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Previous studies have indicated that cell sorting and tissue spreading are caused by cell combination-specific differences in intercellular adhesive energies, acting in a system of motile cells. We wished to determine whether these adhesive energies could drive cell rearrangements as well as guide them. Accordingly, aggregates of intermixed embryonic cells were cultured in solutions of the drug cytochalasin B (CCB) at a concentration shown to inhibit the locomotion of cells on a solid surface. In addition, spherical aggregates of several kinds were cultured in mutual contact under similar conditions. Both cell sorting and tissue spreading were found to be inhibited. The prompt release of this inhibition upon removal of the CCB showed that the inhibited cells were not merely injured. Moreover, aggregation experiments showed that CCB did not prevent cells of several kinds from initiating mutual adhesions. In fact, heart cell aggregation was enhanced by CCB. We conclude that interfacial forces, originating outside the cell, act together with forces originating inside it in bringing about the morphogenetic movements of cell sorting and tissue spreading. We propose the term "cooperative cell locomotion" to describe translational movements of cells arising from such a combination of intrinsic and extrinsic forces.

The ability of cytochalasin B to inhibit ruffled membrane activity and cellular locomotion of vertebrate cells in monolayer culture prompted its use to study the necessity for this kind of active cellular locomotion in cell sorting in heterotypic cell aggregates. Cell sorting was inhibited in chick embryo heart-pigmented retina aggregates but a remarkable degree of sorting did occur in neural retina-pigmented retina aggregates. In these experiments, the levels of cytochalasin B employed (5 or 10 microg/ml) are sufficient to inhibit completely locomotion of these cell types in monolayer culture. It is proposed that the degree of cell movement achieved during sorting in neural retina-pigmented retina aggregates in the presence of cytochalasin B is the result of changes in cell contact resulting from adhesive interaction of cells. The effect of cytochalasin B on the initial aggregation of dissociated cells was also tested. With the cell types used in this study (chick embryo neural retina and limb bud), aggregation was not affected for a period of several hours.

Mixed suspensions of cells obtained by dissociation of 7 day chicken embryo heart and pigmented retina were allowed to reaggregate in tissue culture. The reaggregates which resulted contained both kinds of cells. Establishment of homogeneous tissues by cell sorting out in these reaggregates was advanced by 20 hr in culture and was complete within 2 days. When sorting out was advanced, heterotypic aggregates were fixed, sectioned, and examined in the electron microscope. Particular attention was paid to the morphology of regions of contact between cells. No qualitative differences were observed in the contact junctions between like cells (heart-heart or pigmented retina-pigment retina junctions) and unlike cells (heart-pigmented retina junctions). Broad areas of undifferentiated cell contact with cell membranes separated by a 100-200 A gap were formed regardless of cell type. Specialized junctions of the fascia and macula adherens type were also present, not only between like cells but also between unlike cells.