In this article we will discuss about the selective affinities of cells as a determining factor in cellular rearrangements of an embryo.
We may ask what is the nature of the forces which control the rearrangement of cells resulting from morphogenetic movements in gastrulation and also in other morphogenetic processes; we may ask whether the new positions of the cells are the result of their own special properties, or whether the organization of the system as a whole (i. e., the whole embryo) is decisive in bringing each kind of cell into its appropriate position.
These questions have been investigated by separating the cells of the embryo and allowing them to interact in vitro. The cells of amphibian embryos placed in water, which had been made alkaline (pH 9.6 to 9.8) by adding potassium hydroxide, lose cohesion, and the embryo or parts of it treated with alkali dis-aggregate into a mass of disconnected single cells.
Masses of cells prepared in this fashion from different embryonic tissues (gastrula ectoderm, mesoderm or endoderm, neurula epidermis, neural plate, and others) may then be mixed together in various combinations and returned to a fresh solution of pH 8.0, in which the cells return to their normal state and again join together.
After clumps of cells are formed, the cells begin to sort themselves out according to their properties. The ectodermal and epidermal cells combined with mesodermal or neural plate cells always ended up by assembling on the surface of the aggregate. The mesodermal and neural plate cells combined either with epidermis or with endoderm disappeared from the surface and were to be found forming solid masses in the interior.
The masses of neural plate cells became hollowed out later and formed structures resembling brain vesicles. Masses of mesodermal cells arranged themselves around “coelomic cavities”. If epidermis, mesoderm, and endoderm were present in an aggregate, the epidermis cells concentrated on the exterior, and mesodermal cells took up a position between the epidermis and the endoderm; the latter, rather unexpectedly, also occupied part of the external surface of the aggregate.
This is, however, not unlike the position of the endoderm in the case of exo-gastrulation. In this way, arrangements of cells similar to those produced at the end of normal gastrulation and neurulation are brought about by the activities of individual cells beginning from a completely abnormal starting point.
In later work, more refined methods have been used for disaggregating cells of embryonic tissues. The result is achieved either by a short treatment with trypsin or by placing the tissues in a medium devoid of Ca and Mg ions and with EDTA (ethylenediaminetetra-acetic acid, a chelating agent) added. Use of the latter method is based on the discovery that Ca and Mg ions play an important part in holding cells together; EDTA binds these ions, removing them from intercellular connections.
The sorting out of mixtures of different kinds of cells occurs not only in cells taken from embryos in stages of gastrulation and neurulation, but also with cells taken from older embryos. Cells of the heart rudiment, pro-cartilage, the liver, the pigmented retina, and others sort themselves out, after having been combined at random, and take up positions either on the surface or in the interior of the clump. Three different tissues may be combined, and they will rearrange themselves into three concentric layers.
Some of these combinations copy the normal arrangement, as when muscle cells surround cartilages. In other cases, when the cells are of such a nature that they do not normally occur together, entirely new combinations are produced. Thus, when limb-bud pro-cartilage, heart muscle, and pigmented retina are combined, pro-cartilage is located in the interior and is surrounded by retina, with heart cells forming the outer layer.
The behavior of the cells under these conditions—and by inference also in normal development—is presumably due to their selective affinities. When cells touch one another as a result of their random movement, they tend to remain in contact.
It is supposed that the forces holding the cells together vary, depending on the kind of cells involved. According to its special properties, a cell may either adhere to another cell or stay in contact with it or move away from a position in which the surroundings are not of such a nature that they keep the cell bound.
Generally speaking, the result of these selective affinities would lead to like cells being brought together. This does not necessarily mean that the bonding between such cells is of a strictly specific nature; to allow similar cells to sort themselves out from among cells with differing properties, it would suffice that the adhesive properties of the former, at any given time, be equal in grade or intensity.
A cell capable of adhering to another cell or cells may be said to possess a certain amount of free energy, the energy that would be used to establish and maintain a contact. General principles require that a system—in our case and aggregate of cells— reach an equilibrium when its free energy is at a minimum or when the adhesiveness of the cells is used to a maximal extent or is “maximalized”.
As cells on the surface of the aggregate do not use their adhesiveness to a maximal capacity (their outer surfaces not being in contact with other cells), the requirements of a system with a minimum of free energy dictate that the aggregate have the least possible surface area. A body with the least possible surface for a given volume is, of course, a sphere, and we see that the aggregates generally assume a spherical shape.
In tending to form a sphere, a clump of cells resembles a drop of liquid suspended in another liquid with which it does not mix—as a drop of oil in water. In fact, a mass of re-aggregating cells does behave as if it were a liquid, in which the surface tension is the force that gives a drop of liquid its spherical form. Different liquids have different surface tensions, depending on their chemical compositions.
Oil, for instance, has a much higher surface tension than water; as a result, water envelops oil, and not vice versa. In the case of tissue aggregates, the surface tension, which is the result of the adhesiveness of the cells to one another, could be directly measured by exposing tissue aggregates to high centrifugal force. The greater the affinity of the cells for one another, the greater is the resistance of the cell aggregate to depart from the spherical shape and to being flattened under the influence of centrifugal force.
The differences in this respect found between different tissues were in correspondence to their behavior in combined cell aggregates- the tissues flattening less under the influence of the centrifugal force, and thus having the greater adhesiveness, occupied internal positions in combined cell aggregates; the tissues flattening to a greater extent, thus having less adhesiveness, occupied external positions in mixed cell aggregates.
If cells with different degrees of adhesiveness are available, the least free energy requirement dictates that only those cells should stay on the surface whose adhesiveness is least; the cells with greater adhesiveness will withdraw into the interior.
In the interior these cells will maximalize their adhesiveness if they appose their surfaces to cells of the same kind, rather than to cells whose adhesiveness is lower. As a result, whenever such highly adhesive cells meet, they will become drawn to each other and eventually will form large clumps within the aggregate.
There seems to be no attraction of like cells from a distance (no evidence for a chemotaxis among cells), and the ability of individual cells to move within the aggregate, once it is formed, is limited to 10 to 30 µ. This was established by direct observation of pigmented retina cells which were mixed with transparent cells of the heart.
However, some contacts could be established as a result of even this limited mobility. The sphere of activity of cells could perhaps be extended still further by filopodia-like extensions of the cytoplasm. The facilities for contacts between like cells of an aggregate must be fairly great, however, since the eventual separation and stratification of unlike cells usually are exceedingly clear-cut.
A complication of the processes governing the arrangement of cells in aggregates is brought about if only part of the surface of the cells involved is adhesive and another part is non-adhesive. In the amphibian neurula the free outer surface of epidermal cells becomes non-adhesive, and this may be the case in some other epithelial cells as well.
When such cells participate in the aggregate they end up forming a layer on the outer surface. If the number of such partially non-adhesive cells were large in relation to the volume of the whole, the surface area would no longer need to be the least possible one. In fact, the surface may be thrown into folds to accommodate all the partially non-adhesive cells.
The theory of differential adhesiveness does not tell us anything about the physical nature of the forces that keep the cells together. These may be of a different nature in different cases, or different forces may cooperate in joining cells, especially joining cells of a similar nature.
It has been suggested that surfaces of tells are provided with special molecules serving for the recognition of similar cells. It is of course a well-known fact that surfaces of cells may be highly specific, supplied with specific antigens in the form of protein molecules protruding from the cell membrane, or attached to it, or in the form of glycoprotein molecules attached to the cell membrane.
To link cells together, however, it is not enough that they should carry specific antigen molecules on their surface; it would be necessary for the molecules in cells which are to be held together to have “complementary” molecules, which could fit and link to each other. In a particular tissue both types of such pairs of “complementary” molecules could be present on the cell surface, so that the linking together would be on a “mutual” basis.
Attempts have been made to detect such “ligand” molecules, and in fact a chemical could be isolated from the supernatant fluid in which certain cells were cultured (cells of the neural retina), or from isolated membranes of the same cells, which greatly accelerates the aggregation of the same kind of cells, after these have been dis-aggregated. The purified “ligand” substance has a molecular weight of 50,000 ± 500.
The “ligand” molecules are supposed to be responsible for the first contact of cells, before they firmly associate. Subsequently, morphologically discernible junctions are formed between the adjacent cells. These may appear very rapidly, in a matter of seconds for gap junctions.
There is one very obvious discrepancy between the results of re-aggregation of isolated embryonic cells and the processes of gastrulation in intact normal embryos. In re-aggregates, the endoderm in combination with mesoderm becomes arranged outside, and the mesoderm takes up a position inside the endoderm, while the reverse happens in normal development. Even if cells of all three germ layers are present in the re-aggregate, the endoderm does not take up a position inside but forms part of the surface on one side of the newly formed complex.
The reason for this discrepancy is that in the stage preceding gastrulation (the blastula stage) the cells are in the form of an epithelium (the blastoderm) which is more or less distinctly polarized. As in typical epithelia, it has a distal surface, in contact with the external environment, which is different from the proximal surface, facing the inner milieu of the embryo.
It has been noticed that when an epithelial layer is bent, or when cells escape from an epithelium as ameboid or mesenchyme cells, the direction of movement is nearly always in the proximal direction, toward the internal milieu of the embryo. It is the proximal end of the cell that is more likely to start forming pseudopods and embarking on ameboid locomotion and this is why in an intact embryo the movement of both mesoderm and endoderm is inward and not outward.
Under experimental conditions, especially if ectoderm is prevented in some way from covering the endoderm (as when extensive parts of the ectoderm and mesoderm are removed by an operation), the endoderm flows to the exterior, and once on the surface it remains there.