Immigration of mesodermal cells from the primitive streak in amniotes and migration of mesenchyme cells in sea urchin gastrulation can be regarded as examples of an important step in morphogenesis – the creation of mesenchyme-type tissues, which are a new structural element of an animal’s body.
The mesenchyme cells are not linked together, as are epithelial cells. They are free to move, and as a result the morphogenetic processes in mesenchyme are somewhat different from those occurring in epithelial cell masses. It is surprising, however, that some of the cellular mechanisms involved in the change in shape of epithelial cells recur in mesenchyme-type cells as well.
The movements of mesenchyme cells have often been described as “ameboid movements,” but this statement must be qualified. The better known type of movement of free-living amebae involves streaming of cytoplasm in the interior of the ameba’s body. When pseudopods are formed at the progressing end of the ameba, internal cytoplasm flows into the pseudopod, and enlargement of the pseudopod is supported or accompanied by this forward streaming of cytoplasm.
When mesenchyme cells (fibroblasts and similar types of cells) are observed in vitro, they are found to move by a completely different mechanism. No internal streaming of cytoplasm can be observed. Instead, the cytoplasm at one end of the cell becomes extended into a very thin edge which is seen to be in continuous undulating movement.
This edge has been described as the “ruffled membrane.” The ruffled membrane intermittently makes and breaks contact with the substrate over which the cell is moving and progressing forward. The rest of the cell trails behind, forming an attenuated “tail” at its posterior end.
The same elements that are involved in the change in shape of epithelial cells are found in migrating mesenchyme cells. The electron microscope shows a mass of microfilaments in the ruffled membrane arranged in the form of a lattice, with the filaments running in different directions. Where the edge of the membrane protrudes, the microfilaments converge to the protruding point.
The trailing “tail” of the cell is supported by longitudinally arranged microtubules. If the cells are treated with cytochalazin B, the movements of the ruffled membrane stop immediately, and the microfilaments are found to have been disrupted and converted into granular cytoplasm.
Colchicine, on the other hand, causes the disintegration of the microtubules, the result being that the “tail” of the cell collapses and is withdrawn into the cell body. Although the movement of the ruffled membrane continues, the cell seems to have become depolarized and cannot progress in one direction, as if the “tail” has been a sort of rudder keeping the cell on course.
The preceding description of the movement of cells refers to observations and experiments on cells cultivated in vitro. The mechanism of cell movements in the organism need not necessarily be the same. In vitro the cells move over a hard flat surface (the bottom of the culture dish), and their movements are thus dominated by cell-to-substrate contacts.
In the organism, and especially in the case of an early embryo, the contacts are cell-to-cell. The study of movements of individual cells in the intact organism involves considerable difficulties, since the object being observed must be sufficiently small and sufficiently transparent for the cells inside the body to be accessible for microscopic observation. For this reason very few reliable studies of this kind are available at present. After becoming free of the epithelial blastoderm, the primary mesenchyme cells start sending out long thin pseudopods, rather than ruffled membranes.
These pseudopods may come into contact with the inner surface of the ectodermal cells and may make firm connection with them. If this occurs, the pseudopod contracts and pulls the mesenchyme cell away from the original site of its immigration into the blastocoele. If the pseudopod fails to make a firm contact or if it does not reach an ectodermal cell but is surrounded by the blastocoele fluid, the pseudopod is withdrawn and new pseudopods are formed.
By this mechanism of “trial and error” the mesenchyme cells gradually disperse from their point of origin and take up positions on the inner surface of the ectodermal layer. The pseudopods, being elongations of the cell cytoplasm, are supported by bundles of microtubules.
Another case in which the transparency of the early embryo was used for observing movements of cells is the blastodisc of a bony fish. The surface cells of the blastodisc form an immobilized continuous layer, but the deeper-lying cells in the blastula and gastrula stages are in a state of continuous movement and can be photographed on cine film through the surface layer.
The locomotion of the deep cells is more similar to the classic type of ameboid movement. The movement starts by the cell’s forming a broad rounded “bleb” on its surface which appears to consist of hyaline cytoplasm, without the coarser cell inclusions (mitochondria, yolk granules, etc.). The bleb may be extended into a tongue-like lobopodium.
As the lobopodium is formed and extended, more cytoplasm flows into it. The bleb or lobopodium (the terms are interchangeable) can sometimes be seen to flatten and attach itself to adjoining cells. When it subsequently contracts, it pulls the cell forward. The cell may, however, progress forward as a whole, without a visible contraction or withdrawal of the lobopodium.
Cell movements somewhat similar to those of the primary mesenchyme cells of the sea urchin gastrula play an important part in amphibian gastrulation as well. While “bottle cells” are responsible for the formation of the invagination at the surface of the embryo — for the formation of the pit or groove, the beginning of the archenteron—cells of the “inner marginal zone” initiate the move forward of the mesoderm and endoderm along the inner surface of the future ectoderm.
The moving cells have been studied after fixation and examination with the scanning electron microscope, as well as by direct observation after opening the gastrula and by filming the inner surface of the blastoderm. The cells at the tip of the invading mass were seen to develop a broad lobopodium, at their anterior ends, which is equivalent to the “ruffled membrane”, and also a slender process posteriorly (the “tail”!), which is attached to one of the cells that had not yet advanced as far.
It appears that the cells move forward with the aid of the ruffled membrane flattened against the inner surface of the future ectoderm, and by way of the posterior process the advancing cells drag behind them other cells of the mesoderm and endoderm. This mechanism would account for the gliding movement of the masses of endo-mesodermal cells along the inner surface of the blastoderm.
Microfilaments have not, as yet, been seen in the blebs or lobopodia of early embryonic cells, but there is some evidence that they may be present. This can be concluded from the fact that gastrulation in sea urchins is suppressed by cytochalazin B. Also, movements of cells of four- to seven-day-old chick embryos are suppressed by the same drug.
The latter observation was made in the course of an experiment designed to test whether active movements of cells are the means by which the re-aggregation and “sorting out” of different kinds of cells are achieved after embryonic tissues have been dis-aggregated.
It was found that cytochalazin B does not prevent the cells from adhering to one another on contact but that the aggregates formed do not attain the regular spherical shape, and the sorting out of different kinds of cells in the interior of the aggregates does not take place.
These results show that the active movement of cells is directly involved in their taking up the correct positions in the aggregates (and thus also in the normal embryo!). At the same time, the results indicate that the movement of embryonic cells, as well as the movement of fibroblasts in tissue culture, depends on the presence and activity of microfilaments.
To become a morphogenetic factor, the movement of cells must be directed in definite pathways. By moving in an organized way, the mesenchyme-type cells form accumulations in specific parts of the embryo, which become rudiments of organs (e.g., rudiments of many skeletal parts), or the cells accumulate around epithelial organ rudiments, forming connective tissue or skeletal capsules of internal organs.
The properties of the substratum along which the cells migrate are among the major factors affecting the direction of migration. The cells cannot move through space filled only with fluid. In the parts of the embryo occupied by mesenchyme, however, the intercellular spaces are filled with a colloidal solution which is in part gelated, so that fibers of molecules are stretched through the space in various directions.
These fibers serve as a substratum for the migration of mesenchyme cells. If the fibers of the intercellular substratum do not show any orientation, the migration of mesenchyme cells is disorganized, and the cells become scattered evenly in space. If the fibers of the intercellular substratum become oriented in any particular direction, the mesenchyme cells migrate along the oriented bundles of fibers.
This can be proved experimentally with cells in tissue cultures. The fibers of the plasma clot on which the cells are cultivated may be caused to be stretched in a certain direction by various means, for instance by allowing the clot to set under mechanical tension. The cells will then follow the direction of the plasma clot fibers in their migration.
If the mesenchyme cells aggregate around an epithelial vesicle, this aggregation may be the result of the orienting influence of the epithelium on the fibers of the intercellular substrate.
If these fibers are stretched perpendicularly to the surface of the epithelial vesicle, the migrating mesenchyme cells will converge toward the vesicle. Having reached the surface of the epithelium, the cells of the mesenchyme may be held there by intercellular bonds whose nature is not well understood but whose existence can hardly be doubted.
The influences of adjoining parts on the movements of migrating cells are not the only form of interaction of parts playing an important role in the development of organ rudiments. Influences of a nature similar to the induction of the neural plate by the roof of the archenteron have been discovered in the development of organ rudiments. Such influences may modify the movements of cells, but not directly. The direct result is a modification of the properties of cells, and consequently the movements of cells are also altered.
This can be illustrated by the development of the neural crest which differentiates together with the neural plate as a result of an induction from the roof of the archenteron. Once induced, the neural crest cells acquire new properties. They become mesenchyme cells and start migrating away from their source of origin, while both the cells of the epidermis and the cells of the neural tube remain relatively stationary.