A correct interpretation of gastrulation is impossible without a knowledge of the position which the presumptive germinal layers occupy in the blastula. This position may be ascertained in various ways. A chart, showing the fate of each part of an early embryo, in particular, a blastula, is called a fate map.

In tracing the fate of various parts of the blastoderm, it is sometimes possible to make use of the peculiarities of the cytoplasm in certain parts of the egg, such as the presence of pigment granules. In the developing amphibian egg, for instance, one may trace in which part of the differentiated embryo the black pigment comes to lie.

Originally this pigment is restricted to the animal hemisphere of the egg. However, peculiarities of pigmentation are seldom sufficient to make it possible to reconstruct the fate map in any great detail. Recourse must be had to artificial marking of parts of the blastoderm.

A satisfactory method of marking was devised for this purpose by Vogt (1925). The method consists in soaking a piece of agar in a vital dye (Nile blue sulfate, neutral red, Bismarck brown) and then applying the piece of agar to the surface of the embryo in the necessary position.

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The dye diffuses from the agar, and in a matter of minutes the cells of the embryo to which the agar has been applied take up sufficient dye to produce a stain on the surface of the embryo. This marking-can is done without removing the vitelline membrane, since it is permeable to the vital dyes, and thus the embryo continues to develop normally.

The presence of the stain does not change the normal development of the embryo, and the position of the stained cells in the differentiated embryo clearly shows the fate of the stained area. It has been established by trial that the vital dye remains, on the whole, restricted to the cells which had originally taken up the dye and to their descendants.

The diffusion of the stain, if the staining has been done correctly, is negligible and does not interfere with interpreting the results. Several stain marks may be made on the surface of the same embryo, using different colors (red, blue, brown). In this way one experiment may disclose the fate of many parts of the early embryo at the same time.

It was later found that cellophane can also be used as a stain carrier instead of agar and that it is actually a more convenient one, as cellophane comes in thin sheets from which it is easy to cut out pieces of the desired size and shape. All vital staining is now done with cellophane as the stain carrier.

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Independently of vital staining, another method has also been devised for marking cells of a developing embryo. This consists in applying tiny particles of carbon to the 196 surface of the embryo. Carbon particles stick to the surface of the cells and can thus be used as markers enabling the investigator to follow the movements of the cells and to draw up fate maps.

The vital stain marking method was first applied to the reconstruction of the fate map in the amphibian embryo, and the original investigations have subsequently been checked by many embryologists. It is most advantageous, therefore, that the fate map of an amphibian embryo, such as the embryo of a newt (Triturus) or axolotl (Ambystoma), be described first.

The Fate Map of a Urodele Amphibian Embryo:

The whole surface of the blastula of an amphibian may be roughly divided into three main regions:

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(1) A large area on and around the animal pole,

(2) An intermediate zone, also known as the marginal zone, extending all around the equator of the blastula, and

(3) The area on and around the vegetal pole.

These three main regions coincide approx­imately with areas which differ in their pigmentation. The whole of the animal region is deeply pigmented. The marginal zone, which is much broader on one side of the embryo, is pigmented but not so deeply as the region around the animal pole. The vegetal region has very little pigment.

Inside each region we find areas corresponding to the future organs of the animal. The animal region consists of two main areas- the area whose fate is to develop into the nervous system of the embryo, and the area which is to become the skin epidermis of the embryo. The material for the sense organs is also contained in these two areas.

Inside the nervous system area, a small subarea may be traced which is to participate in the formation of the eyes of the embryo; inside the epidermis area, the material for the nose, the ears, and the ectodermal part of the mouth may similarly be traced. In the inter­mediate or marginal zone, we find the material for the notochord occupying a large area on the dorsal side of the blastula.

This is followed by an area lying nearer to the vegetal pole and containing the material for the prechordal connective tissue; this area is known as the prechordal plate. Farther down, toward the vegetal pole but still inside the marginal zone, lies the material for the anterior parts of the alimentary canal – the endodermal lining of the mouth, gill region, and pharynx.

The parts of the marginal zone on both sides of the notochordal area are taken up by the material for the segmental muscles of the body. The lateral and ventral parts of the marginal zone give rise to the mesodermal lining of the body cavity, the kidneys, etc. (ventrolateral mesoderm). The vegetal region is composed of cells which are later found in the midgut and hindgut.

Local vital staining of the surface of the embryo, if performed carefully, leaves the color only in the superficial cells; strictly speaking, the fate map shows only the destina­tion of the cells of the superficial layer.

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The blastoderm of the amphibian embryo in the blastula stage, however, is not a simple (that is, single-layered) epithelium but a stratified epithelium, and the nearer it is to the vegetal pole, the greater the number of layers of cells. Yolk-laden cells are stacked in large numbers on top of one another in the vegetal hemisphere of the blastula.

The number of cell layers is least at the animal pole, but in the marginal zone the blastoderm is many cells thick. The cells lying in the interior in the marginal zone are intermediate in size and yolk content between the cells of the vegetal field and the cells of the animal hemisphere, and form a ring in the interior of the blastula, called the inner marginal zone.

The destiny of the cells not reaching the surface need not be the same as those on the exterior, and their fate cannot be ascertained from surface color marks. In fact, Vogt could not detect the location in the blastula of the material for some mesodermal structures- the cells going into the formation of the heart, and of the blood islands. He concluded that these parts develop from cells of the inner marginal zone.

Vogt, using the same methods, also constructed a fate map for a frog blastula, that of Bombina. In Fig. 127 is presented the fate map of another frog, Discoglossus, worked out by Pasteels (1942), which embodies some minor corrections to Vogt’s map. The general arrangement of the various areas is the same as in the fate map for a urodelean blastula, but all presumptive areas are more concentrated toward the marginal zone. In particular, the area for the neural plate does not reach, by far, the animal pole.

A fate map has been worked out also for an earlier stage of an amphibian egg, namely for the 32-blastomere stage of the frog Xenopus laeuis. At the 32-cell stage the blastomeres are arranged in four tiers, each tier consisting of eight cells. For reference the blastomeres belonging to each tier are designated by capital letters –  A for the topmost tier (the tier at the animal pole), B, C, and D for the following tiers in descending order.

The tier C forms the marginal zone, and the tier D consists of the vegetal pole blastmeres. Within each tier the blasto­meres are designated by the numbers 1-4, on each side, starting from the mid-dorsal plane. Each individual blastomere was stained with Nile blue sulfate, following Vogt’s method. The essential results of the staining were as follows.

Blastomeres A1, right and left, give rise to the anterior end of the neural plate, including the eye rudiments, and the transverse neural fold. The rest of the neural plate is contained in the blastomeres B1 B2 and C2. The epidermis is produced from blasto­meres A2 A3 A4 B2 B3 B4 and C3 C4. Most of the notochord is derived from the blasto­meres B1, right and left. The posterior part of the notochord is derived from blastomeres C1, right and left.

The same blastomeres also contain the material for the foregut. Most of the somites are derived from the blastomeres C2 and C3. The blastomeres of the D tier go entirely into the formation of the gut, the blastomeres D1 D2 and D3 forming the midgut, and the blastomeres D4 forming the hindgut. Blastomeres C3 and C4 also contribute to a slight extent to the formation of mid- and hindgut.

Comparing the fate map arising from this experiment with the fate map for a frog blastula two essential differences may be noted. Firstly, the neural plate area reaches further toward the animal pole in the morula stage; in fact, the transverse neural fold lies at the animal pole. This shows that between cleavage stages and the beginning of gastrulation there is a shifting downward of the presumptive neural material, toward the equator.

This shifting was actually noted by Nakamura and Kishiyama. The second difference concerns a much greater concentration of the material for the notochord toward the mid-dorsal plane. It is thus possible that between cleavage and the beginning of gastrulation, while the presumptive neural material shifts downward toward the equator, the equatorial material, the presumptive notochord in particular, spreads out sideways.

In the 32-cell stage all blastomeres lie superficially, and are presumably stained right through. There are no blastomeres in the interior. Thus parts of the cells which will later become internal, in particular cells of the inner marginal zone, would all be stained in the present experiment. Rudiments later located in the superficial layer, and those derived later from cells lying in the interior, May well be derived from the same blastomeres of the 32-cell embryo.

It will be noticed that the areas destined to develop into the organs of the mid-dorsal part of the animal lie on one side of the blastula, on that side where the marginal zone is taken up by the notochord area. Nearer to the animal pole the area of the neural system is situated. This side of the blastula corresponds therefore to the dorsal side of the embryo.

Similarly, parts of the head (eye, nose, ears) develop from areas near the animal pole of the blastula, which therefore corresponds to the anterior end of the embryo. Since the materials of the egg have not been displaced to any great extent during the cleavage, it may be inferred that the animal pole of the fertilized egg corresponds to the anterior end of the embryo.

The side where the marginal zone is the broadest is the dorsal side; the opposite side may be considered as ventral, and the vegetal pole as the posterior. We find, however, that the foregut area (pharynx, part of the epithelium of the mouth) is also situated on what we have agreed to call the dorsal side of the egg.

On the whole, however, the location of the areas destined to develop into most of the organs does not seem to have anything in common with the position of the same organs in the adult animal.

Organs which later are situated in the interior of the animal’s body—such as the notochord, the gut, or the brain—are represented by areas laid out on the surface of the blastula. The cells destined to cover the whole of the animal’s body, such as the epidermis of the skin, occupy only a limited area on the surface of the blastula.

Parts of the three germinal layers—ectoderm, endoderm, and mesoderm—are all located on the surface of the embryo, instead of being in a concentric arrangement, with the ectoderm on the outside, the endoderm in the middle, and the mesoderm in between. It is obvious that a far-reaching displacement or reshuffling of the parts of the blastoderm must take place before each cell can arrive at its final position.

This displacement of parts of the blastoderm, which are eventually rearranged in a system of concentric layers of cells, is the essence of the process of gastrulation. We shall now, for a time, leave the amphibians and consider the process of gastrulation in an animal with a more simple organization, a representative of lower chordates, the Amphioxus.

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