellular adhesiveness. Our earlier measurements of liquid-like cell aggregate surface tensions have shown that, without exception, a cell aggregate of lower surface tension tends to envelop one of higher surface tension to which it adheres. We here measure the surface tensions of L cell aggregates transfected to express N-, P- or E-cadherin in varied, measured amounts. We report that in these aggregates, in which cadherins are essentially the only cell–cell adhesion molecules, the aggregate surface tensions are a direct, linear function of cadherin expression level. Taken together with our earlier results, the conclusion follows that the liquid-like morphogenetic cell and tissue rearrangements of cell sorting, tissue spreading and segregation represent self-assembly processes guided by the diminution of adhesive-free energy as cells tend to maximize their mutual binding. This conclusion relates to the physics governing these morphogenetic phenomena and applies independently of issues such as the specificities of intercellular adhesives.

Cadherin-mediated cell adhesion and tissue segregation: qualitative and quantitative determinants

It is widely held that segregation of tissues expressing different cadherins results from cadherin-subtype-specific binding specificities. This belief is based largely upon assays in which cells expressing different cadherin subtypes aggregate separately when shaken in suspension. In various combinations of L cells expressing NCAM, E-, P-, N-, R-, or B-cadherin, coaggregation occurred when shear forces were low or absent but could be selectively inhibited by high shear forces. Cells expressing P- vs E-cadherin coaggregated and then demixed, one population enveloping the other completely. To distinguish whether this demixing was due to differences in cadherin affinities or expression levels, the latter were varied systematically. Cells expressing either cadherin at a lower level became the enveloping layer, as predicted by the Differential Adhesion Hypothesis. However, when cadherin expression levels were equalized, cells expressing P- vs E-cadherin remained intermixed. In this combination, “homocadherin” (E-E; P-P) and “heterocadherin” (E-P) adhesions must therefore be of similar strength. Cells expressing R- vs B-cadherin coaggregated but demixed to produce configurations of incomplete envelopment. This signifies that R- to B-cadherin adhesions must be weaker than either “homocadherin” adhesion. Together, cadherin quantity and affinity control tissue segregation and assembly through specification of the relative intensities of mature cell–cell adhesions.

Formation and maintenance of distinctive cell patterns by coexpression of membrane-bound ligands and their receptors.

We show that graded or checkerboard-like cell patterns, and segmental domains along a body axis, can be generated by cell behaviors involving differences in intercellular repulsion. A membrane-bound signal transduction system mediating contact-dependent cell interactions includes membrane-bound ligands (ephrins) and their receptors with tyrosine-kinase activity (Eph proteins). These molecules mediate both repulsive and attractive interactions under bilateral threshold control, i.e., cells expressing the receptors adhere to a surface bearing a critical density of ligand reciprocal to the density of receptor but are repelled by a surface with other densities of ligand (Honda [1998] J. Theor. Biol. 192:235-246). We extend this model. General membrane-bound ligands (not always ephrins) and their receptors are presumably coexpressed in a single cell under bilateral threshold control. Computer simulations of cell pattern formation showed that when coexpression of the ligand and receptor is reciprocal, the cells self-organize into a pattern of segmental domains or a graded cell arrangement along the body axis. The latter process interprets positional information in terms of protein molecules. When coexpression of the two species of molecules is not always reciprocal, the cells generate various patterns including checkerboard and kagome (star) patterns. The case of separate expression of ligands and receptors in different cells is also examined. The mechanism of differences in cell repulsion is compared with the differential cell adhesion hypothesis, which has been used to explain cell sorting.

Cell sorting is analogous to phase ordering in fluids.

Morphogenetic processes, like sorting or spreading of tissues, characterize early embryonic development. An analogy between viscoelastic fluids and certain properties of embryonic tissues helps interpret these phenomena. The values of tissue-specific surface tensions are consistent with the equilibrium configurations that the Differential Adhesion Hypothesis predicts such tissues reach after sorting and spreading. Here we extend the fluid analogy to cellular kinetics. The same formalism applies to recent experiments on the kinetics of phase ordering in two-phase fluids. Our results provide biologically relevant information on the strength of binding between cell adhesion molecules under near-physiological conditions.

The mechanics of cell sorting and envelopment

Aggregates of embryonic cells undergo a variety of intriguing processes including sorting by histological type and envelopment of cell masses of one type by another. It has long been held that these processes were driven by differential adhesions, as embodied in the famous differential adhesion hypothesis (DAH). Here, we use analytical mechanics to investigate the forces that are generated by various sub-cellular structures including microfilaments, cell membranes and their associated proteins, and by sources of cell–cell adhesions. We consider how these forces cause the triple junctions between cells to move, and how these motions ultimately give rise to phenomena such as cell sorting and tissue envelopment. The analyses show that, contrary to the widely accepted DAH, differential adhesions alone are unable to drive sorting and envelopment. They show, instead, that these phenomena are driven by the combined effect of several force generators, as embodied in an equivalent surface or interfacial tension. These unconventional findings follow directly from the relevant surface physics and mechanics, and are consistent with well-known cell sorting and envelopment experiments, and with recent computer simulations.

Embryonic tissues are viscoelastic materials

Early embryonic development is characterized by spectacular morphogenetic processes such as sorting or spreading of tissues. Analogy between viscoelastic fluids and certain properties of embryonic tissues turned out to be useful in interpreting some aspects of these morphogenetic phenomena. In accordance with the differential adhesion hypothesis, the values of tissue-specific surface tensions have been shown to be consistent with the equilibrium configurations such tissues reach in the course of sorting and spreading. A method to measure tissue surface tension and viscoelastic properties is described. Notions like the Laplace's equation relating surface tension to radii of curvature, or the Kelvin model of viscoelasticity are used to analyze the results of these measurements. The fluid analogy is extended to time-dependent phenomena, in particular, to the analysis of cellular pattern evolution in the course of spreading. On the basis of recent experimental findings, we demonstrate that the kinetics of spreading and nucleation in binary fluids can be analyzed using the same formalism. We illustrate how our results can be used to obtain biologically relevant information on the strength of binding between specific cell adhesion molecules under near physiological conditions. We also suggest a diagnostic application of our method to monitor the metastatic potential of tumors. PACS No.: 03.65Ge

Existence of gradient in cell adhesiveness along the developing Xenopus hind limb bud, shown by a cellular sorting-out experiment in vitro.

To examine the possibility of a difference in cell adhesiveness along the developing Xenopus hind limb bud axes, single mesenchymal cells from developing hind limb buds were cultured, allowing them to form an aggregate in a gyratory culture system. By observing the distribution of cells within aggregates, it was found that sorting-out occurred between cells from different positions and different stages. Cells derived from more distal positions tended to be situated interiorly in the aggregates. According to Steinberg's differential adhesion hypothesis, these results support the idea that there is a graded difference in cell adhesiveness along the proximo-distal axis of the developing limb, with adhesiveness increasing distally. Although similar sorting-out was observed between anterior and posterior cell populations, it could not be determined which cell populations were definitely more cohesive. These properties may be correlated with the experimentally demonstrated 'positional value', which should be different among cells located at different positions along the axes of the developing vertebrate limb bud.

Germ-Layer Surface Tensions and “Tissue Affinities” inRana pipiensGastrulae: Quantitative Measurements

The morphogenetic properties causing germ-layer spreading and stratification in amphibian gastrulation were called “tissue affinities” by Holtfreter. The differential adhesion hypothesis (DAH) attributes such liquidlike tissue rearrangements to forces generated by intercellular adhesions within and between the migrating cell populations. This theory predicts that, among the primary germ layers, the cohesiveness of deep ectoderm should be the greatest, that of deep mesoderm should be intermediate, and that of deep endoderm should be the least. Also, the cohesiveness of differentiating neural ectoderm should increase after induction, causing it to internalize and segregate from epidermis. The DAH also explains why the cohesiveness of “liquid” tissues, whose cells are free to rearrange, should be measurable as tissue surface tensions. Using a specially designed tissue surface tensiometer, we demonstrate that (i) aggregates ofRana pipiensdeep germ layers do possess liquid-like surface tensions, (ii) their surface tension values lie in precisely the sequence necessary to account for germ-layer stratificationin vitroandin vivo,and (iii) the surface tension of deep ectoderm just underlain by the archenteron roof is twice that of not-yet-underlain deep ectoderm. These measurements provide direct, quantitative evidence that the “tissue affinities” governing germ-layer flow during early stages of vertebrate morphogenesis are reflected in tissue surface tensions.

Surface tensions of embryonic tissues predict their mutual envelopment behavior

During embryonic development, certain tissues stream to their destinations by liquidlike spreading movements. According to the 'differential adhesion hypothesis', these movements are guided by cell-adhesion-generated tissue surface tensions (sigmas), operating in the same manner as surface tensions do in the mutual spreading behavior of immiscible liquids, among which the liquid of lower surface tension is always the one that spreads over its partner. In order to conduct a direct physical test of the 'differential adhesion hypothesis', we have measured the sigmas of aggregates of five chick embryonic tissues, using a parallel plate compression apparatus specifically designed for this purpose, and compared the measured values with these tissues' mutual spreading behaviors. We show that aggregates of each of these tissues behave for a time as elasticoviscous liquids with characteristic surface tension values. Chick embryonic limb bud mesoderm (sigma = 20.1 dyne/cm) is enveloped by pigmented epithelium (sigma = 12.6 dyne/cm) which, in turn, is enveloped by heart (sigma = 8.5 dyne/cm) which, in turn, is enveloped by liver (sigma = 4.6 dyne/cm) which, in turn, is enveloped by neural retina (sigma = 1.6 dyne/cm). Thus, as predicted, the tissues' surface tension values fall in the precise sequence required to account for their mutual envelopment behavior.

Cellular segregation and engulfment simulations using the cell programming language

In developmental biology, modeling and simulation play an important role in understanding cellular interactions. In this paper a simple language, the Cell Programming Language(CPL), is suggested for writing programs that describe this behavior. Using these programs, it is possible to simulate and visualize intercellular behavior. CPL is used to model cellular segregation based upon the differential adhesion hypothesis. Results indicate that a high degree of segregation can be produced in a mixture of cells by allowing random motion. The engulfment of a tissue by a less adhesive tissue is also observed when the two tissues are placed in contact. Both these simulations utilize only local interactions and random motion of cells. Earlier simulations used long-range interactions to observe similar effects. The present simulations prove that random motion of cells can produce long-range effects.

Cell sorting out: the self-assembly of tissues in vitro.

The question posed by the science of analytical histology is how the properties and interactions of the components of the tissues determine their organization in the organs. The relevant components of the tissues are the cells and the extracellular matrix. The ability of cohering populations of cells to self-assemble structured tissues by cell sorting out offers an important opportunity for the experimental study of the mechanisms by which the cells and extracellular matrix interact to determine structure. The investigator can manipulate the initial organization and the cellular composition of the system and, in favorable situations, the composition of the extracellular matrix and the activities of candidate adhesive molecules. It can reasonably be expected that the recent progress in the characterization of the molecular species involved in cell-cell and cell-extracellular matrix interaction will allow the analysis of the molecular basis of tissue organization, with study of the self-assembly of tissue structure during sorting out playing an important role in this analysis. The importance of the differential adhesion hypothesis is its success in describing the rules by which macroscopic tissue structure is governed by the adhesive interactions of cell with cell and cell with extracellular matrix. The DAH describes how the physical forces of cell-cell and cell-matrix adhesion determine structure. Elucidation of the particular adhesive molecules involved in these interactions (e.g., the CAMs, junctional proteins, and matrix adhesion molecules) will yield an explanation at the biochemical level. A complete understanding of structure requires both levels of explanation.

Can retinal adhesion mechanisms determine cell-sorting patterns: a test of the differential adhesion hypothesis.

Embryonic chick neural retina cells possess two classes of adhesion mechanism, one Ca2+-independent, one Ca2+-dependent, responsible for short-term cell aggregation. This study investigates the role of these mechanisms in the long-term cell sorting potentially relevant to in vivo histogenesis. Retina cells are prepared either with both (E cells) or with only one mechanism (TC cells, CD; LTE cells, CI), respectively. The two types of cell preparations are differentially labelled using fluorescein or rhodamine isothiocyanate, mixed and allowed to aggregate in the presence or absence of cycloheximide at 0.5 microgram ml-1 to retard metabolic recovery of the removed adhesive mechanism. When observed by fluorescence and phase-contrast microscopy, the aggregates formed in cycloheximide show cell sorting, the cells with both mechanisms assuming a more interior position relative to those with a single adhesion mechanism. In parallel hanging-drop experiments, preformed aggregates of cells with a single adhesion mechanism are seen to spread upon aggregates of cells with both mechanisms. No sorting occurs amongst cells from a given stage prepared using any single dissociation protocol. The observed cell sorting would thus seem to derive exclusively from differential cell adhesiveness dependent upon the different dissociation conditions and maintained in the presence of cycloheximide. The experiments support the hypothesis that the dual CI and CD adhesion mechanisms in question can play a central role in governing cell-sorting behaviour during normal histogenesis.

Strategies for specifying form and pattern: adhesion-guided multicellular assembly.

We define a material pattern as a particular arrangement of material elements in space. We then make an effort to categorize the developmental strategies that underlie the emergence of multicellular patterns. These strategies are divided into three broad categories according to whether cell position influences or is influenced by cell fate. In that category of strategies in which cell fate influences cells to move to particular positions, we focus our attention upon morphogenetic and patterning phenomena that appear to be determined by adhesion-mediated interactions of cells with each other and with their surroundings. The differential adhesion hypothesis details how cellular adhesive properties can guide tissue movements and specify patterns of cell association. Motile, adhesive cells will naturally tend to group so as to maximize their adhesive interactions (minimize interfacial free energy). A homogeneous population of uniformly adhesive (isotropic) cells will tend toward spherical form. Cell surface adhesive anisotropies can determine other most-stable (equilibrium) configurations of the population, such as cell sheets, tubes and vesicles. Heterogeneous cell populations may preferentially either intermix or sort out, depending upon the balance of adhesive forces between like and unlike elements. The precise configuration adopted will depend upon the particular adhesive relationships that prevail. Both this end state and the approach toward it arise from the adhesive relationships among the interacting cells. Such morphogenetic phenomena as tissue spreading and the segregation of organ primordia are probably brought about in this way. We outline here some results of our recent experiments on the morphogenesis of the salamander pronephric duct. These illustrate the reality of emergent adhesion-generated tissue immiscibility as a cause of organ segregation and point toward a craniocaudally travelling adhesion gradient as the information that guides the migrating pronephric duct to the cloaca.

The sorting out of embryonic cells in monolayer, the differential adhesion hypothesis and the non-specificity of cell adhesion.

It has been reported previously that sorting out of chick embryonic liver parenchyma and limb bud mesenchymal cells would take place in monolayer culture. The distribution of cell types obtained (liver formed the internal, discontinuous phase) was interpreted in terms of the differential adhesion hypothesis. It was suggested that, in monolayer, liver cells were more cohesive than limb bud cells. In this paper we set out to extend the previous observations with 2 particular questions in mind: (i) Is sorting out in monolayer a general phenomenon occurring between a wider range of cell types? (ii) Can evidence be provided for or against the interpretation of results in terms of the differential adhesion hypothesis? Sorting-out experiments were conducted on circular hydrophilic islands, on an otherwise hydrophobic substratum. Under these conditions, sorting-out in monolayer was obtained with binary combinations of 4 chick embryonic tissue types: liver parenchyma, limb bud mesenchyme, pigmented epithelium of the eye and corneal epithelium. With every combination but one, the cells of one type surrounded the cells of the other type, generating what we have called a 'circle-within-a-circle' configuration. With the remaining combination, liver parenchyma and corneal epithelium, only localized sorting was obtained. The 'circle-within-a-circle' configuration is consistent with an interpretation in terms of the differential adhesion hypothesis, according to which the distribution of cells is determined by the relative strengths of cohesions between their lateral surfaces. In direct support of this is the finding from plating the different cell types at sub-confluent density on hydrophilic substrata that limb bud is the cell tye having the weakest lateral cohesion in monolayer. Limb bud surrounded the other 3 tissues on hydrophilic island. A hierachy of lateral cohesiveness between the 4 cell types has been constructed. It is unlikely that the results can be explained in terms of specific cohesion. When plated together at subconfluent density, the 3 epithelial cell types aggregate together to form mixed monolayered islands, suggesting that they share common adhesive mechanisms.

Can the differential adhesion hypothesis explain how an aggregate of strongly cohesive cells can be penetrated by more weakly cohesive cells?

The differential adhesion hypothesis, as advanced by Steinberg [Steinberg (1963). Science 14, 401–408; (1964). In “Cellular Membranes in Development” (M. Locke, ed.), pp. 321–366. Academic Press, New York; (1970). J. Exp. Zool. 173, 395–434; (1975). J. Theor. Biol. 55, 431–443], predicts that, in an aggregate composed of cells from two different kinds of tissue, more cohesive cells will tend to become enveloped or covered by an external layer of less cohesive cells. Both embryonic chick heart and liver cells sorted out externally in every case to embryonic limb bud cells and are, therefore, according to the hypothesis, less cohesive than limb bud cells. However, when a few of the less cohesive heart or liver cells were seeded onto the surfaces of aggregates of the more cohesive limb bud cells, about half of the less cohesive cells assumed subsurface positions within several days of culture. The penetration of an aggregate of cohesive cells by less cohesive cells may indicate that the differential adhesion hypothesis will require modification for universal applicability to the cell level.

Self vs nonself in tissue assembly: Correlated changes in recognition behavior and tissue cohesiveness

Vertebrate embryonic cell populations of unlike kind, when combined in vitro, typically spread around and sort out from one another in combination-specific patterns, whereas like cell populations merely coalesce. These differing responses to self and nonself constitute one form of morphogenetic self-recognition behavior. Prolonged shaker-flask culturing and dissociation and reaggregation of embryonic chick heart tissue were both previously shown to reverse the tissue's spreading behavior with liver. Here, we show that these treatments simultaneously initiate, in heart tissue, a “foreign” spreading reaction toward untreated heart. Moreover, the direction of this heart-heart spreading can be deduced from the change in direction of heart-liver spreading. This suggests that certain properties of heart tissue participate in the determination of both the foreign- and the self-recognition behaviors studied here. The differential adhesion hypothesis postulates that these properties are the intensities of tissue cohesion, with less cohesive tissues enveloping more cohesive ones. If so, our observations imply that heart fragments precultured 1 2 day should be more cohesive than 1 2 - day precultured heart reaggregates, but less cohesive than heart fragments precultured 2 1 2 days. With our centrifugation assay, in which relative tissue cohesiveness is assessed by the relative roundness of centrifuged aggregates at shape equilibrium, we confirm this prediction.

Is cell sorting caused by differences in the work of intercellular adhesion? A critique of the steinberg hypothesis

The differential adhesion hypothesis, developed by Malcolm Steinberg, proposes that the histotypic sorting out behavior of aggregated cells is mechanistically equivalent to certain aspects of liquid surface tension, specifically the spontaneous separation of immiscible liquids of differing surface tension. According to Steinberg's hypothesis, the adhesive forces between aggregated cells play essentially the same role in cell sorting as are played by intermolecular attractive forces in liquid surface tension. In this paper I discuss a number of crucial distinctions between intermolecular attraction (in liquids) and intercellular adhesion (in aggregates). First, liquid drops are closed systems thermodynamically whereas aggregates of living cells can generate an indeterminate amount of metabolic energy capable of altering cell positions and adhesions. Secondly, intercellular adhesions are more than just close range attractions since cells can be held together by forces in addition to those which originally pulled them together. Third, the breakage of intercellular adhesions need not be simply the reverse, thermodynamically, of the formation of those adhesions. And fourthly, because intercellular adhesion is generally concentrated at relatively small foci such as desmosomes, a maximization of intercellular adhesion does not necessarily require a maximization of intercellular contact area, or vice versa. In addition, several alternative hypotheses are proposed, each of which is theoretically capable of explaining cell sorting and the other surface tension-like aspects of cell aggregate behavior which Steinberg has sought to explain as consequences of differential adhesion. In particular, a differential surface contraction hypothesis is proposed, according to which cell sorting and related phenomena are the results of cell surface contractions induced to occur over those portions of the cell surface which are exposed to the surrounding culture medium. Because of the evidence that various invagination type movements of embryonic epithelia are caused by cell surface contractions, it is suggested that differential surface contraction is the most likely explanation of histotypic cell sorting. A number of experiments are suggested by which these various hypotheses might be tested.

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.

Contact inhibitions of overlapping and differential cell adhesion: a sufficient model for the control of certain cell culture morphologies.

ABSTRACT Using intuitive arguments, several investigators have proposed that the relative strengths of adhesion of cell to cell and of cell to substratum could determine whether or not monolayering – and specifically contact inhibition of cell overlapping – will occur. In the present communication, these ‘strengths of adhesion’ are given precise physical definitions, and the adhesive relationships which would promote spontaneous cell monolayering are rigorously derived, using the thermodynamic approach embodied in the differential adhesion hypothesis. This analysis verifies that contact inhibition of overlapping could, in principle, be a result solely of differential adhesion. In addition, it is demonstrated that for homogeneous populations of uniform cells cultured on a solid, uniform substratum, eleven distinct equilibrium configurations (cell population morphologies) could be generated merely by varying the relative values of cell-to-cell and cell-to-substratum adhesiveness. Most of these configurations have been observed previously in actual cell cultures.