In this article we will discuss about the process of determining primary organ rudiments of embryos.
The activation of genes at the beginning of gastrulation and the resulting synthesis of new kinds of proteins is an important step in development, but in itself it would not be sufficient for initiating the formation of different organs and organ systems of the embryo. In order for this to occur, the new proteins must be placed in different parts of the embryo, so that these different parts may each undergo a particular type of differentiation.
Displacing the masses of cells of the embryo in gastrulation is a means of attaining the same result, some groups of cells remaining on the surface, as ectoderm, and some sinking into the interior, as mesoderm and endoderm. The difference in the positions taken up by cells or groups of cells in the embryo becomes a factor in guiding the cells to different goals.
Also, the change in the relative positions of cells brings some of the groups of cells into the proximity of other groups, which were distant from one another in their original positions, prior to gastrulation. This change in position provides possibilities for new interactions between parts of the embryo.
The experiments of local vital staining and similar methods, allowing the experimenter to reconstruct fate maps, do not furnish any information as to whether or not the cells in the various areas actually have different potentialities for development. The only information that these experiments give concerns the eventual destination of each part of the early embryo in normal development.
Certain distinctions between cells belonging to the various areas are apparent, such as differences in yolk content, pigmentation, and so on. There is, however, no a priori reason to believe that such differences are actually connected with the future development of each part.
To find out whether the presumptive areas of a blastula are actually predestined for a specific part in the future development, methods other than observing normal development are necessary. One of the methods used is the method of transplantation.
Small pieces may be cut out of embryos in various stages of development and inserted into suitably prepared wounds of the same or another embryo. In the case of transplantation of a piece of an embryo to another place in the same embryo, the transplantation is said to be autoplastic. If the transplantation is from one individual to another of the same species, the transplantation is homoplastic.
If the transplantation is to an individual of another species belonging to the same genus, the transplantation is heteroplastic. Transplantation to an individual more distantly related than species of one genus is called xenoplastic. The animal (embryo) from which a part is taken is referred to as the donor; the animal to which the part is transplanted is called the host or recipient.
In adult animals, especially highly organized animals such as vertebrates, transplantation is not easy, and only autoplastic and homoplastic transplantations are usually successful. In embryos, however, and in lower invertebrates (such as the coelenterates), the grafts may heal successfully even after xenoplastic transplantations. Successful transplantations have been carried out between embryos of frogs and salamanders and between mammals and birds.
It is usually important to know later which tissues and cells are derived from the graft and which from the host. The distinction is easy if donor and host are sufficiently different from each other. The cells may be distinguished by their size, staining properties, and other factors.
Sometimes differences may be found even between cells belonging to closely related animals. Differences in pigmentation may be very useful. The eggs of Triturus (Triton) cristatus, for instance, lack pigment, whereas eggs of other species of Triturus (T. taeniatus, T. alpestris) have fine granules of black pigment in their cytoplasm.
These pigment granules are distributed to all the cells during cleavage. If tissues of T. taeniatus and T. cristatus are joined in transplantation, the cells of T. taeniatus can be distinguished both macroscopically and in sections by the presence of pigment granules.
If no natural differences can be discovered between the two animals (embryos) used for transplantation, artificial differences may be introduced by staining one of the embryos with vital dyes. The presence or absence of the dye will indicate the position of the graft. In some cases the vital staining may be preserved in sections of the operated embryo by special methods.
Heteroplastic transplantation between Triturus cristatus and Triturus taeniatus embryos has been used to investigate the potency of the presumptive ectodermal areas—the epidermis area and the neural system area. A small piece of the epidermis area of an early gastrula was transplanted into the neural system area of another embryo in the same stage of development.
The result was that the grafted material developed in conformity with its new surroundings and became first a part of the neural plate of the host and later a part of the neural tube. There was every reason to conclude that it was differentiating as nervous tissue. In the case of a reverse transplantation, presumptive neural material differentiates into skin epidermis in conformity with its new surroundings.
Presumptive ectoderm (either from the epidermis area or from the neural system area) was also transplanted into the marginal zone of an early gastrula. In this case, the graft was drawn into the blastopore in the course of gastrulation and was later found in different places in the interior of the host. In every case, the graft differentiated in conformity with its surroundings and took part in the development of various organs of the host – the notochord, somites, lateral plate mesoderm, kidney tubules, and wall of the gut.
It is thus evident that the fate of presumptive nervous system and presumptive epidermis is not fixed at the time of the stages used in the preceding experiments. Besides having a definite normal fate—called the prospective significance—the parts tested possess abilities to develop in various other ways, under experimental conditions. This ability of the parts of an early embryo to develop in more than one way is called the prospective potency of these parts.
The prospective potency of the neural area in the early gastrula stage is therefore shown to include not only epidermis but also mesodermal and endodermal tissues. The prospective potency of the epidermis area includes nervous system and mesodermal and endodermal tissues as well. The two prospective potencies are practically identical, in spite of the different prospective significance.
The potencies of the marginal zone and the vegetal region could not be tested as easily as those of the presumptive ectoderm, because they both tended to invaginate into the interior from every position in which they were placed. However, in exceptional cases parts of the transplanted marginal zone may remain on the surface at the end of gastrulation, and then they also conform to their surroundings in their development and differentiate as epidermis or neural system.
An entirely different result is observed if the transplantation of pieces of ectoderm is performed at the end of the period of gastrulation. A transplanted piece of presumptive neural system area will differentiate as brain or spinal cord in whatever parts of the embryo it has been placed.
Usually in this case, the transplanted tissue sinks from the surface of the embryo—this would correspond to neurulation in normal development— and develops into a vesicle with thickened walls. Sometimes definite parts of the neural system can be recognized, such as one or other of the brain vesicles or an eye.
The presumptive epidermis of a completed gastrula likewise loses the ability to differentiate as nervous system. If it is transplanted into the nervous system area it does not conform to its surroundings and may interfere with the closing of the neural tube, but even if it does become enclosed inside the neural tube it differentiates into epidermis and not into nervous tissue.
Obviously some change has occurred between the early gastrula and the completion of gastrulation. The prospective potencies of presumptive neural tissue and epidermis have been narrowed down and have become the same as their prospective significance, as far as these experiments go. This narrowing down of the prospective potency, which is equivalent to fixing the fate of a part of the embryo, is known as determination.
After the process of determination has taken place the respective parts are said to be determined. The experiments described so far show that the neural system as a whole is determined by the end of gastrulation. They do not show, however, whether every part of the neural system is determined at the same time.
The experiments just described also show that the determination of parts of the ectoderm does not take place from causes inherent in the ectoderm itself. The differentiation of parts of the ectoderm is dependent on the surroundings in which the ectodermal cells find themselves.
Further experiments have proved that the condition which is decisive in determining the development of the neural plate is contact of the ectoderm with the roof of the archenteron—i.e., with the sheet of cells representing mainly the presumptive notochord and somites—which shifts forward underneath the ectoderm during gastrulation. Parts of the ectoderm in contact with the archenteron roof differentiate into neural plate and nervous tissue.
One of the most spectacular experiments showing the role of the archenteron roof was performed by Spemann’s pupil, Hilde Mangold. H. Mangold transplanted heteroplastically (from Triturus cristatus to Triturus taeniatus) a piece of the dorsal lip of the blastopore of an early gastrula.
The graft was placed near the lateral lip of the blastopore of the host embryo (also an early gastrula), and subsequently most of the graft invaginated into the interior, leaving, however, a narrow strip of tissue on the surface. When the host embryo developed further, it was found that an additional whole system of organs appeared on the side where the graft had been placed.
The additional organs together comprised an almost complete secondary embryo. This secondary embryo lacked the anterior part of the head. The posterior part of the head was present, however, as indicated by a pair of ear rudiments. There was a neural tube flanked by somites and a tail-bud at the posterior end.
A microscopic examination revealed the presence of a notochord, kidney tubules, and an additional lumen in the endoderm representing the gut of the secondary embryo. All the parts of the secondary embryo were found to lie at about the same level as corresponding parts of the host.
Since heteroplastic transplantation was used in this experiment, it was possible to determine which parts of the secondary embryo were developed from the graft and which were developed from the cells of the host. It was found that the notochord of the secondary embryo consisted exclusively of graft cells; the somites consisted partly of graft and partly of host cells.
A small number of graft cells were present in the neural tube; these were certainly the cells that had not invaginated. The bulk of the neural tube, part of the somites, the kidney tubules, and the ear rudiments of the secondary embryo consisted of host cells. The additional lumen in the endoderm was also surrounded by host cells.
All these latter parts would not have developed if the graft had not been present. It follows that their development was due to some influence of the graft. This influence, causing the development of certain organs of the embryo, is called embryonic induction. The part which is the source of the influence is called the inductor.
From the experiment just described, it may be concluded that the induction of the neural system is due to the activity of the underlying tissues, the presumptive notochord and the presumptive somites in particular. The issue is somewhat complicated by the participation of both graft and host cells in some of the organs of the secondary embryo.
This was later shown not to be essential; in other experiments the whole of the induced neural tube was developed exclusively from host tissue. Furthermore, the anterior parts of the brain could also be induced by the roof of the archenteron, so that the induced nervous system might be complete.
In special experiments, it has been ascertained which parts of the gastrula are capable of inducing the neural system. Pieces were taken from all parts of the early gastrula and each slipped into the blastocoele of an embryo in the early gastrula stage. Only grafts taken from the dorsal lip of the blastopore and the adjoining parts of the marginal zone were found to be able to induce. The area capable of induction coincided, in fact, almost exactly with the presumptive areas of notochord, somites, and prechordal plate.
Likewise, it has been ascertained which embryonic tissues are able to react to the induction by developing as nervous system. The tissue in question is the ectoderm or presumptive ectoderm of the gastrula.
The reactive ability is highest in the early gastrula stage, is still high in the mid-gastrula stage, begins to decline in the late gastrula, and fades away with the beginning of neurulation. This can be shown by transplanting the inductor underneath the ectoderm in successive stages, and the result may be expressed as a percentage of successful inductions.
In later stages, not only does the percentage of inductions diminish, but the volume and degree of differentiation are also reduced. Complete neural systems or large brain vesicles may be induced by transplantation of inductors in the early gastrula stage, but in the early neurula inductions are very weak; they may consist of only a few cells differentiating as nervous tissue.
The presumptive ectoderm of the blastula is not able to react to the inductive stimulus; if the inductor is transplanted in the blastula stage, the ectoderm reacts by the formation of a neural plate only after the gastrulation is completed. In all cases, the induced neural system develops simultaneously with the neural system of the host.
All this shows that the reacting cells must be in a particular state to be able to differentiate into nervous system under the influence of the inductor. This particular state of reactivity is referred to as competence. The competence for neural induction is restricted to the ectoderm and is present during a short period only.
If during this period the presumptive ectoderm is not stimulated to differentiate as neural tissue, it differentiates as epidermis. No special stimulation (induction) is necessary to cause the latter differentiation, though it has been found that certain conditions must be satisfied if the epidermis is to develop progressively and to produce normal skin epithelium.
The presumptive ectoderm isolated in the early gastrula stage loses the regular epithelial arrangement of its cells, and becomes an irregular spongy mass of cells. The differentiation of the cells, however, proceeds as in normal epidermis. In particular, part of the cells differentiates as ciliary cells, found in normal neurula and early tadpole embryos.
The cells also develop tight junctions and desmosome junctions found in normal epidermis. The lack of regular stratified arrangement is the result of the absence of the underlying mesoderm—a dependence which is found, in a number of epithelium-mesenchyme systems.